Citations

Abstract The importance of the blood–brain barrier in preventing effective pharmacotherapy of glioblastoma has been controversial. The controversy stems from the fact that vascular endothelial cell tight junctions are disrupted in the tumor, allowing some systemic drug delivery. P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) efflux drugs from brain capillary endothelial cells into the blood. We tested the hypothesis that although the tight junctions are “leaky” in the core of glioblastomas, active efflux limits drug delivery to tumor-infiltrated normal brain and consequently, treatment efficacy. Malignant gliomas were induced by oncogene transfer into wild-type (WT) mice or mice deficient for Pgp and BCRP (knockout, KO). Glioma-bearing mice were orally dosed with dasatinib, a kinase inhibitor and dual BCRP/PgP substrate that is being currently tested in clinical trials. KO mice treated with dasatinib survived for twice as long as WT mice. Microdissection of the tumor core, invasive rim, and normal brain revealed 2- to 3-fold enhancement in dasatinib brain concentrations in KO mice relative to WT. Analysis of signaling showed that poor drug delivery correlated with the lack of inhibition of a dasatinib target, especially in normal brain. A majority of human glioma xenograft lines tested expressed BCRP or PgP and were sensitized to dasatinib by a dual BCRP/Pgp inhibitor, illustrating a second barrier to drug delivery intrinsic to the tumor itself. These data show that active efflux is a relevant obstacle to treating glioblastoma and provide a plausible mechanistic basis for the clinical failure of numerous drugs that are BCRP/Pgp substrates. Mol Cancer Ther; 11(10); 2183–92. ©2012 AACR.

Abstract A hypoxic microenvironment is believed to be a characteristic of solid tumors including pancreatic adenocarcinoma (PAC) and is associated with resistance to chemotherapy. The hypoxia-activated prodrug, TH-302, is designed to specifically target the hypoxic microenvironment. Combining G with TH-302 may enable the targeting of both the normoxic and hypoxic regions of PAC. TH-302 was investigated with full-dose G as part of a Phase 1/2 study with a 21% response, median PFS and OS of 5.9 and 8.5 months, respectively, and one-year survival of >40%. Based upon these data, a randomized Phase 2B study (NCT01144455) was conducted to assess the benefit of G+T to standard dose G for first-line therapy of PAC. An open-label multi-center study of two dose levels of TH-302 (240 mg/m2 or 340 mg/m2) in combination with G versus G alone (randomized 1:1:1) was initiated in June 2010. G (1000 mg/m2) and T were administered IV over 30-60 minutes on Days 1, 8 and 15 of a 28-day cycle. Patients on the G arm could crossover after progression and be randomized to a G+T arm. The primary efficacy endpoint is a comparison of progression-free survival (PFS) between the combination arms and G alone (80% power to detect 50% improvement in PFS with one-sided alpha of 10%). 214 pts (69 G: 71 G+T240: 74 G+T340) with advanced PAC including 77% distant metastases, 62% involving liver and 11% with prior adjuvant tx; median age: 65 (range 29-86); 126 M/88 F; 79/128 ECOG 0/1. Median cycles received: G - 4, G+T240 - 5, G+T340 - 6. Median PFS was 3.6 mo in G vs 5.6 mo in G+T arms with HR of 0.61 (95% CI: 0.43 - 0.87) and logrank p-value of 0.005. Median PFS in G + T340 was 6.0 mo. RECIST best response was 12% in G, 17% in G+T 240 and 27% in G+T340. 153 (71%) patients had elevated CA19-9 and follow-up. CA19-9 decreases were significantly greater in the G+T groups and greatest in the G+T340 arm which had 37 of 53 (70%) pts with a greater than 50% CA19-9 decrease. One death (suicide) was considered possibly related to study drug. Adverse events leading to discontinuation were: 16% G, 15% G+T240 and 11% G+T340. Serious adverse events were balanced across the treatment arms. The most common non-laboratory events, fatigue (49%), nausea (43%), constipation (34%) and peripheral edema (38%), were similar across groups. Rash (14% G, 39% G+T240 and 45% G+T340) and stomatitis (6% G, 17% G+T240 and 36% G+T340) were significantly greater with G+T combination but no Grd 4 (4 pts with Grade 3). Grd 3/4 thrombocytopenia (11% G, 39% G+T240 and 59% G+T340) and Grd 3/4 neutropenia (28% G, 56% G+T240 and 59% G+T340) were higher with G+T. G+T combination with full dose G improved the efficacy of G with significantly longer PFS, higher response rate and greater CA19-9 declines. The G+T combination was well tolerated. Skin and mucosal toxicity and myelosuppression were the most common related adverse events with no increase in treatment discontinuation. 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 LB-121. doi:1538-7445.AM2012-LB-121

Abstract Background: Therapy with proteasome inhibitors (PIs) is an effective strategy against multiple myeloma (MM), but a significant proportion of patients is primarily resistant or eventually becomes refractory. Paradoxical activation of phospho-Akt, induction of HSP27 and excessive proteasome activity in relation to polyubiquitinated protein burden are all mechanisms for bortezomib resistance in MM cells in vitro. Preclinical data showed investigational drug MLN9708, a specific, reversible, orally bioavailable, small molecule PI, to be active in MM cell lines, patient cells and mouse models and is currently under clinical investigation. We compared the signaling pathways induced by MLN2238, the biologically active form of MLN9708, and bortezomib to outline differences that could be employed to design rational combinations to overcome resistance. Results: We performed time course experiments in MM.1S cells with EC50 doses of MLN2238 and bortezomib and analyzed signaling pathways via western blotting. Compared to bortezomib, MLN2238 induced earlier cleavage of caspase 8, 9, 3 and PARP and a significant downregulation of XIAP starting at 12 hours. Early and persistent phosphorylation of Bad, known to protect from mitochondrial apoptosis, was observed in bortezomib-, but not MLN2238-treated cells, in which phospho-Bad was reduced at 24 hours. Similarly, MLN2238 decreased phosphorylated Akt and mTOR at 24 hours, while bortezomib-treated cells only showed subtle changes. Interestingly, both drugs caused downregulation of the mTOR downstream targets phospho-p70 S6 kinase and phospho-4E-BP1, albeit of different intensity. Induction of heat shock proteins was comparable with both PI, opening the possibilities for combination therapy of MLN2238 with HSP90 inhibitors. Importantly, the inhibitory effect on phosphorylation of Akt, mTOR and Bad persisted when MM.1S cells were treated with MLN2238 in the presence of bone marrow stromal cells (BMSC). Conclusion: We observed differences in several crucial signaling pathways upon treatment of MM.1S cells with bortezomib or MLN2238. In particular, treatment with the latter inhibited phosphorylation of Bad, Akt and mTOR, suggesting a broader pro-apoptotic and anti-proliferative profile, compared to bortezomib. MLN2238 retained the capability of targeting these substrates also in the presence of BMSC. Confirmatory data on patient-derived cells are under current investigation in our lab. We believe that these data help understanding the differences in PI pharmacodynamics and bortezomib escape. This will allow us to design new drug combinations in the future that overcome clinical resistance to PIs and improve MM patient survival. 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 5669. doi:1538-7445.AM2012-5669

Abstract Purpose: Surrogate biomarkers are lacking for predicting or monitoring response to anti-angiogenesis therapies. We investigated whether circulating biomarkers (CBMs) of angiogenesis (i.e., bFGF, SDF-1α, HGF, sKIT, Ang-2, and PlGF) correlated with treatment efficacy in a pooled analysis of metastatic colorectal cancer (mCRC) and recurrent glioblastoma multiforme (rGBM) patients (pts) treated with bevacizumab/sorafenib (BEV/SOR). Experimental procedures: Peripheral blood samples were obtained for CBM analyses from pts enrolled in the North Central Cancer Treatment Group (NCCTG) phase II studies N054C (mCRC, n=75, Grothey, ASCO 2010) and N0776 (rGBM, n=54, Galanis, ASCO 2010). Blood collection time points were: baseline (BL), cycle 1 day 3 (C1D3), prior to treatment cycles 2, 3, 5, 7, 9, 11, 13, and after the pt went off study. Plasma CBM levels were determined by commercially-available ELISAs (R&D Systems) and performed according to manufacturer's instructions. For each CBM, a linear regression model that adjusted for study was used to evaluate the correlation with either progression-free survival (PFS) success/failure or progression within 14 days of the last blood draw for the following measures: absolute BL levels, log2 BL levels; C1D3 log2-fold change (FC) from BL, prior to cycle 2 log2-FC from BL, last draw log2-FC from BL, and last draw log2-FC from prior draw. Results summary: There appears to be no significant difference in the PFS or overall survival (OS) between the pts on these two studies; therefore, we pooled the patients from both studies for a combined analyses. We found no difference in absolute or log2 levels for any of the CBMs during the course of BEV/SOR treatment. In these pooled analyses, we found increased absolute and increased log2 BL SDF-1α levels were significantly correlated with PFS success (p=0.023 and p=0.02, respectively). After correcting for multiple comparisons (method of Benjamini & Hochberg) the p-values <0.05 had a false-discovery-rate of 41.4%. None of the other CBM measures were significantly correlated with either PFS success/failure or progression within 14 days of the last blood draw (all p>0.10). Conclusions: This study provides preliminary evidence that SDF-1α may be a useful CBM for determining treatment efficacy in these pts treated with BEV/SOR. Further prospective validation of SDF-1α for predicting pt progression with this treatment combination in advanced disease (e.g., mCRC and rGBM) appears warranted. 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 5562. doi:1538-7445.AM2012-5562

Abstract INTRODUCTION: The L-Type amino acid transporter 1 (LAT1) transports large neutral amino acids across the plasma membrane in a variety of cells. Positron emission tomography (PET) imaging with the amino acid tracer 6-18F-fluoro-L-dopa (18F-FDOPA) provides useful histological information in human gliomas in addition to providing better localizing information then CT or MRI alone. This study is the first to correlate LAT1 expression with uptake of the 18F-FDOPA analog, 3H-L-DOPA in human glioblastomas. METHODS: Endogenous expression of LAT1 was evaluated in two established glioblastoma multiforme (GBM) cell lines and three primary GBM xenografts using Western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Uptake of FDOPA was assessed in vitro using 3H-L-DOPA as an analog to 18F-FDOPA. Knockdown of LAT1 expression was achieved by lentiviral expression of anti-LAT1 shRNA in GBM lines with high LAT1 expression. The effect of LAT1 knockdown on 3H-L-DOPA uptake was then quantified. RESULTS: Results from Western blotting and qRT-PCR demonstrate that LAT1 is variably expressed in GBM. Levels of 3H-L-DOPA uptake were directly related to LAT1 expression (p<0.001) across the 5 lines. Knockdown of LAT1 was achieved in T98 cells (knockdown efficiency = 82.8%) and xenograft GBM 28 (knockdown efficiency = 73.7%) as confirmed by qRT-PCR and Western blotting. Knockdown of LAT1 reduced 3H-L-DOPA uptake by 57.2% (p<0.0001) in T98 and 52.1% in GBM 28 (p<0.001). CONCLUSIONS: This is the first report of 3H-L-DOPA uptake being driven by LAT1 in human GBM. Our findings suggest LAT1 as a transporter of 18F-FDOPA in GBM. Additional studies are underway to correlate LAT1 expression with PET 18F-FDOPA uptake in patients with GBM. 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 4543. doi:1538-7445.AM2012-4543

Abstract Pancreatic cancer is the fourth leading cause of cancer deaths in the United States and continues to be a disease with few options for treatment. Current therapeutic regimens for pancreatic cancer often include the nucleoside analog gemcitabine (GEM), but while it has been in use for many years, the overall five year survival rate continues to be about 5%. New therapies are urgently needed to treat this disease. The nutritional supplement gamma-linolenic acid (GLA) is a poly-unsaturated fatty acid (PUFA) of the n-6 PUFA family, and has garnered attention for its cytotoxic effects against tumor cell lines while not affecting normal cells. In this study we look at the effect GLA has on Panc-1 cells and its ability to increase the sensitivity of these cells to treatment with GEM. Through the use of cell viability assays, immunofluorescence, 3-D growth assays, and western blotting, our results show that GLA alone will inhibit growth and induce apoptosis in Panc-1 cells. When GLA is combined with GEM, the results are synergistically enhanced. In addition, our results show that GLA may act through disruption of lipid rafts in the cellular membrane and may activate the extrinsic apoptotic pathway causing cell death. These results suggest that the nutritional supplement GLA could be used in combination with GEM to increase the therapeutic effectiveness of GEM while treating pancreatic cancer. 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 3854. doi:1538-7445.AM2012-3854

Abstract In this study we demonstrate that gain of HER-2/Neu expression in human breast cancer xenografts harboring constitutive active Raf-1 induces Aurora-A over-expression that ultimately drives the transition from a luminal to a highly invasive basal-like phenotype. Over-expression of Aurora-A was induced by phosphorylation and protein stabilization by HER-2/Neu rather than increased Aurora-A gene expression. Moreover, Aurora-A stabilization induced an increase of HER-2/Neu expression in breast cancer cells defining a novel positive feed-back loop between Aurora-A and HER-2/Neu oncogenic signaling pathways. Basal-like tumor cells over-expressing Aurora-A and HER-2/Neu underwent to loss of CD24 surface receptor, expressed epithelial to mesenchymal transition (EMT) markers and acquired stem cell-like properties such as mammospheres formation and self-renewal. Pharmacologic inhibition of HER-2/Neu signaling pathway with lapatinib in cancer cells over-expressing Aurora-A demonstrated that Aurora-A was downstream to HER-2/Neu in the development of EMT and stemness. To determine whether Aurora-A induced a cancer stem cell-like phenotype through development of chromosomal instability and tumor cell phenotypic heterogeneity, we performed a SKY analysis on the breast cancer cell lines employed in this study. Our results demonstrate that invasive breast cancer cells harboring a basal-like phenotype did not develop higher chromosomal instability compared to luminal non-invasive cancer cells, signifying that Aurora-A has a direct effect on the development of basal-like breast cancer cells with stemness properties. Importantly, pharmacologic inhibition of Aurora-A kinase activity with the small molecule MLN8237 restored CD24 expression and reversed EMT leading to the re-establishment of a luminal phenotype and suppression of distant metastases. Our findings implicate for the first time the mitotic kinase Aurora-A in the development and maintenance of CD24 negative basal-like tumor stem cells responsible for breast cancer recurrence and progression. These observations have also important implications for the development of molecular targeted therapies to inhibit Aurora-A kinase activity and stemness improving the overall survival of breast cancer patients. 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 328. doi:1538-7445.AM2012-328

Abstract Background: The five year survival rate for women with ovarian cancer (OC) is less than 40%. Crucial to improving the outcomes for ovarian cancer patients is the development of accurate pre-clinical models of human epithelial OC, which can be used to unravel the mechanisms underlying its evolution and behaviour. Methods: We have generated a novel xenograft model of human high-grade serous OC (HG-SOC) in order to enable preclinical molecular and drug response assessment of individual HG-SOC: this includes a comparison of standard OC xeno-transplantion approaches (intra-peritoneal, subcutaneous and sub renal capsular), with the novel use of the rodent ovarian bursa, the orthotopic site. Utilising the rodent bursa may better replicate the relevant microenvironment and increase the success of EOC xeno-transplantion. Other aspects designed to optimize the model include the use of NOD-SCID-IL-2rg recipient mice, systemic estrogen supplementation and transplantation of fresh human HG-SOC fragments which have had no prior in vitro culture. Histological, functional and molecular analysis of the novel xenograft cohort (at baseline and following xenotransplantation) includes histological review; documentation of in vitro Homologous Recombination (HR) DNA repair and drug response capabilities (using novel α-irradiation and explant drug assays); classification according to molecular subtype (Tothill classfier); documentation of NHEJ pathway status, BRCA1/2 status and other DNA repair gene status. In vivo drug treatment studies are being performed, with the choice of treatment targeted to the specific molecular characteristics of the HG-SOC in question. Results: Fourteen consecutive chemotherapy naive potentially HG-SOC samples have been collected. Corresponding clinical data has been collected. Data concerning DNA repair capability and response to DNA damaging agents will be presented, including IHC for markers of DNA damage (γH2AX), DNA repair (RAD51) and apoptosis (capsase 3 cleavage). HG-SOC in this cohort have been classified according to Tothilll (Tothill et al 2008). Eight appropriate HG-SOC have been transplanted and 5 of the first 6 have successfully xenografted, with phenotyping of xenografts underway. In vivo analysis of response to cisplatin treatment and other relevant therapeutics will be presented. Conclusions: A novel xenograft model has been developed of human HG-SOC, which includes comparative characterization of important prognostic features both in the baseline panel of fresh human HG-SOC and in subsequent xenografts. This clinically, functionally and molecularly annotated consecutive xenograft cohort of HG-SOC will provide outstanding utility for the development of improved therapeutic approaches. 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 3276. doi:1538-7445.AM2012-3276