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PDF file - 40K, Figure S2: Biomarkers of in vivo response to inhibitors of PI3K/mTOR signaling. Reverse Phase Protein Analysis (RPPA) of KPL-1 tumor samples taken after 7 days treatment with the molecules indicated. Tumors taken 24 hours post final dose. A, pAKT Ser473. B, pS6 Ser235/236.

PDF file - 36K, Figure S3. Xenograft response data for 7-day tissue acquisition studies. A-C, SUM190, UACC812 and KPL-1 xenografts (6 mice per arm) were treated with BKM120 at 35 mg/kg, BYL719 at 50 mg/kg, RAD001 at 10 mg/kg and BEZ235 at 30 mg/kg, all daily PO. Trastuzumab was given at given at 10 mg/kg IP twice weekly. For SUM190 (A), tumors were collected 2.5 hours post final dose, for UACC812 (B) and KPL-1 (C), samples were collected 24 hours post final dose.

PDF file - 772K, Figure S1: Targeting the PI3K/mTOR pathway in HER2-amplified breast cancer cell lines. Effect of A, RAD001, B, BEZ235, C, BKM120 or D, BYL719 treatment on PI3K/mTOR signaling in two representative cell lines; SKBR3 and MDA453 (PIK3CA mutant H1047R). All lysates taken 24 hours post-treatment.

XLSX file - 213K, Table S2. Summary of in vivo activity of BKM120, BYL719, RAD001 and BEZ235 in breast cancer tumor models. T/C: Ratio of mean tumor volume of treated (T) to control (C) at final day of treatment. Final day was determined as the final day of treatment for the vehicle control arm for each study in Figure 5. Comparisons between groups were all made by Repeated Measures ANOVA (RMANOVA) followed by Dunnett test for multiple comparisons. BW: body weight; TR: treatment related; NTR: non-treatment related.

XLSX file - 14K, Table S1: Calculated IC50s for the PI3K/mTOR pathway inhibitors against the breast cancer cell line panel. ER: Estrogen Receptor. N/A: IC50 not achieved, N/D: IC50 not determined

Introduction: TPI is a hallmark of cancer and plays a pivotal role in tumor initiation, development, progression, and invasiveness. High levels of tumor-promoting inflammatory cytokines e.g. IL-1β and IL-6 are associated with advanced malignancies and reduced survival. Selective IL-1β inhibitors like canakinumab (CAN) and gevokizumab (GEV) aim to target TPI within the tumor microenvironment (TME) by reducing tumor-associated immune suppression. In phase III CANTOS study, CAN significantly reduced lung cancer (LC) incidences and LC related mortalities in atherosclerosis patients, providing the first clinical evidence for the role of IL-1β inhibition in LC. We have utilized preclinical models to gain further insights into the mechanistic role of CAN and GEV in cancer development and progression. Methods: Preclinical efficacy of anti-human IL-1β antibodies, CAN and GEV was assessed using humanized mouse models of non-small cell lung cancer (NSCLC, H358), triple-negative breast cancer (TNBC, MDA-MB-231) and colorectal cancer (CRC, SW480) either as single agents or in combination with other immunomodulatory agents including PD-1 inhibitor pembrolizumab (PEM) and anti-VEGF therapy. A murine surrogate IL-1β blocking antibody clone 01BSUR was used to evaluate efficacy in murine syngeneic models of breast (4T1) and lung (LL-2) cancer either as single agent or with PD-1 inhibitor and docetaxel (DOC). Tumor infiltrating lymphocytes (TIL) characterization was carried out by flow cytometry and IHC in syngeneic and humanized models, respectively. Results: The IL-1β inhibitors showed anti-tumor activity in both murine syngeneic and humanized models as single agents or with other agents. In the H358 NSCLC mouse model, CAN + PEM significantly inhibited tumor growth and was associated with >50% regression vs control. Decreased tumor growth was also observed with GEV treatment in the SW480 CRC mouse model, both alone and with anti-VEGF therapy. In GEV+anti-VEGF arm, a decrease in tolerogenic DC-10 immune populations and an increase in CD45+ immune cells and CD68+ myeloid cells were also observed. In the MDA-MB-231 TNBC mouse model, CAN single-agent showed a decrease in tumor growth vs control. In murine syngeneic 4T1 and LL-2 models, anti-IL-1β alone and anti-IL-1β + anti-PD-1 resulted in increased presence of CD8+ T cells and reduction of immunosuppressive myeloid-derived suppressor cells, neutrophils, and regulatory T cells in the tumor, suggesting that the TME may be shifting to a less suppressive phenotype after IL-1β blockade. Similar changes were observed with combination of anti-IL-1β with DOC and anti-VEGF in murine syngeneic models. Conclusions: These data provide key insights into the biology behind IL-1β blockade currently being pursued in clinical trials with CAN and GEV. Additional preclinical studies to investigate mechanism of action of these agents and other novel combinations are planned. Citation Format: Pushpa Jayaraman, Vanessa Rodrik-Outmezguine, John Millholland, Neil O'Brien, Connie Wong, Rohan Diwanji, Muchun Wang, Elizabeth Choi, Ronald Linnartz, Kristine Rose, Marc Pelletier, Alexander Savchenko, Tong Luo, Valerie Rezek, Scott Kitchen, Jason Baum, Catherine Sabatos-Peyton, Anne-Marie Martin, Glenn Dranoff. Targeting tumor-promoting inflammation (TPI) via the IL-1βpathway for cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5640.

Background: Altered PI3K/mTOR signaling, through activating mutations in PIK3CA and/or PTEN loss, has been implicated in resistance to both hormonal and HER2-directed therapeutics for breast cancer. Recent data from the BOLERO-1 phase III clinical trials demonstrate an improvement in progression free survival (PFS) of 7.2 months in patients with HER2+/estrogen receptor negative (HER2+/ER-) advanced breast cancer when treated with trastuzumab and paclitaxel plus the mTORC1 inhibitor, everolimus (Afinitor ®). However, improvement in PFS was not observed in patients with HER2+/ER+ positive disease. These data suggest that hormone receptor levels may influence the response of HER2-amplifed cells to both HER2 and PI3K/mTOR directed therapies in the absence of hormonally directed therapies. In this study, we investigated the role of ER in HER2+ breast cancer by screening a large panel of HER2+/ER- and HER2+/ER+ breast cancer cell lines for responses to single agent and combined treatment with PI3K, mTOR, HER2 and ER-directed therapeutics. Materials and Methods: The anti-proliferative activity of BKM120 (pan-PI3K inhibitor), BYL719 (p110α-specific) and everolimus were assessed as single agents or in combination with trastuzumab and/or tamoxifen in a panel of six HER2+/ER+ versus six HER2+/ER- breast cancer cell lines in two-dimensional culture. Drug response IC50s were generated from actual cell counts as measured by Z2-particle counting. Biomarker analyses were conducted using baseline mRNA microarray (Agilent) and reverse phase protein array (RPPA) profiling of each of the cell lines to determine associations with response or resistance data. Results: RPPA analysis confirmed the presence of higher levels of ER protein in the cell lines designated as ER+ and significantly higher levels of PTEN protein were detected in those cell lines. Interestingly, each of the PI3K/mTOR pathway inhibitors tended to have increased single agent activity in the ER+ relative to the ER- lines. Combined activity with trastuzumab and either BKM120 or BYL719 was observed in 6 of the 12 cell lines tested and occurred independent of ER status. Similarly, combined activity of everolimus and trastuzumab was also observed in both HER2+/ER+ and HER2+/ER- cell lines. In 3/6 HER2+/ER+ cell lines the addition of tamoxifen provided no benefit to the combination of trastuzumab and PI3K/mTOR pathway inhibitor, whereas in two cell lines mild antagonism was observed with the triple combination. Finally, one cell line did show significant potentiation from the addition of endocrine therapy on top of HER2/PI3K/mTOR targeting. Discussion: These data confirm levels of estrogen receptor are likely playing a role in response to single agent PI3K/mTOR pathway inhibition and highlight the potential utility of combining endocrine therapy with HER2/PI3K/mTOR-directed therapeutics in a sub-group of HER2-amplified breast cancers. Further in depth biomarker analyses may reveal additional molecular alterations responsible for this differential sensitivity to the double and triple combinations and is underway. Citation Format: O'Brien NA, Nichols CM, Thomas J, Conklin D, Kalous O, Linnartz R, Di Tomasso E, Hurvitz SA, Hirawat S, Slamon DJ. Single agent and combined targeting of PI3K, mTOR, HER2 and ER signaling in a panel of HER2+/ER+ versus HER2+/ER- breast cancer cell lines. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-14-25.

Everolimus (RAD001, Afinitor(®)) is an oral, selective mTOR inhibitor recently approved by the US-FDA in combination with exemestane for treatment of hormone receptor positive advanced breast cancer. To date, no molecular predictors of response to everolimus in breast cancer have been identified. We hypothesized predictive markers could be identified using preclinical models. Using a molecularly characterized panel of human breast cancer and immortalized breast epithelial cell lines, we determined sensitivity to everolimus alone or in combination with ER- or HER2- targeted therapy. Gene expression microarrays and comparative genomic hybridization were performed on the cell lines to identify predictors of response to everolimus. Among 13 everolimus-sensitive cell lines, 10/13(77 %) were luminal, while in 26 resistant cell lines, 16/26(62 %) were non-luminal, and 10/26(38 %) were luminal. Only 3/24 non-luminal lines were sensitive, two of which were HER2+. Everolimus enhanced the anti-proliferative effect of both tamoxifen (TAM) and fulvestrant (FUL) in ER+ breast cancer cell lines, as well as trastuzumab in HER2+ cell lines. Everolimus + FUL but not everolimus + TAM reversed acquired resistance to TAM. Everolimus inhibited mTOR in tested cell lines by decreasing S6 phosphorylation, mediating its anti-proliferative effect by G0/G1 cell cycle arrest and induction of apoptosis. Chromosomal amplifications of AURKA (p value = 0.04) and HER2 (p value = 0.03) were each associated with increased sensitivity to everolimus. Transcript expression microarrays identified GSK3A, PIK3R3, KLF8, and MAPK10 among the genes overexpressed in sensitive luminal lines, while PGP, RPL38, GPT, and GFAP were among the genes overexpressed in resistant luminal cell lines. These preclinical in vitro data provide further support for continued clinical development of everolimus in luminal (ER+ or HER2+) breast cancer in combination with targeted therapies. We identified several potential molecular markers associated with response to everolimus that will require validation in clinical material.

Background: To study the molecular mechanism regulating sensitivity to MEK inhibition in pancreatic cancer cell lines. Methods: A growth inhibition assay determined sensitivity to MEK162 in a panel of 29 pancreatic cancer cell lines. For the same panel, KRAS mutational status and copy-number variation (CNV) was determine using PCR, array CGH and FISH. Two sensitive and two resistant cell lines were further interrogated for difference in baseline and MEK162-induced gene expression, as well as signal transduction using microarray and western blotting. Cell cycle and apoptosis analysis was measured by flow cytometry. Results: We report a strong correlation between both specific KRAS mutational subtype and CNV, and sensitivity to MEK inhibition. Cell lines with a KRAS (V12) mutation and KRAS gains or loss (n=7) are ∼10 times more resistant than those having neither a KRAS (V12) mutation nor KRAS CNV (n=14). Significant differences in baseline and MEK162-induced gene expression exist between the sensitive and resistant lines, especially in genes involved in RAS, EGF receptor and PI3K pathways. This was further supported by difference in signal transduction. MEK 162 blocked ERK1/2, as well as inhibited PI3K and S6 and increased p27KIP1 levels in the sensitive lines. Conclusions: Given the potency of MEK162, it may be a promising new therapy for patients with pancreatic cancer and KRAS mutational subtypes, and CNV may serve as important biomarkers for selecting patients that benefit from MEK-targeting based on these preclinical data.

Introduction: Inflammation is a hallmark of cancer wherein diverse immune cells exert either pro- or antitumor properties and affect therapeutic resistance (Ritter B et al, JEM 2019). High concentrations of tumor-promoting inflammatory cytokines such as IL-1β (Litmanovich A et al, Oncol Ther 2018) and IL-6 (Altundag O et al, J Clin Oncol 2005) correlate with advanced malignancies and are associated with reduced survival. A recent phase III clinical trial (CANTOS) demonstrated that canakinumab, a selective IL-1β inhibitor, could significantly reduce lung cancer incidences and lung cancer related mortalities as a secondary endpoint in atherosclerosis patients, providing the first clinical evidence for the role of IL-1β inhibition in lung cancer. Canakinumab and other selective IL-1β inhibitors such as gevokizumab aim to target tumor promoting inflammation (TPI) within the tumor microenvironment (TME) by reducing tumor-associated immune suppression. In an effort to gain further insights into the clinical mechanistic role of canakinumab and gevokizumab in cancer development and progression, we have utilized preclinical models to understand the immunological events following IL-1β blockade. Methods: Preclinical efficacy of anti-human IL-1β antibodies, canakinumab and gevokizumab, was assessed in humanized mouse models of NSCLC (H358) and CRC (SW480) respectively. Anti-mouse IL-1β blocking antibody was used to evaluate efficacy in murine syngeneic models of breast (4T1) and lung (LL-2) cancer either as a single agent or in combination with PD-1 inhibitor and docetaxel. Tumor infiltrating lymphocytes (TIL) characterization was carried out either by flow cytometry or by IHC in syngeneic and humanized models. Results: Using both murine syngeneic and humanized mouse models, we demonstrate that IL-1β inhibition remodels the TME and slows tumor growth either alone or in combination with other agents. In the humanized H358 NSCLC mouse model, the combination of canakinumab and the anti-PD-1 monoclonal antibody, pembrolizumab, significantly slowed tumor growth by more than 50% vs control antibodies. Furthermore, canakinumab alone and in combination resulted in increased CD8 and CD3 positive TIL infiltration within the tumor. Decreased tumor growth was also observed with another IL-1β inhibitor, gevokizumab, in a humanized SW480 CRC mouse model, both alone and in combination with anti-VEGF therapy. In syngeneic 4T1 and LL-2 models, anti-IL-1β alone and in combination with anti-PD-1 resulted in increased infiltration of CD8+ cells along with the reduction of immunosuppressive myeloid derived suppressor cells (MDSCs), neutrophils and regulatory T cells (Tregs) into the tumor, suggesting that the TME may be shifting to a less suppressive phenotype after IL-1β blockade. Similar changes to the tumor microenvironment were observed with the combination of anti-IL-1β with docetaxel. Additional combinations are being explored in other tumor models reflecting different indications. Conclusions: Our results highlight the role for IL-1β in tumor immunomodulation and that the pathophysiological role of the IL-1β pathway in innate immunity might have important consequences on T cell function and checkpoint blockade in cancer. These results support the ongoing clinical evaluation of IL-1β inhibitors such as canakinumab and gevokizumab and in combination with other therapeutic agents across several cancers. Citation Format: Pushpa Jayaraman, John Millholland, Neil O’Brien, Connie Wong, Rohan Diwanji, Muchun Wang, Elizabeth Choi, Ronald Linnartz, Kristine Rose, Vanessa Rodrik-Outmezguine, Marc Pelletier, Alexander Savchenko, Tong Luo, Valerie Rezek, Scott Kitchen, Jason Baum, Catherine Sabatos-Peyton, Anne-Marie Martin, Glenn Dranoff . Targeting IL-1β pathway for cancer immunotherapy [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 C103. doi:10.1158/1535-7163.TARG-19-C103

Addition of CDK4/6 inhibitors such as palbociclib, ribociclib or abemaciclib to endocrine-based therapies significantly improves progression-free and in some subgroups, overall survival in patients with advanced estrogen receptor-positive (ER+) breast cancer. However, acquired resistance to CDK4/6 inhibitors remains a significant unmet clinical need. It is critical to ascertain the mechanisms by which tumor cells evade CDK4/6 based therapy. In this study we screen multiple in vitro and in vivo models of acquired resistance to CDK4/6 inhibitors to identify potential resistance/escape pathways and targets for pharmaceutical intervention to overcome resistance. ER+ breast cancer cell lines of diverse molecular backgrounds were conditioned to acquire resistance to CDK4/6 inhibitors through either long-term culture in the presence of clinically relevant concentrations of inhibitors or as cell line xenografts treated through progression on a CDK4/6 inhibitor plus fulvestrant. Baseline and pharmacodynamic changes in cell signaling were measured using reverse phase protein array (RPPA) and RNAseq analysis. Acquired resistance to CDK4/6 inhibitors was associated with decreases in phosphorylated-Rb (pRb) and ER-alpha protein and increases in pAKT and pS6 relative to isogenic controls. The p110α-selective PI3K-inhibitor, alpelisib, in combination with fulvestrant or ribociclib/fulvestrant blocked pAKT signaling in xenografts progressing on palbociclib/fulvestrant and induced significant tumor regressions. Apelisib plus fulvestrant also produced robust anti-tumor responses in xenografts progressing on ribociclib/fulvestrant. Triple combination treatment with ribociclib/alpelisib/fulvestrant induced significant regressions in resistant tumors and in treatment naïve models, where complete tumor regressions occurred regardless of PIK3CA mutation status. Regressions were maintained for >9 weeks post withdrawal of treatment, indicating that therapeutic resistance may be prevented by this triple combination approach. RPPA analysis of responding tumors identified sustained inhibition of both PI3K- and CDK4/6:Rb-pathway signaling accompanied by activation of pro-apoptotic proteins. These data support clinical investigation of targeting PI3K with alpelisib in breast cancers progressing on CDK4/6 based therapies and investigation of upfront triple combination therapy prior to acquisition of resistance to CDK4/6. Citation Format: Neil A. O'Brien, Martina SJ McDermott, Dylan F. Conklin, Alex Gaither, Tong Luo, Raul Ayala, Suruchi Salgar, Emmanuelle DiTomaso, Naveen Babbar, Faye Su, Sara A. Hurvitz, Ronald Linnartz, Kristine Rose, Samit Hirawat, Dennis J. Slamon. Targeting activated PI3K/mTOR signaling overcomes resistance to CDK4/6-based therapies in preclinical ER+ breast cancer models [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 3825.

Background: The selective mTORC1 inhibitor everolimus (RAD001/Affinitor®) has recently been approved, in combination with exemestane, for treatment of advanced hormone receptor positive breast cancer after demonstrating significantly improved progression free survival for patients having progressed on a previous non-steroidal aromatase inhibitor. However, despite these improved outcomes many tumors go on to develop resistance to everolimus. Alterations in PI3K/AKT signaling, either through activating mutations in PIK3CA or loss of PTEN, have been shown to influence responses to everolimus treatment in vitro (Weigelt et al., 2011). In this study we used cell line xenograft models of everolimus resistance to assess the potential of a pan-PI3K inhibitor (BKM120) or a p110a-specific inhibitor (BYL719) to reverse resistance either as single agents or in combination with anti-ER therapy (Fulvestrant). Materials and Methods: Everolimus resistant xenografts were developed from three originally everolimus sensitive ER+ cell lines carrying distinct genetic alterations that confer aberrant PI3K/AKT signaling; ZR75-1 (PTEN null), MCF7 (PIK3CA mutant) and KPL1 (wild-type). Mice were treated by daily oral everolimus (10 mg/kg) until tumors began to progress on treatment (∼ 35 days of daily treatment). Mice that developed everolimus resistant tumors were randomized into six treatment groups (n = 6) as follows: continued on everolimus (10 mg/kg), fulvestrant (5 mg/wk), BKM120 (35 mg/kg), BYL719 (50 mg/kg), fulvestrant + BKM120 or BYL719. Results: All three cell line xenografts were initially responsive to everolimus. ZR75-1 xenografts were allowed to progress on everolimus until they reached a mean tumor volume greater than that on day zero, at which point mice were randomized into treatment groups. Tumor selected to continue on everolimus gained a mean tumor volume of 550 mm3 over the six-week duration of study, demonstrating resistance to everolimus. These tumors showed a partial response to Fulvestrant (28% TGI), BYL719 alone (40% TGI) and BYL719 combined with fulvestrant (61% TGI). In contrast, over 100% tumor growth inhibition (tumor regression) was observed with both single agent BKM120 or BKM120 plus fulvestrant. This is consistent with our in vitro findings that PTEN null tumor cells might be less sensitive to inhibition by BYL719 yet sensitive to BKM120. Data from the ongoing PIK3CA mutant and wild-type KPL1 models will inform whether these molecular subtypes of ER+ breast cancer are more sensitive to the pan-PI3K inhibitor or the p110α-specific inhibitor. Comprehensive pharmacodynamic analyses of these tumor tissues may identify the additional molecular determinants responsible for resistance to everolimus and help guide the design of future clinical studies. Discussion: These pre-clinical data indicate that targeting PI3K either alone or in combination with fulvestrant overcomes resistance to everolimus in ER+ breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD1-5.

Background: Aberrant activity of the PI3K/mTOR pathway has been implicated in resistance to trastuzumab and anti-hormonal therapy for HER2-amplified and ER-positive breast cancer, respectively. Previous studies in our laboratory and others have shown that increased PI3K/mTOR signaling, either through PTEN loss or activating PIK3CA mutations, can confer resistance to trastuzumab therapy. In this study, we assessed the potential of targeting the PI3K/mTOR pathway in overcoming both de novo and acquired trastuzumab resistance. Materials and Methods: The in vitro activity of the pan-PI3K inhibitor BKM120, the mTORC1 inhibitor RAD001 and the dual PI3K/mTORC1/2 inhibitor BEZ235 were evaluated in a panel of 49 human breast cancer and immortalized cell lines. The in vivo activity of these molecules was assessed in six cell line xenografts models representing, ER+/HER2− (KPL-1, ZR75-1), ER+/HER2+ (UACC812, MDA361), ER−/HER2+ (SUM190), PIK3CA mutant (SUM190, MDA361), PTEN-null (ZR75-1) and trastuzumab resistant (BT-TR) breast cancer. Finally, the effect of PI3K/mTOR inhibition on feedback activation of PI3K signaling and compensatory pathways was measured by Western blot, immunohistochemistry (IHC) and reverse phase protein analysis (RPPA) of control and treated cell lysates/tumors. Results: Using a sensitivity cut-off of an IC50 of < 1 µmol/L, 16 of the 18 HER2-amplified breast cancer cell lines and 8 of 10 cell lines with activating mutations in PIK3CA were sensitive to the pan-PI3K inhibitor BKM120. BEZ235 showed the most potent efficacy across the panel with IC50s < 100 nmol/L for each of the 49 cell lines tested. The HER2-amplified/PIK3CA mutant cell lines were also unexpectedly sensitive to the mTORC1 inhibitor RAD001, this was despite the silencing of mTORC1 signaling being followed by a feedback increase in phospho-AKT signaling. Furthermore, each of these molecules showed remarkable in vivo activity across the panel of xenografts models. BKM120, RAD001 and BEZ235 induced both tumor stabilization and regression independent of PTEN, PI3K, ER and HER2 status. Pharmacodynamic analysis of tumor tissue revealed that BEZ235 and RAD001 both inhibited mTORC1 signaling as indicated by a reduction in the levels of phosphorylated ribosomal protein S6 (pS6). However, in contrast to BKM120 and BEZ235, RAD001 did not induce a reduction in the levels of pAKT (S473 or TH308) yet showed comparable in vivo efficacy to each of these molecules. Finally, combined targeting of HER2 and PI3K/mTOR in vitro increased the anti-proliferative activity of the molecules and led to an increased induction of apoptosis. The efficacy of these molecules (alone or in combination with trastuzumab) was assessed in a model of in vivo trastuzumab resistance generated through long-term treatment of the trastuzumab sensitive BT474 cells (BT-TR). All PI3Ki induced complete inhibition of tumor proliferation in monotherapy, while the combination of trastuzumab and each of these molecules induced tumor regression in the trastuzumab resistant tumors. Discussion: These pre-clinical data indicate that targeting the PI3K/mTOR pathway either alone or in combination with trastuzumab is effective strategy for overcoming trastuzumab resistance. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-08-01.

Background & Aims Hepatocellular carcinoma (HCC) is the third leading cause of cancer death. Although sorafenib has been shown to improve survival of patients with advanced HCC, this improvement is modest and patients eventually have refractory disease. This study aims at investigating the antitumor, antiangiogenesis and antimetastatic activities of dovitinib in preclinical models of HCC. Methods 21-0208 and SK-HEP1 cells as well as patient-derived HCC models were employed to study the antitumor effect of dovitinib. Changes of biomarkers relevant to FGFR/VEGFR/PDGFR pathways were determined by Western blotting. Microvessel density, apoptosis and cell proliferation were analyzed by immunohistochemistry. Results Treatment of SK-HEP1 cells with dovitinib resulted in G2/M cell cycle arrest, inhibition of colony formation in soft agar and blockade of bFGF-induced cell migration. Dovitinib inhibited basal expression and FGF-induced phosphorylation of FGFR-1, FRS2-α and ERK1/2. In vivo , dovitinib potently inhibited tumor growth of six HCC lines. Inhibition of angiogenesis correlated with inactivation of FGFR/PDGFR-β/VEGFR-2 signaling pathways. Dovitinib also caused dephosphorylation of retinoblastoma, upregulation of p-histone H2A-X and p27, and downregulation of p-cdk-2 and cyclin B1, which resulted in a reduction in cellular proliferation and the induction of tumor cell apoptosis. In an orthotopic model, dovitinib potently inhibited primary tumor growth and lung metastasis and significantly prolonged mouse survival. Conclusions Dovitinib demonstrated significant antitumor and antimetastatic activities in HCC xenograft models. This study provides a compelling rationale for clinical investigation in patients with advanced HCC.

Objective Gastric cancer is a major gastrointestinal malignancy for which targeted therapies are emerging as treatment options. This study sought to identify the most prevalent molecular targets in gastric cancer and to elucidate systematic patterns of exclusivity and co-occurrence among these targets, through comprehensive genomic analysis of a large panel of gastric cancers. Design Using high-resolution single nucleotide polymorphism arrays, copy number alterations were profiled in a panel of 233 gastric cancers (193 primary tumours, 40 cell lines) and 98 primary matched gastric non-malignant samples. For selected alterations, their impact on gene expression and clinical outcome were evaluated. Results 22 recurrent focal alterations (13 amplifications and nine deletions) were identified. These included both known targets ( FGFR2 , ERBB2 ) and also novel genes in gastric cancer ( KLF5 , GATA6 ). Receptor tyrosine kinase (RTK)/RAS alterations were found to be frequent in gastric cancer. This study also demonstrates, for the first time, that these alterations occur in a mutually exclusive fashion, with KRAS gene amplifications highlighting a clinically relevant but previously underappreciated gastric cancer subgroup. FGFR2 -amplified gastric cancers were also shown to be sensitive to dovitinib, an orally bioavailable FGFR/VEGFR targeting agent, potentially representing a subtype-specific therapy for FGFR2 -amplified gastric cancers. Conclusion The study demonstrates the existence of five distinct gastric cancer patient subgroups, defined by the signature genomic alterations FGFR2 (9% of tumours), KRAS (9%), EGFR (8%), ERBB2 (7%) and MET (4%). Collectively, these subgroups suggest that at least 37% of gastric cancer patients may be potentially treatable by RTK/RAS directed therapies.

Introduction: The objective of this study was to evaluate the efficacy of dovitinib (TKI258), a small-molecule dual inhibitor of fibroblast growth factor receptor (FGFR) and vascular endothelial growth factor receptor (VEGFR) in mouse models of human breast cancer. Molecular epidemiological studies have implicated that FGFR1 and FGFR2 gene amplification plays a critical role in breast cancer tumorigenesis. FGFR1 gene amplification correlates with gene expression and is associated with poor prognosis in patients with estrogen receptor-positive (ER+) tumors. Dual FGFR/VEGFR inhibitory activities of dovitinib make it an attractive molecule for targeting breast cancer. Experimental Design: Dovitinib was evaluated in 3 primary human breast carcinoma xenograft models with different histological and molecular profiles: a triple-negative breast cancer model (HBCx-15), a HER2+ model (HBCx-5), and an ER+ model (HBCx-3). Dovitinib was administered orally at 20- and 40-mg/kg dose levels (n = 8–10/group), once daily for 21 consecutive days. Results: At a dose of 40 mg/kg, dovitinib demonstrated strong antitumor efficacy as a single agent, particularly in the ER+ HBCx-3 and the HER2+ BCx-5 tumor models (ΔT/T0 = −26.42% and ΔT/ΔC = 5.09%, respectively), whereas the response was less significant in the triple negative breast cancer HBCx-15 model (ΔT/ΔC = 31.7%). Conclusion: Dovitinib showed significant antitumor effect in human breast cancer xenograft mouse models. This study provides a strong rationale for clinical investigation of dovitinib in patients with advanced breast cancer, particularly in patients with ER+ and HER2+ disease. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-17-11.

Fibroblast growth factor receptors 1 (FGFR1) belongs to a subfamily of receptor tyrosine kinases (RTKs), which are involved in proliferation and apoptosis. FGFR1 amplification and mRNA overexpression occurs in ∼10% of breast cancers and has been associated with poor outcome. However, studies assessing FGFR1 amplification and protein overexpression in a large collection of breast tumors are lacking. In this prospective, we examined a series of primary breast cancers samples derived from the BCIRG trials assembled into 15 tissue microarrays. FGFR1 gene amplification was studied using chromogenic in situ hybridization (CISH) and evaluated with respect to association with level of protein expression and clinicopathological parameters. FGFR1 gene amplification was significantly associated with high protein levels as determined by immunohistochemistry (P Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-24.

Purpose To describe the natural history of nonmetastatic prostate cancer and rising prostate-specific antigen (PSA) despite androgen deprivation therapy. Patients and Methods: The 201 patients in this report were the placebo control group from an aborted randomized controlled trial to evaluate the effects of zoledronic acid on time to first bone metastasis in men with prostate cancer, no bone metastases, and rising PSA despite androgen deprivation therapy. Relationships between baseline covariates and clinical outcomes were assessed by Cox proportional hazard analyses. Covariates in the model were baseline PSA, Gleason sum, history of bilateral orchiectomies, regional lymph node metastases at diagnosis, prior prostatectomy, time from androgen deprivation therapy to random assignment, time from diagnosis to random assignment, and PSA velocity. Results At 2 years, 33% of patients had developed bone metastases. Median bone metastasis–free survival was 30 months. Median time to first bone metastases and overall survival were not reached. Baseline PSA level greater than 10 ng/mL (relative risk, 3.18; 95% CI, 1.74 to 5.80; P < .001) and PSA velocity (4.34 for each 0.01 increase in PSA velocity; 95% CI, 2.30 to 8.21; P < .001) independently predicted shorter time to first bone metastasis. Baseline PSA and PSA velocity also independently predicted overall survival and metastasis-free survival. Other covariates did not consistently predict clinical outcomes. Conclusion Men with nonmetastatic prostate cancer and rising PSA despite androgen deprivation therapy have a relatively indolent natural history. Baseline PSA and PSA velocity independently predict time to first bone metastasis and survival.

The selective cyclin dependent kinases 4 and 6 (CDK-4/6) inhibitor, palbociclib, has recently been approved in combination with endocrine-based therapies for the treatment of advanced ER+/HER2- breast cancer (BC). Despite the improvements in progression-free and overall survivals, a significant number of patients on CDK-4/6 therapy will go on to develop progressive disease. As such, it is critical to identify the mechanisms by which tumor cells evade CDK-4/6 targeted therapy and to develop therapeutic strategies to overcome resistance. In this study, we evaluated the efficacy of a p110α-selective PI3K inhibitor, alpelisib (BYL719) and the mTORC1 specific inhibitor, everolimus (RAD001), in ER+/HER2- xenograft tumors conditioned in vitro and in vivo to acquire resistance to combined palbociclib + endocrine-based therapy. The EFM19 (ER+/HER2-, PIK3CA mt), MDA134 (ER+/HER2-, PIK3CA wt), and MDA361 (ER+/HER2+, PIK3CA mt) BC cell lines were conditioned to acquire palbociclib resistance through long-term culture in the presence of increasing concentrations of drug. Baseline total/phosphoprotein levels of over 280 cancer-associated analytes were measured in the parental and resistant cell lines by Reverse Phase Protein Array (RPPA). In vivo resistant models were developed from xenografts of the MCF7 and HCC1500 ER+ BC cell lines, treated until progression with palbociclib (75 mg/kg) + fulvestrant (5 mg/mse). EFM19-PR cells maintained palbociclib resistance in vivo and were included in the xenograft studies. Resistance to palbociclib was confirmed in vitro by a shift in growth inhibitory IC50 from an average of less 50nM to over 1 µM. Significantly reduced Rb and ER protein levels were detected in each of these palbociclib resistant cell lines by RPPA. Cross-resistance to an alternative CDK-4/6 inhibitor (ribociclib) was found in in vitro studies. In addition, switching treatment of the xenografts progressing on palbociclib + fulvestrant to ribociclib (75mg/kg) + fulvestrant did not impact tumor progression. However, when progressing tumors were switched to BYL719 (30mg/kg) + fulvestrant or RAD001 (10mg/kg) + fulvestrant, sustained tumor regression was observed for over 45 days of treatment in every model tested. The triplet combination of ribociclib + fulvestrant + BYL719 or RAD001 provided marginal additional benefit over the doublet combination of BYL719 or RAD001 + fulvestrant. RPPA analysis of xenograft tissue collected from these studies will help to identify additional molecular alterations involved in resistance to CDK-4/6 targeted therapy. These preclinical data indicate that acquired resistance to CDK-4/6 inhibition occurs through a loss of dependence on Rb-signaling. However, targeting an alternative pathway like PI3K/mTOR with molecules such as BYL719 or RAD001 may be a viable strategy for further clinical investigation. Citation Format: Neil A. O'Brien, Dylan Conklin, Tong Luo, Raul Ayala, Shawnt Issakhanian, Ondrej Kalous, Erika Von Euw, Sara A. Hurvitz, Emmanuelle DiTomaso, Faye Su, Ronald Linnartz, Stefan Scherer, Samit Hirawat, Dennis J. Slamon. Anti-tumor activity of the PI3K/mTOR pathway inhibitors alpelisib (BYL719) and everolimus (RAD001) in xenograft models of acquired resistance to CDK-4/6 targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4150. doi:10.1158/1538-7445.AM2017-4150

Purpose: Altered PI3K/mTOR signaling is implicated in the pathogenesis of a number of breast cancers, including those resistant to hormonal and HER2-targeted therapies. Experimental Design: The activity of four classes of PI3K/mTOR inhibitory molecules, including a pan-PI3K inhibitor (NVP-BKM120), a p110α isoform–specific PI3K inhibitor (NVP-BYL719), an mTORC1-specific inhibitor (NVP-RAD001), and a dual PI3K/mTORC1/2 inhibitor (NVP-BEZ235), was evaluated both in vitro and in vivo against a panel of 48 human breast cell lines. Results: Each agent showed significant antiproliferative activity in vitro, particularly in luminal estrogen receptor–positive and/or HER2+ cell lines harboring PI3K mutations. In addition, monotherapy with each of the four inhibitors led to significant inhibition of in vivo growth in HER2+ breast cancer models. The PI3K/mTOR pathway inhibitors were also effective in overcoming both de novo and acquired trastuzumab resistance in vitro and in vivo. Furthermore, combined targeting of HER2 and PI3K/mTOR leads to increased apoptosis in vitro and induction of tumor regression in trastuzumab-resistant xenograft models. Finally, as previously shown, targeting mTORC1 alone with RAD001 leads to consistent feedback activation of AKT both in vitro and in vivo, whereas the dual mTOR1–2/PI3K inhibitor BEZ235 eliminates this feedback loop. However, despite these important signaling differences, both molecules are equally effective in inhibiting tumor cell proliferation both in vitro and in vivo. Conclusion: These preclinical data support the findings of the BOLERO 3 trial that shows that targeting of the PI3K/mTOR pathway in combination with trastuzumab is beneficial in trastuzumab-resistant breast cancer. Clin Cancer Res; 20(13); 3507–20. ©2014 AACR.

Introduction: NSCLCs harboring rearrangements of the anaplastic lymphoma kinase (ALK) gene are highly sensitive to ALK inhibition. Ceritinib is a novel ALK inhibitor that demonstrates significant preclinical and clinical antitumor potency, including in tumors that have developed resistance to the approved ALK inhibitor crizotinib. Upregulation of the phosphatidylinositol 3-kinase (PI3K) pathway may be involved in ALK inhibitor resistance, suggesting that combining ceritinib with a PI3K inhibitor may provide synergistic antitumor activity in ALK-rearranged NSCLC. Here, we assess the synergy and antiproliferative activity of PI3K inhibitor/ceritinib combinations in crizotinib-naïve primary preclinical models of NSCLC. Methods: In vitro experiments used the EML4−ALK-translocated lung cancer cell line H2228 to screen for synergistic combinations of ceritinib with 18 other compounds (PI3K, MEK, and CDK4/6 inhibitors, among others). In vivo experiments used a human-mouse primary xenograft lung cancer model with similar EML4−ALK translocation (LUF1656). Tumor fragments (diameter 2−3 mm) from stock mice inoculated with LUF1656 lung cancer tissue were harvested and used for inoculation into nu/nu mice for tumor development. Once mean tumor size had reached ∼150 mm3, treatment was initiated with single-agent or combination regimens of buparlisib (pan-PI3K inhibitor; 35 mg/kg QD), alpelisib (PI3Kα inhibitor; 30 mg/kg QD), or ceritinib (25 mg/kg [low dose] or 50 mg/kg [full dose] QD). All doses used were equivalent to known therapeutic doses in patients. Results: In vitro experiments revealed the strongest antiproliferative activity when ceritinib was combined with either buparlisib or alpelisib versus the other compounds tested. In vivo, low-dose ceritinib in combination with buparlisib improved tumor growth delay over single-agent, full-dose ceritinib. Full-dose ceritinib plus alpelisib showed no significant difference in tumor growth delay versus full-dose ceritinib alone. Low-dose ceritinib plus alpelisib appeared to be better tolerated than full-dose ceritinib plus alpelisib, but with similar efficacy to low-dose ceritinib alone. A delayed tumor growth rate after treatment interruption was noted in all ceritinib combinations. Conclusion: Synergy was observed between ceritinib and PI3K inhibitors in a crizotinib-naïve ALK-translocated lung model in vitro. In the in vivo EML4−ALK lung preclinical cancer models, low-dose ceritinib (25 mg/kg) combined with buparlisib showed improved efficacy versus full-dose ceritinib (50 mg/kg) alone. Preclinical experiments exploring combinations of PI3K- and ALK-targeted therapies in crizotinib-resistant ALK-rearranged tumors are also ongoing. Citation Format: Emmanuelle Di Tomaso, Ronald Linnartz, Fang Li, Cristian Massacesi, Samit Hirawat. Combination of ceritinib (LDK378) with PI3K inhibitors (buparlisib [BKM120] and alpelisib [BYL719]) demonstrates synergistic preclinical antitumor activity in ALK-rearranged non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4698. doi:10.1158/1538-7445.AM2015-4698

Background: The addition of the selective mTORC1 inhibitor everolimus (RAD001/Afinitor®), to exemestane significantly prolongs progression free survival in patients with advanced hormone receptor (ER+) positive breast cancer. However, despite these improved outcomes, the majority of patients that achieve an initial clinical benefit will eventually go on to develop progressive disease. In order to prevent or delay the onset of this acquired resistance, it is essential to identify the mechanisms by which continued proliferation is being driven in these resistant tumors. In this study, we used reverse phase protein array (RPPA) technology to compare cell signaling in ER+ breast cancer cell line xenografts that were responsive to everolimus versus those that had acquired resistance to everolimus. Materials and Methods: Xenograft models were established from three ER+ breast cancer cell lines containing different molecular alterations that confer activated PI3K/mTOR signaling; MCF7 and KPL-1 (both PIK3CA mutant) and ZR75-1 (PTEN-null). Mice were treated daily with either vehicle or 10 mg/kg everolimus until progression. Snap frozen tissue samples (n = 3) were collected from vehicle control tumors, from everolimus resistant tumors and from everolimus responsive tumors. Whole cell lysates were prepared from tumor tissues and processed for RPPA analysis. A customized panel of 27 known cancer/PI3K pathway associated signaling proteins were selected for the RPPA analysis. Results: In the vehicle controls, significantly higher levels of pAKT, pPRAS40 and p4EBP1 were detected in the PTEN-null ZR75-1 tumors compared to the PIK3CA mutant tumors, indicating potential variations in intensity of PI3K/AKT pathway signaling depending on the initiating molecular alteration. Complete inhibition of tumor proliferation was observed in response to everolimus in all 3 xenograft models for up to 7-weeks of treatment, until tumors showed signs of progression. Significantly lower S6 phosphorylation was observed in tumors responding to everolimus, which was accompanied by feedback activation of pAKT. Significant inhibition of S6 phosphorylation was also observed in each of the ZR75-1 and MCF7 tumors that were progressing on everolimus, indicating that although the tumor cells have acquired resistance to the anti-proliferative activity of everolimus, mTORC1 signaling is still being successfully blocked. Feedback activation of pAKT was observed in the ZR75-1 resistant model, however no feedback activation of AKT occurred in the MCF7 resistant model. The KPL-1 model showed the expected loss of S6 pathway inhibition upon resistance. No significant signaling changes were observed in any of the other signaling proteins measured, including ER, HER2, EGFR, MEK and ERK. Ongoing studies to measure the mRNA expression changes in these tumors using mRNA microarrays may identify signaling pathways that are driving the proliferation of these resistant tumors. Discussion: These preclinical data show that multiple pathways of resistance can develop in response to long-term everolimus treatment and resistance can occur despite continued inhibition of PI3K/mTOR signaling. Data from mRNA microarray experiments will be presented at the time of the meeting. Citation Format: Neil A O'Brien, Dylan Conklin, Michaela Ching, Luo Tong, Raul Ayala, Shawnt Issakhanian, Sara A Hurvitz, Emmanuelle di Tomaso, Ronald Linnartz, Richard S Finn, Samit Hirawat, Dennis J Slamon. Acquired resistance to everolimus occurs independently of mTORC1 inhibition in preclinical in vivo models of ER+ breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-04.

BACKGROUND: MAPK pathway dysregulation is frequently seen in cancer. For NSCLC, this may be due to driver mutations or gene amplification in EGFR, KRAS, or BRAF, though tumors without such mutations frequently display pathway hyperactivity. Aberrant EGFR signaling in SCCHN may lead to hyperactivity of the MAPK or PI3K pathways. MEK162 (ARRY-438162) is a potent, tight binding, uncompetitive MEK inhibitor currently in clinical development for treatment of solid tumors. MEK162 has an IC50 of 10nM against purified MEK1/2. We evaluated whether MEK162 would inhibit NSCLC and SCCHN cell lines and whether specific molecular alterations predicted anti-tumor activity. We also evaluated the combination of MEK162 in combination with BYL719, a potent PI3K alpha inhibitor. METHODS: The 50% inhibitory concentration (IC50) of MEK162, alone or in combination with BYL719, was determined in 40 human NSCLC and 30 SCCHN cell lines in vitro. Effects on MAPK and PI3K/AKT pathway signaling were studied by western blot analysis. Effects of MEK162 alone or in combination with BYL719 on cell cycle progression were also determined by flow cytometry. RESULTS: Using previously published cut-offs for sensitivity in cell lines, among NSCLC cell lines, 20/40 (50%) were sensitive to MEK162 (IC50 ≤ 500nM), 9/40 were intermediately sensitive (IC50 500nM-1µM), and 11/40 were resistant (IC50 ≥ 1µm). Using the same cut-offs, 80% (24/30) of SCCHN cell lines were sensitive, 2/30 were intermediately sensitive, and 4/30 were resistant to MEK162. MEK162 exposure resulted in increased cells in G0/G1 and decreased cells in S-phase. MEK162 decreased phosphorylated ERK (p-ERK) in all cell lines treated and resulted in an increase in phosphorylated AKT (p-AKT), presumably as a compensatory result from MAPK pathway inhibition. Combined treatment with MEK162 and BYL719 resulted in synergistic growth inhibition for all cell lines and decreased levels of both p-ERK and p-AKT levels at 24 hours. CONCLUSION: MEK162 potently inhibited growth of human NSCLC and SCCHN cell lines in most cell lines tested and led to synergistic inhibition when combined with BYL719. The compensatory increase in pAKT seen with MEK162 was abrogated with dual treatment with MEK162 and BYL719, demonstrating that synergy may be mediated by simultaneous blockade of MAPK and PI3K/AKT pathways. These data provide a preclinical rationale for evaluating MEK162, alone or in combination with BYL719, in metastatic NSCLC and SCCHN. Supported by 1K23CA149079, P50 CA090440, V Foundation for Cancer Research, Jonsson Comprehensive Cancer Center, Wolfen Family Lung Cancer Research Program, Stiles Program in Oncology, National Lung Cancer Partnership and One Ball Matt Memorial Golf Tournament. Citation Format: Deborah JL Wong, Edward B. Garon, Danielle D. Silveira, Naeimeh Kamranpour, Sharon Pitts, Meenal Chalukya, Habib Hamidi, Steven Dubinett, Ronald Linnartz, Richard S. Finn, Dennis J. Slamon. Potent anti-tumor activity of the MEK1/2 inhibitor MEK162 in human non-small cell lung cancer (NSCLC) and squamous cell carcinoma of the head and neck (SCCHN) cell lines. [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 743. doi:10.1158/1538-7445.AM2014-743

Approximately 60-70% of invasive breast cancers express estrogen receptor (ER) and/or progesterone receptor and are termed hormone receptor positive (ER+). Endocrine therapy remains the therapeutic backbone for the treatment of ER+ cancers and although anti-estrogen therapies are initially frequently effective, 50% of ER+ patients develop resistance to hormonal manipulation within their lifetime, ultimately leading to therapeutic failure. Two emerging mechanisms of endocrine resistance include activation of growth signaling pathways such as the phosphatidylinositol 3-kinase (PI3K) pathway and more recently, the decoupling of cell cycle control from ER-signaling, via deregulation of the cyclinD-cyclin dependent kinase (CDK-4/6:INK4:Rb) pathway. In this study we hypothesized that combining an anti-estrogen (letrozole or fulvestrant) with a CDK-4/6 inhibitor (LEE011) and PI3K inhibitors (buparlisib [BKM120; pan-PI3K inhibitor] or BYL719 [α-specific PI3K inhibitor] would elicit an improved tumor response over agents inhibiting either pathway alone. Four ER+ BC mouse models including, an ER+ patient primary human letrozole sensitive model (HBX34, PTEN/PIK3CA wild-type) and three ER+ cell lines; ZR75-1 (PTEN null), MCF7 (PIK3CA mutant) and KPL1 (PTEN/PIK3CA wild-type) were used for this study. Treatment was carried out daily at doses relevant to clinical exposures for a period of 4 weeks with fulvestrant combinations and 8 weeks with letrozole combinations followed by observation for tumor progression. Treatment doses were as follows: LEE011 (75 mg/kg), BKM120 (30mg/kg), BYL719 (35mg/kg), Letrozole (2.5mg), Fulvestrant (5mg/wk) either as single agents or in combinations (Hormone therapy (HT)+LEE011, HT+PI3Ki, HT+LEE011+PI3Ki). Significant tumor growth inhibition (TGI) was observed for single agent treatment with the CDK-4/6 inhibitor in each of the four ER+ xenograft models. The addition of letrozole (HBX34) or fulvestrant (MCF7, ZR751 and KPL1) with LEE011 increased the TGI observed with single agent. The triplet combination with each PI3K inhibitor increased the (TGI) even further, inducing tumor regressions in each of the four models. Response was independent of PI3K/PTEN mutation status. Complete tumor regressions were observed for a subset of mice within each of the triple combination arms. Tumor regressions were maintained for up to four weeks post-interruption of treatment. No significant toxicities were observed with any of the triplet combinations. These preclinical data highlight the potential efficacy and safety of targeting the ER, CDK-4/6 and PI3K signaling pathways in ER+ breast cancer. LEE011 in combination with an anti-estrogen is sufficient to inhibit tumor growth in vivo, while the addition of a PI3K inhibitor results in robust tumor regressions. The combination of LEE011 with an anti-estrogen and a PI3K inhibitor is a rational therapeutic approach that should be investigated in the clinic. Citation Format: Neil A. O'Brien, Emmanuelle Di Tomaso, Raul Ayala, Luo Tong, Shawnt Issakhanian, Ronald Linnartz, Richard S. Finn, Samit Hirawat, Dennis J. Slamon. In vivo efficacy of combined targeting of CDK4/6, ER and PI3K signaling in ER+ breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4756. doi:10.1158/1538-7445.AM2014-4756

SAM486A (previously termed CGP 48664), a potent inhibitor of S-adenosylmethionine decarboxylase, is under clinical development for the treatment of advanced refractory malignancies. Hematological toxicity manifested by dose-dependent neutropenia has been observed in phase I studies. Population methods were used to investigate pharmacokinetics (PK) as a prognostic factor for safety end point (hematological toxicity) in patients with advanced cancers. SAM486A plasma concentrations and neutrophil counts were collected from three ascending-dose tolerability and PK studies (study 1: single 5-day continuous intravenous (IV) infusion with doses ranging from 24-700 mg/m(2)/cycle; study 2:10-minute to 3-hour IV infusion once weekly with doses ranging from 16-325 mg/m(2)/week; study 3:1-hour IV infusion once daily for 5 days with doses ranging from 3.6-202.8 mg/m(2)/day). The FK of SAM486A were best estimated by a population linear three-compartment model with NONMEM (version 5) using data from 79 patients in studies 1 through 3. The population pharmacokinetic parameters (SD) were CL = 6.2 (0.4) l/h/m(2), Q(2) = 15.4(1.5) l/h/m(2), Q(3) = 33.6 (5.3) l/h/m(2), V(1) = 9.5 (1.6) l/m(2), V(2) = 672 (52) l/m(2), and V(3) = 39.9 (8.3) l/m(2), and the corresponding intersubject variability was 45.4%, 74.0%, 85.3% 80.1%, 37.0%, and 103%, respectively where CL is total body clearance, Q(2) and Q(3) are intercompartmentaI clearances, and V(1), V(2), and V(3) are the volumes of distribution in central and peripheral compartments, respectively The intrasubject variability was 24.0%. The cumulative AUC before the onset of neutrophil nadir count (AUC) and the duration of exposure over threshold SAM486A concentrations in the range of 0.05 to 0.1 mu M based on Bayesian PK parameter estimates significantly correlated with absolute neutrophil count nadir (

Background: Activating mutations in KRAS have been observed in over 90% of pancreatic tumors and are thought to be a major mechanism of oncogenesis in pancreatic cancer. The EGFR signaling pathway serves as a potential target for cancer therapies. Although targeting KRAS itself has been difficult, targeting the downstream effector MEK1/2, a dual specific kinase required for activation of ERK1/2 has proven to be worthy in both preclinical cell lines and animal studies. MEK162 (ARRY-438192) is a potent, selective, ATP-uncompetitive inhibitor of MEK1/2 and was found to be effective at inhibiting growth in a cohort of solid tumors. The objective of this preclinical study was to assess the antitumor activity of MEK162 in pancreatic cancer comprehensively using a panel of 29 pancreatic cancer cell lines. Methods: MEK162 sensitivity in 29 pancreatic cancer cell lines was examined using a five day proliferation assay to assess growth inhibition in vitro. Array-comparative genomic hybridization (array-CGH) was performed on each cell line to determine genomic copy number variation (CNV). KRAS mutational status at codon 12 and 13 was determined by PCR. Results: Sensitivity of pancreatic cancer cell lines to MEK162 varied. There were 15 cell lines with IC50 values less than 500nm and 14 cell lines with IC50s greater than 500nM and 1 cell line (PATU8988T) in which the IC50 was not achieved at the maximum dose of 10uM. Although significant CNV was observed in many genes, only KRAS CNV was found to be significantly associated with sensitivity to MEK162 (p =0.007). In 15 cell lines with IC50 500nM (resistant lines), 10 had detectable KRAS copy number variations (gains and losses), and 4 had normal levels of KRAS. KRAS mutational subtypes were also associated with sensitivity. Although, the average IC50 of cells with wildtype KRAS (n=4) was not statistically different from those with mutant KRAS (n=25), those cell lines with a KRAS(V12) mutation (n=12) were significantly more resistant to MEK 162 than those with wildtype (n=4) or KRAS(D12) mutations (n=10). Activating mutations resulting in a polar amino acid [KRAS(D12), KRAS(R12) or KRAS(C12)] were more sensitive to MEK162 than nonpolar amino acids [KRAS(V12) (p-value=0.01466)]. Finally, we showed that cells with a KRAS CNV and KRAS(V12) mutation (n=7) were less sensitive than cells having neither KRAS CNV or KRAS(V12) mutation (n=14) (p-value=0.0007). The remaining 8 cell lines had moderate sensitivity to MEK inhibition. Conclusions: Our study has established that MEK162 sensitivity in pancreatic cancer cells lines is associated with KRAS CNV and mutational subtype. Clinical validation of these markers is required. Citation Format: Habib R. Hamidi, Richard Finn, Lee Anderson, Ming Lu, Marlena Fejzo, Charles Ginther, Ronald Linnartz, Angela Zubel. KRAS mutational subtypes and copy number variations are predictive of response of human pancreatic cancer cell lines to MEK162 in vitro. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 936. doi:10.1158/1538-7445.AM2013-936

Background and aims: Hepatocellular carcinoma (HCC) is the third leading cause of cancer death. Although sorafenib has been shown to improve survival of patients with advanced HCC, this improvement is modest and patients eventually have refractory disease. This study aims to investigate the antitumor, antiangiogenesis and antimetastatic activities of dovitinib in preclinical models of HCC. Methods: 21-0208 and SK-HEP1 cells as well as patient-derived HCC models were employed to study of the antitumor effect of dovitinib. Changes of biomarkers relevant to FGFR/VEGFR/PDGFR pathways were determined by Western blotting. Microvessel density, apoptosis and cell proliferation were analyzed by immunohistochemistry. Results: Treatment of SK-HEP1 cells with dovitinib resulted in G2/M cell cycle arrest, inhibition of colony formation in soft agar and blockade of bFGF-induced cell migration. Dovitinib inhibited basal expression and FGF-induced phosphorylation of FGFR-1, FRS2-α and ERK1/2. In vivo, dovitinib potently inhibited tumor growth of six HCC lines. Inhibition of angiogenesis correlated with inactivation of FGFR/PDGFR-α/VEGFR-2 signaling pathways. Dovitinib also caused dephosphorylation of retinoblastoma, upregulation of p-histone H2A-X and p27, and downregulation of p-cdk-2 and cyclin B1, which resulted in a reduction in cellular proliferation and the induction of tumor cell apoptosis. In an orthotopic model, dovitinib potently inhibited primary tumor growth and lung metastasis and significantly prolonged mouse survival. Conclusion: Dovitinib demonstrated significant antitumor and antimetastatic activities in HCC xenograft models. This study provides a compelling rationale for clinical investigation in patients with advanced HCC. 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 2314. doi:1538-7445.AM2012-2314

Introduction: The aim of this study was to evaluate the efficacy of dovitinib (TKI258), a small molecule dual inhibitor of fibroblast growth factor receptor (FGFR) and vascular endothelial growth factor receptor (VEGFR) in mouse models of human bladder cancer. Molecular epidemiological studies have implicated FGFR and VEGFR pathways in the pathogenesis of high grade, muscle invasive bladder cancer. Somatic activating mutations in FGFR3 have been identified in 16%-20% of muscle-invasive bladder cancer and FGFR3 overexpression has also been documented in a high fraction of bladder cancers. High microvessel density, serum VEGF levels and urine FGF2 levels have also been associated with high grade bladder cancer and poor disease-free survival. Dual FGFR/VEGFR inhibitory activities of dovitinib make it an attractive molecule targeting advanced bladder cancer. Experimental Design: Dovitinib was evaluated in the RT112 human bladder carcinoma xenograft model. Dovitinib was administered orally at three dose levels (n = 10/group), once daily for 28 consecutive days (qd × 28). A rat RT112 xenograft study was also carried out to evaluate the PK/PD effect of single dose dovitinib administration. Results: Dovitinib at 15, 30, and 60 mg/kg resulted in tumor growth suppression, with mean changes of 60% T/C, 6% T/C (P < 0.01), and -71% T/T0 (P < 0.001), respectively. The 60 mg/kg regimen yielded 6 partial regressions out of 10 animals by day 28. In the rat RT112 model, dovitinib caused dose- and time-dependent inhibition of pFRS2 at 25 and 50 mg/kg after 3, 7 and 24 h of a single oral dose administration. Conclusion: Dovitinib demonstrated significant antitumor effect in a mouse model of human bladder cancer xenograft. This study provides a strong rationale for clinical investigation of dovitinib in patients with advanced bladder cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3575. doi:10.1158/1538-7445.AM2011-3575

Background: Endometrial carcinoma is the most common gynecological malignancy in the western world. Activating mutations of the fibroblast growth factor receptor 2 (FGFR2) have been described in 12-16% of endometrial cancers. Moreover, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) and their receptors are involved in the neovascularization, invasiveness, and metastatic potential of endometrial cancer. Therefore, suppression of FGFR/VEGFR/PDGFR signaling by TKI258 may represent a novel approach for treatment of endometrial cancer. Experimental Design: The aims of this study were 1.) to assess the effect of TKI258 on tumor cell growth in two-dimensional (2D) and three-dimensional (3D) culture assays using a panel of 20 human endometrial cancer cell lines, 2.) to identify candidate molecular markers predicting sensitivity using baseline gene expression profiling and mutational analyses, and 3.) to determine the in vivo antineoplastic activity of TKI258 in endometrial cancer xenograft models. In addition, we screened 200 fresh frozen endometrial cancer specimens to comprehensively assess the distribution pattern of PI3K, PTEN, and FGFR mutations in endometrial cancer. Results: Concentration-dependent anti-proliferative effects of TKI285 using 2D assays were seen in all endometrial cancer cell lines tested, but varied significantly between individual cell lines (IC50 range: 0.42µM – 3.06 µM). The three most sensitive endometrial cancer cell lines demonstrated activating FGFR2 mutations (MFE296: N549K, IC50 0.42µM; AN3CA: N549K and K310R, IC50 0.50µM; MFE280: S252W, IC50 0.66µM). Assessment of TKI258 responses in 3D assays demonstrated similar results to those observed in 2D culture assays in that the 3 cell lines harboring FGFR2 mutations were the most sensitive to TKI258 achieving 100% growth inhibition at a concentration of 1µM. AN3CA and MFE296 endometrial cancer cells formed xenografts in nude mice and antitumor activity was studied in both models. Inhibition of p-FGFR, p-PDGFR, p-VEGFR-2, pAKT and p-ERK1/2 were observed. Comprehensive data on the pattern of PI3K, PTEN and FGFR mutations in endometrial cancer will be provided. Conclusion: TKI258 demonstrates significant antitumor activity in endometrial cancer cells with FGFR2 mutations. This study provides a strong rationale for the clinical evaluation of TKI258 in patients with endometrial cancer, specifically in those harboring FGFR2 mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3589. doi:10.1158/1538-7445.AM2011-3589

Background. During the performance of the BCIRG trials primary patient tumor samples were obtained from paraffin blocks and assembled into 15 tissue microarrays, including 3000 samples from trial 005 (adjuvant taxol in Her2- node positive patients), 2200 in 006 (adjuvant herceptin in Her2+ high risk patients) and 300 in 007 (addition of platinum in Her2+ first line therapy). This array set was prepared prospectively, in anticipation of molecular epidemiologic studies of a variety of targets in relation to outcome. In order to address the potentially important role of the PIK3CA pathway in modulating outcome in different clinical situations we assessed components of the PIK3CA pathway by various methods.Methods. We analysed the expression of PTEN, Cyclin D1, p53 and Stathmin by immunohistochemistry using standard methods. All markers were scored by histoscore. Phospho S6 240 and phospho Akt 473 were assessed simultaneously by quantum dot immunofluorescence using automated image capture and segmentation. PIK3CA mutations were evaluated using SnaPshot analysis of laser captured TMA spots in a subset of approximately 2000 samples.Results. In the samples analyzed PIK3CA mutation had an incidence of 23%, with 9% being mutations in exon 9 and 13% in exon 20, with 0.5% being mutant in both exons. Initial analysis of the results prior to outcome analysis indicated that Stathmin expression, while being a robust marker with good dynamic range, did not appear to correlate with PTEN loss by IHC or with PIK3CA mutation.Conclusions. PIK3CA mutational analysis is feasible from TMA cores and gives mutation incidences similar to the published literature for archival material. Relationships of markers with outcome will be presented. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4043.