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Supplementary Fig. 6. Histone marks expression in sunitinib resistant RP-R-01 tumors

Supplementary Fig. 5. Histone marks expression associated with resistance to sunitinib

Supplementary Fig 4. Intratumoral and plasma sunitinib concentrations

Supplementary Fig. 7. EZH2 and E-cadherin mRNA expression in 786-0shRNA scramble, 786-0shEZH2 (clone a and d), and 786-0 treated with GSK126

Supplementary Figure legend

Supplementary Fig. 2. Tumor growth of RP-R-01 ccRCC xenograft treated with sunitinib and associated changes in EZH2, H3K27me3 and E-cadherin expression.

Supplementary Fig 3. Liver histology and body weights of mice on sunitinib dose escalation.

Supplementary Fig. 1. Characterization of RP-R-01 and RP-R-02 PDX models

Supplementary Figure Legends from Aflibercept Exerts Antivascular Effects and Enhances Levels of Anthracycline Chemotherapy In vivo in Human Acute Myeloid Leukemia Models

Supplementary Figures 7-8 from Aflibercept Exerts Antivascular Effects and Enhances Levels of Anthracycline Chemotherapy In vivo in Human Acute Myeloid Leukemia Models

Additional Prussian blue and CD68 staining in a tumor core biopsy after FMX dosing. (A, B) Serial tumor sections from formalin-fixed, paraffin-embedded biopsies of liver lesions were stained for (A) FMX (Prussian blue) and (B) macrophages (CD68). FMX deposition is detectable primarily in vascular-accessible macrophages in stromal areas surrounding tumor lesions.

We examined whether potent vascular endothelial growth factor (VEGF) blockade mediated by aflibercept, a decoy VEGF receptor (VEGFR) 1/2 moiety with stronger affinity for VEGF than bevacizumab, resulted in antileukemia effects and enhanced the efficacy of systemic chemotherapy. The efficacy of aflibercept alone and in combination with doxorubicin was evaluated in human VEGF-expressing acute myeloid leukemia (AML) cell lines and primary cells xenotransplanted into immunodeficient mice. Aflibercept reduced primary VEGF/VEGFR-positive AML colony formation growth in vitro and inhibited AML xenograft growth up to 93% in association with antiangiogenic and antiproliferative effects, hypoxia, and VEGF sequestration in multiple models. High VEGF-A expression by AML cells promoted in vivo xenograft growth and aflibercept sensitivity. Aflibercept therapy slowed disease progression in two systemic human AML xenograft models and reduced peripheral leukemia disease in a primary relapsed AML model in NOD/SCID/IL2Rγnull mice. Combination aflibercept and doxorubicin enhanced antitumor effects in local xenograft models. Sequential aflibercept followed by doxorubicin resulted in progressive anthracycline accumulation in marrow and extramedullary AML sites and resulted in 2-fold higher drug levels 24 hours after administration. In contrast, tissues (tumor, plasma, marrow) treated with chemotherapy only showed progressive drug clearance over time. Combination aflibercept and doxorubicin also resulted in vascular narrowing, decreased vessel number, and perivascular apoptosis. These data suggest that inefficient drug delivery by leukemia-associated vasculature may mediate chemoresistance and support further clinical evaluation of combination aflibercept and anthracycline therapy in refractory/relapsed AML patients. Mol Cancer Ther; 9(10); 2737–51. ©2010 AACR.

Correlation between FMX 72-hour signals and binding constant. (A) Mechanistic PK model for tumor deposition of FMX driven by permeability and binding parameters; example for lesion fits for low permeability/low signal retention is shown. (B) Correlation for tissue binding parameter B to FMX signal measured at 72 hours. The normalized FMX ratio between tumor and plasma values is shown to account for plasma FMX PK variability.

Example patient (patient 009) with widespread hepatic metastasis who demonstrated a treatment response following therapy. (A) Selected axial images from FMX-MRI acquired from the FSPGR Fat-Sat breath-hold images (TE = 13.2 milliseconds). The lesion outlined by the red box highlights one of the target lesions that underwent biopsy analysis and subsequent response assessment by RECIST v1.1. The values above each of the axial images are the estimated iron concentrations. (B) Axial contrast-enhanced CT images demonstrating tumor shrinkage (red boxes with reduction in lesion size by 67.3% at cycle 8).

Sequence of study procedures.

Correlation between the average irinotecan concentration of the biopsied lesion of that patient and the patient's time on treatment. Biopsy samples were obtained 72 hours after nal-IRI infusion. Time on treatment is measured from the date of first nal-IRI dose to the treatment termination date.

Includes additional detail concerning methods

Supplementary Tables 1-3 from Aflibercept Exerts Antivascular Effects and Enhances Levels of Anthracycline Chemotherapy In vivo in Human Acute Myeloid Leukemia Models

ROC analysis of FMX lesion response. Receiver operating characteristics for lesion classification according to best lesion size reduction, either as overall lesion shrinkage or partial response criteria, had an AUC {greater than or equal to}0.8 for early FMX measurements at 1 hour (A) or 24 hours (B).

Supplementary Methods and References from Aflibercept Exerts Antivascular Effects and Enhances Levels of Anthracycline Chemotherapy In vivo in Human Acute Myeloid Leukemia Models

Supplementary Figures 1-6 from Aflibercept Exerts Antivascular Effects and Enhances Levels of Anthracycline Chemotherapy In vivo in Human Acute Myeloid Leukemia Models

Purpose: To determine whether deposition characteristics of ferumoxytol (FMX) iron nanoparticles in tumors, identified by quantitative MRI, may predict tumor lesion response to nanoliposomal irinotecan (nal-IRI).Experimental Design: Eligible patients with previously treated solid tumors had FMX-MRI scans before and following (1, 24, and 72 hours) FMX injection. After MRI acquisition, R2* signal was used to calculate FMX levels in plasma, reference tissue, and tumor lesions by comparison with a phantom-based standard curve. Patients then received nal-IRI (70 mg/m2 free base strength) biweekly until progression. Two percutaneous core biopsies were collected from selected tumor lesions 72 hours after FMX or nal-IRI.Results: Iron particle levels were quantified by FMX-MRI in plasma, reference tissues, and tumor lesions in 13 of 15 eligible patients. On the basis of a mechanistic pharmacokinetic model, tissue permeability to FMX correlated with early FMX-MRI signals at 1 and 24 hours, while FMX tissue binding contributed at 72 hours. Higher FMX levels (ranked relative to median value of multiple evaluable lesions from 9 patients) were significantly associated with reduction in lesion size by RECIST v1.1 at early time points (P < 0.001 at 1 hour and P < 0.003 at 24 hours FMX-MRI, one-way ANOVA). No association was observed with post-FMX levels at 72 hours. Irinotecan drug levels in lesions correlated with patient's time on treatment (Spearman ρ = 0.7824; P = 0.0016).Conclusions: Correlation between FMX levels in tumor lesions and nal-IRI activity suggests that lesion permeability to FMX and subsequent tumor uptake may be a useful noninvasive and predictive biomarker for nal-IRI response in patients with solid tumors. Clin Cancer Res; 23(14); 3638–48. ©2017 AACR.

Purpose: To determine whether deposition characteristics of ferumoxytol (FMX) iron nanoparticles in tumors, identified by quantitative MRI, may predict tumor lesion response to nanoliposomal irinotecan (nal-IRI).Experimental Design: Eligible patients with previously treated solid tumors had FMX-MRI scans before and following (1, 24, and 72 hours) FMX injection. After MRI acquisition, R2* signal was used to calculate FMX levels in plasma, reference tissue, and tumor lesions by comparison with a phantom-based standard curve. Patients then received nal-IRI (70 mg/m2 free base strength) biweekly until progression. Two percutaneous core biopsies were collected from selected tumor lesions 72 hours after FMX or nal-IRI.Results: Iron particle levels were quantified by FMX-MRI in plasma, reference tissues, and tumor lesions in 13 of 15 eligible patients. On the basis of a mechanistic pharmacokinetic model, tissue permeability to FMX correlated with early FMX-MRI signals at 1 and 24 hours, while FMX tissue binding contributed at 72 hours. Higher FMX levels (ranked relative to median value of multiple evaluable lesions from 9 patients) were significantly associated with reduction in lesion size by RECIST v1.1 at early time points (P < 0.001 at 1 hour and P < 0.003 at 24 hours FMX-MRI, one-way ANOVA). No association was observed with post-FMX levels at 72 hours. Irinotecan drug levels in lesions correlated with patient's time on treatment (Spearman ρ = 0.7824; P = 0.0016).Conclusions: Correlation between FMX levels in tumor lesions and nal-IRI activity suggests that lesion permeability to FMX and subsequent tumor uptake may be a useful noninvasive and predictive biomarker for nal-IRI response in patients with solid tumors. Clin Cancer Res; 23(14); 3638–48. ©2017 AACR.

Interstitial fluid pressure (IFP) is elevated in tumors and high IFP, a negative cancer prognosticator, is known to limit the uptake and efficacy of anti-tumor therapeutics. Approaches that alter the tumor microenvironment and enhance uptake of therapeutics are collectively referred to as tumor “priming”. Here we show that the cytotoxic biological therapy Apo2L/TRAIL can prime the tumor microenvironment and significantly lower IFP in three different human tumor xenograft models (Colo205, MiaPaca-2 and a patient gastrointestinal adenocarcinoma tumor xenograft). We found that a single dose of Apo2L/TRAIL resulted in a wave of apoptosis which reached a maximum at 8 hrs post-treatment. Apoptotic debris subsequently disappeared concurrent with an increase in macrophage infiltration. By 24 hrs post-treatment, treated tumors appeared less condensed with widening of the stromal areas which increased at 48 and 72 hrs. Analysis of tumor vasculature demonstrated a significant increase in overall vessel size at 48 and 72 hrs although the number of vessels did not change. Notably, IFP was significantly reduced in these tumors by 48 hrs after Apo2L/TRAIL treatment. Administration of gemcitabine at this time resulted in increased tumor uptake of both gemcitabine and liposomal gemcitabine and significantly improved anti-tumor efficacy of liposomal gemcitabine. These results suggest that Apo2L/TRAIL has potential as a tumor priming agent and provides a rationale for developing a sequencing schema for combination therapy such that an initial dose of Apo2L/TRAIL would precede administration of gemcitabine or other therapies.

No evaluation of sex and race influences on mycophenolic acid (MPA) pharmacokinetics and adverse effects (AEs) during enteric-coated mycophenolate sodium (ECMPS) and tacrolimus immunosuppression are available. The primary objective of this study was to investigate the influence of sex and race on MPA and MPA glucuronide (MPAG) pharmacokinetics in stable renal transplant recipients receiving ECMPS and tacrolimus The pharmacokinetics of MPA and MPAG and their associated gastrointestinal AEs were investigated in 67 stable renal transplant recipients: 22 African American males (AAMs), 13 African American females (AAFs), 16 Caucasian males (CMs), and 16 Caucasian females (CFs) receiving ECMPS and tacrolimus. A validated gastrointestinal AE rating included diarrhea, dyspepsia, vomiting, and acid-suppressive therapy was completed. Apparent clearance, clearance normalized to body mass index (BMI), area under the concentration–time curve from time zero to 12 h (AUC12) and dose-normalized AUC12 (AUC*) were determined using a statistical model that incorporated gastrointestinal AE and clinical covariates. Males had more rapid apparent MPA clearance (CMs 13.8 ± 6.27 L/h vs. AAMs 10.2 ± 3.73 L/h) than females (CFs 8.70 ± 3.33 L/h and AAFs 9.71 ± 3.94 L/h; p = 0.014) with a race–sex interaction (p = 0.043). Sex differences were observed in MPA clearance/BMI (p = 0.033) and AUC* (p = 0.033). MPA AUC12 was greater than 60 mg·h/L in 57 % of renal transplant recipients (RTR) with 71 % of patients demonstrating gastrointestinal AEs and a higher score noted in females. In all patients, females exhibited 1.40-fold increased gastrointestinal AE scores compared with males (p = 0.024). Race (p = 0.044) and sex (p = 0.005) differences were evident with greater MPAG AUC12 in AAFs and CFs. Sex and race differences were evident, with females having slower MPA clearance, higher MPAG AUC12, and more severe gastrointestinal AEs. These findings suggest sex and race should be considered during MPA immunosuppression.

Preclinical activity of irinotecan has been seen in glioma models, but only modest efficacy has been noted in clinical studies, perhaps related to drug distribution and/or pharmacokinetic limitations. In preclinical testing, irinotecan liposome injection (nal-IRI) results in prolongation of drug exposure and higher tissue levels of drug due to slower metabolism and the effect of enhanced permeability and retention. The objective of the current study was to assess the safety and pharmacokinetics (PK) of nal-IRI and to determine the maximum tolerated dose (MTD) in patients with recurrent high-grade glioma stratified based on UGT1A1 genotyping. This phase I study stratified patients with recurrent high-grade glioma into 2 groups by UGT1A1 status: homozygous WT (“WT”) vs heterozygous WT/*28 (“HT”). Patients who were homozygous *28 were ineligible. The design was a standard 3 + 3 phase I design. WT patients were started at 120 mg/m2 intravenously every 3 weeks with dose increases in 60 mg/m2 increments. HT patients were started at 60 mg/m2, with dose increases in 30 mg/m2 increments. The assessment period for dose-limiting toxicity was 1 cycle (21 days). In the WT cohort (n = 16), the MTD was 120 mg/m2. In the HT cohort (n = 18), the MTD was 150 mg/m2. Dose-limiting toxicity in both cohorts included diarrhea, some with associated dehydration and/or fatigue. PK results were comparable to those seen in other PK studies of nal-IRI; UGT1A1*28 genotype (WT vs. HT) did not affect PK parameters. Nal-IRI had no unexpected toxicities when given intravenously. Of note, UGT1A1 genotype did not correlate with toxicity or affect PK profile.

Purpose: To determine the maximal tolerated dose of capecitabine with oxaliplatin + radiotherapy in a phase I study of localized esophageal cancer. Patients and Methods: Oxaliplatin (85 mg/m2) administered on days 1, 15, and 29. Capecitabine administered twice daily 5 days weekly; dose levels (DL) were 1, 1000; 2, 1250; and 3, 1500 mg/m2 with 50.4 Gy radiation. Results: Dose-limiting toxicity was reached at DL 3. Carboxylesterase expression in day 2 tumor specimens and induction correlated with response (p ≤ 0.05). Conclusion: The maximal tolerated dose was 85 mg/m2 of oxaliplatin, 1,250 mg/m2/day of capecitabine, and 50.4 Gy of radiation.

We conducted a phase I study to determine the recommended dose of selenomethionine (SLM) in combination with irinotecan that consistently results in a protective plasma selenium (Se) concentrations15 microM after 1 week of SLM loading.A 3-3 standard escalation design was followed. SLM was given orally twice daily (BID) for one week (loading) followed by continuous once daily (QD) dosing (maintenance). Seven dose levels of selenomethionine were investigated. Irinotecan was given intravenously at a fixed standard weekly dose, starting on the first day of maintenance SLM.Thirty-one patients were treated on study. Dose limiting diarrhea complicated by sepsis was noted in one of six patients at each of the dose-levels 1 and 7. Dose-levelsor = 5 (4,800 mcg/dose loading maintenance) resulted in day 8 Se concentrations15 microM while dose-level 7 (7,200 mcg/dose loading and maintenance) resulted in day 8 Se concentrations20 muM. No significant variations in SN-38 or biliary index were noted between weeks 1 and 4 of treatment. Despite achieving target Se concentrations, gastrointestinal and bone marrow toxicities were common and irinotecan dose modification was prevalent. Objective responses were seen in two patients and nine patients had disease control for 6 months or longer.Selenomethionine can be escalated safely to 7,200 mcg BID x 1 week followed by 7,200 mcg QD in combination with a standard dose of irinotecan. No major protection against irinotecan toxicity was established; however, interesting clinical benefits were noted-supporting the investigation of this combination in future efficacy trials.

This study was designed to understand the basis for the efficacy of methylselenocysteine (MSC) in increasing the therapeutic index of irinotecan against human tumor xenografts. Nude mice bearing human FaDu and A253 tumors were treated orally with different doses of MSC and irinotecan. Plasma, tumor and normal tissue samples were collected at different times after MSC treatments and were analyzed for selenium (Se) concentration using electrothermal atomic absorption spectrophotometry. MSC is highly effective in modulating the therapeutic index of irinotecan in the treatment of human squamous cells carcinoma of the head and neck xenografts (FaDu and A253). Enhanced irinotecan efficacy was greater in FaDu tumors (100% CR) than in A253 tumors (60% CR), and depended on MSC dose with a minimum effective dose of 0.01 mg/d x 28. The highest plasma Se concentration was achieved 1 h after a single dose and 28 d after daily treatments of MSC. The ability of FaDu tumors to retain Se was significantly better than A253 tumors, and the highest Se concentration in normal tissue was achieved in the liver. Peak plasma and tissue Se concentrations were functions of the dose and duration of MSC treatment. The MSC-dependent increase in Se level in normal tissues may contribute to the protective effect against irinotecan toxicity observed in those tissues. Intratumoral total Se concentration was not found to be predictive of the combination therapy response rates. There is a critical need to develop a method to measure the active metabolite of MSC, rather than total Se.

Irinotecan and topotecan are the only camptothecin analogues approved by the FDA for cancer treatment. However, inherent and/or acquired irinotecan and topotecan resistance is a challenging issue in clinical practice. In this report, we showed that FL118, a novel camptothecin analogue, effectively obliterated human xenograft tumors that acquire irinotecan and topotecan resistance. Consistent with this finding, Pharmacokinetics studies indicated that FL118 rapidly clears from circulation, while effectively accumulating in tumors with a long elimination half-life. Consistent with our previous studies on irinotecan, FL118 exhibited ≥25 fold more effectiveness than topotecan at inhibiting cancer cell growth and colony formation; we further showed that although topotecan can inhibit the expression of survivin, Mcl-1, XIAP or cIAP2, its effectiveness is about 10-100 fold weaker than FL118. Lastly, in contrast to both SN-38 (active metabolite of irinotecan) and topotecan are substrates of the efflux pump proteins P-gp/MDR1 and ABCG2/BCRP, FL118 is not a substrate of P-gp and ABCG2. Consistently, sildenafil, a multiple efflux pump inhibitor, sensitized SN-38 much more than these of the ABCG2-selective inhibitor KO143 in growth inhibition of SW620 and HCT-8 cells. In contrast, both inhibitors showed no effect on FL118 efficacy. Given that both P-gp and ABCG2 express in SW620 and HCT-8 cells and FL118 is not a substrate for P-gp and ABCG2, this suggests that FL118 appears to bypass multiple efflux pump protein-induced resistance, which may contribute to FL118 overcoming irinotecan and topotecan resistance in vivo. These new findings provide renewed perspectives for further development of FL118 for clinical applications.

PURPOSE: We conducted a phase I study to determine the maximum tolerated dose (MTD) of irinotecan with fixed, nontoxic high dose of selenomethionine. EXPERIMENTAL DESIGN: Selenomethionine was given orally as a single daily dose containing 2,200 mug of elemental selenium (Se) starting 1 week before the first dose of irinotecan. Irinotecan was given i.v. once weekly x 4 every 6 weeks (one cycle). The starting dose of irinotecan was 125 mg/m(2)/wk. Escalation occurred in cohorts of three patients until the MTD was defined. Pharmacokinetic studies were done for selenium and irinotecan and its metabolites. RESULTS: Three of four evaluable patients at dose level 2 of irinotecan (160 mg/m(2)/wk) had a dose-limiting diarrhea. None of the six evaluable patients at dose level 1 (125 mg/m(2)/wk irinotecan) had a dose-limiting toxicity. One patient with history of irinotecan-refractory colon cancer achieved a partial response. The long half-life of selenium resulted in a prolonged accumulation towards steady-state concentrations. No significant changes in the pharmacokinetics of CPT-11, SN-38, or SN-38G were identified; however, the coadministration of selenomethionine significantly reduced the irinotecan biliary index, which has been associated with gastrointestinal toxicity. CONCLUSIONS: Selenomethionine at 2,200 mug/d did not allow the safe escalation of irinotecan beyond the previously defined MTD of 125 mg/m(2). None of the patients receiving 125 mg/m(2) of irinotecan had grade >2 diarrhea. Unexpected responses and disease stabilizations were noted in a highly refractory population. Further escalation of selenomethionine is recommended in future trials to achieve defined protective serum concentrations of selenium.

In this report, electrospray ionization tandem mass spectrometry (ESI-MS/MS) for a pharmacokinetic study of IDN 5390, a novel C-seco taxane derivative, which is under preclinical evaluation, has been investigated. Our results showed that IDN 5390 and other taxanes including paclitaxel and IDN 5109 could ionize well in not only positive-, but also in negative-ion mode. Under collision-induced dissociation (CID) conditions, these compounds could fragment into similar M- (molecular), T- (taxane ring) and S- (side chain) series ions. In positive-ion ESI, the formation of both T- and S-series ions involved the breaking of the C-13 ester bond. In negative-ion ESI, however, while the formation mechanism of S-series ions remained the same, the breaking of the C-1' carboxylic ester bond resulted in T-series ions. At optimum collision energy (CE) values, M-, T- and S-series ions of IDN 5390 in both positive- and negative-ion ESI-MS/MS spectra had good intensity. This phenomenon makes both positive- and negative-ion ESI-MS/MS good methods for IDN 5390 metabolite structural characterization, i.e. to reveal the location of modification groups in IDN 5390 metabolites versus IDN 5390 either on the side chain or the taxane ring. A liquid chromatography (LC)/ESI-MS/MS method using the multiple-reaction monitoring (MRM) technique was thereafter developed to quantify IDN 5390 in dog plasma using paclitaxel as internal standard. The method was validated using a concentration range between 5 and 1000 ng/mL and had a limit of detection of 1 ng/mL. The inter-day %CV (%coefficient of variation) of the calibration standards ranged between 4.36 and 9.64%, the intra-day %CV of the calibration standards between 0.61 and 13.44%, and the mean % accuracy of the quality control samples at the low, middle and high end of the concentration curves were 12.5, 6.8 and 9.6%, respectively.

A great deal of experimental evidence connects induction of polyamine catabolism via spermidine/spermine N1-acetyltransferase (SSAT) to antiproliferative activity and apoptosis. Following our initial observation from gene expression profiling that platinum drugs induce SSAT, we undertook this present study to characterize platinum drug induction of SSAT and other polyamine catabolic enzymes and to examine how these responses might be enhanced with the well-known inducer of SSAT and clinically relevant polyamine analogue, N1,N11-diethylnorspermine (DENSPM). The results obtained in A2780 ovarian cancer cells by real-time quantitative RT-PCR and Northern blot analysis show that a 2-hour exposure of A2780 cells to platinum drugs induces expression of SSAT, a second SSAT (SSAT-2), spermine oxidase, and polyamine oxidase in a dose-dependent manner. At equitoxic doses, oxaliplatin is more effective than cisplatin in SSAT induction. The most affected enzyme, SSAT, increased 15-fold in mRNA expression and 2-fold in enzyme activity. When combined with DENSPM to further induce SSAT and to enhance conversion of mRNA to activity, oxaliplatin increased SSAT mRNA 50-fold and activity, 210-fold. Polyamine pools declined in rough proportion to levels of SSAT induction. At pharmacologically relevant oxaliplatin exposure times (20 hours) and drug concentrations (5 to 15 μmol/L), these responses were increased even further. Combining low-dose DENSPM with oxaliplatin produced a greater than additive inhibition of cell growth based on the sulforhodamine-B assay. Taken together, the findings confirm potent induction of polyamine catabolic enzymes, such as SSAT by platinum drugs, and demonstrate that these biochemical responses as well as growth inhibition can be potentiated by co-treatment with the polyamine analogue DENSPM. With appropriate in vitro and in vivo optimization, these findings could lead to clinically relevant therapeutic strategies.

Sunitinib is considered a first-line therapeutic option for patients with advanced clear cell renal cell carcinoma (ccRCC). Despite sunitinib's clinical efficacy, patients eventually develop drug resistance and disease progression. Herein, we tested the hypothesis whether initial sunitinib resistance may be transient and could be overcome by dose increase. In selected patients initially treated with 50 mg sunitinib and presenting with minimal toxicities, sunitinib dose was escalated to 62.5 mg and/or 75 mg at the time of tumor progression. Mice bearing two different patient-derived ccRCC xenografts (PDX) were treated 5 days per week with a dose-escalation schema (40–60–80 mg/kg sunitinib). Tumor tissues were collected before dose increments for immunohistochemistry analyses and drug levels. Selected intrapatient sunitinib dose escalation was safe and several patients had added progression-free survival. In parallel, our preclinical results showed that PDXs, although initially responsive to sunitinib at 40 mg/kg, eventually developed resistance. When the dose was incrementally increased, again we observed tumor response to sunitinib. A resistant phenotype was associated with transient increase of tumor vasculature despite intratumor sunitinib accumulation at higher dose. In addition, we observed associated changes in the expression of the methyltransferase EZH2 and histone marks at the time of resistance. Furthermore, specific EZH2 inhibition resulted in increased in vitro antitumor effect of sunitinib. Overall, our results suggest that initial sunitinib-induced resistance may be overcome, in part, by increasing the dose, and highlight the potential role of epigenetic changes associated with sunitinib resistance that can represent new targets for therapeutic intervention. Mol Cancer Ther; 14(2); 513–22. ©2014 AACR.

We examined whether potent vascular endothelial growth factor (VEGF) blockade mediated by aflibercept, a decoy VEGF receptor (VEGFR) 1/2 moiety with stronger affinity for VEGF than bevacizumab, resulted in antileukemia effects and enhanced the efficacy of systemic chemotherapy. The efficacy of aflibercept alone and in combination with doxorubicin was evaluated in human VEGF-expressing acute myeloid leukemia (AML) cell lines and primary cells xenotransplanted into immunodeficient mice. Aflibercept reduced primary VEGF/VEGFR-positive AML colony formation growth in vitro and inhibited AML xenograft growth up to 93% in association with antiangiogenic and antiproliferative effects, hypoxia, and VEGF sequestration in multiple models. High VEGF-A expression by AML cells promoted in vivo xenograft growth and aflibercept sensitivity. Aflibercept therapy slowed disease progression in two systemic human AML xenograft models and reduced peripheral leukemia disease in a primary relapsed AML model in NOD/SCID/IL2Rγnull mice. Combination aflibercept and doxorubicin enhanced antitumor effects in local xenograft models. Sequential aflibercept followed by doxorubicin resulted in progressive anthracycline accumulation in marrow and extramedullary AML sites and resulted in 2-fold higher drug levels 24 hours after administration. In contrast, tissues (tumor, plasma, marrow) treated with chemotherapy only showed progressive drug clearance over time. Combination aflibercept and doxorubicin also resulted in vascular narrowing, decreased vessel number, and perivascular apoptosis. These data suggest that inefficient drug delivery by leukemia-associated vasculature may mediate chemoresistance and support further clinical evaluation of combination aflibercept and anthracycline therapy in refractory/relapsed AML patients. Mol Cancer Ther; 9(10); 2737–51. ©2010 AACR.

A great deal of experimental evidence connects induction of polyamine catabolism via spermidine/spermine N1-acetyltransferase (SSAT) to antiproliferative activity and apoptosis. Following our initial observation from gene expression profiling that platinum drugs induce SSAT, we undertook this present study to characterize platinum drug induction of SSAT and other polyamine catabolic enzymes and to examine how these responses might be enhanced with the well-known inducer of SSAT and clinically relevant polyamine analogue, N1,N11-diethylnorspermine (DENSPM). The results obtained in A2780 ovarian cancer cells by real-time quantitative RT-PCR and Northern blot analysis show that a 2-hour exposure of A2780 cells to platinum drugs induces expression of SSAT, a second SSAT (SSAT-2), spermine oxidase, and polyamine oxidase in a dose-dependent manner. At equitoxic doses, oxaliplatin is more effective than cisplatin in SSAT induction. The most affected enzyme, SSAT, increased 15-fold in mRNA expression and 2-fold in enzyme activity. When combined with DENSPM to further induce SSAT and to enhance conversion of mRNA to activity, oxaliplatin increased SSAT mRNA 50-fold and activity, 210-fold. Polyamine pools declined in rough proportion to levels of SSAT induction. At pharmacologically relevant oxaliplatin exposure times (20 hours) and drug concentrations (5 to 15 micromol/L), these responses were increased even further. Combining low-dose DENSPM with oxaliplatin produced a greater than additive inhibition of cell growth based on the sulforhodamine-B assay. Taken together, the findings confirm potent induction of polyamine catabolic enzymes, such as SSAT by platinum drugs, and demonstrate that these biochemical responses as well as growth inhibition can be potentiated by co-treatment with the polyamine analogue DENSPM. With appropriate in vitro and in vivo optimization, these findings could lead to clinically relevant therapeutic strategies.

Introduction MM-398, a stable nanoliposomal irinotecan (nal-IRI), is designed to exploit leaky tumor vasculature for enhanced drug delivery to tumors. Tumor deposition of nal-IRI and subsequent irinotecan conversion by CES enzymes in both neoplastic cells and tumor associated macrophages (TAM) may positively correlate with activity. Predictive biomarkers to measure tumor deposition could identify patients likely to benefit from nal-IRI. FMX is a 30nm iron-oxide, superparamagnetic nanoparticle with MRI contrast properties. The particle size, its propensity for uptake by TAMs and similar distribution patterns to nal-IRI in preclinical models led to a clinical study evaluating the feasibility of correlating FMX-based MRI (Fe-MRI) acquisition with tissue drug metabolite levels and other biomarkers to estimate drug delivery to tumor. Patients and methods Eligible patients (n=12) with refractory solid tumors with at least two metastatic lesions >2cm accessible for a percutaneous biopsy were enrolled from one institution. Fe-MRI scans were performed on a 1.5T MRI using T2* iron sensitive sequences prior to and following FMX infusion (0.5h, 24h, 72h). MR images were used to direct biopsies at 72h to FMX high or low regions, permitting intra- and inter-patient comparisons of FMX and nal-IRI tumor levels. Patients continued on nal-IRI at 80mg/m2 q2w until progression. Tissue iron and TAM distribution were assessed by IHC, tissue-bound metabolite levels by mass-spectrometry. T2* signal was used to calculate FMX levels in total lesions along with FMX estimates on biopsy images derived from fused MRI-CT biopsy images. The first 9 patients (2M 7F; median age 57 years, range 28-71 years) are reported. Results There were no safety-related or other potential interactions with nal-IRI and FMX. Adverse events of nal-IRI were consistent with previous studies. FMX levels, quantified in 36 tumor lesions from the first 9 subjects, showed mean FMX accumulation of 37.9 mcg/mL [3.3-101.2 mcg/mL] and 13.2 mcg/mL [0.1-41.0 mcg/mL] at 24h and 72h, respectively. Lesions were localized mostly in liver (67%) and lymph nodes/peritoneal sites (25%). A mechanistic PK model indicated that tissue permeability to FMX contributed to Fe-MRI signals at 24h, while FMX binding contributed at 72h. Levels of irinotecan and SN-38 were 3.59mcg/g [2.29-4.89mcg/g] and 11.43ng/g [4.04-18.8ng/g], respectively, at 72h in biopsies from the first 6 patients. Conclusions This study is one of the first to measure active metabolite SN-38 levels in patient tumors. FMX can be used as a tumor contrast agent prior to nal-IRI treatment. T2* MRI sequences allowed for quantitation of FMX concentrations in tumor and reference tissue. A mechanistic model provided an estimation of FMX tumor tissue permeability and binding that may be useful as a predictive biomarker of nanotherapeutics such as nal-IRI. Citation Format: Ramesh K. Ramanathan, Ronald L. Korn, Jasgit C. Sachdev, Gerald J. Fetterly, Katie Marceau, Vickie Marsh, John M. Neil, Ronald G. Newbold, Natarajan Raghunand, Joshua Prey, Stephan G. Klinz, Eliel Bayever, Jonathan B. Fitzgerald. Pilot study in patients with advanced solid tumors to evaluate feasibility of ferumoxytol (FMX) as tumor imaging agent prior to MM-398, a nanoliposomal irinotecan (nal-IRI). [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 CT224. doi:10.1158/1538-7445.AM2014-CT224

2606 Background: Single nucleotide polymorphisms (SNPs) in carbonyl reductase 3 (CBR3V244M and CBR3C4Y), may impact the variable pharmacodynamics of anthracyclines. For example, homozygosis for the...

Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) has promising anti-tumor therapeutic potential and is in clinical trials. It is known that microenvironmental factors such as elevated interstitial fluid pressure limits uptake and greatly inhibits anti-tumor efficacy of many therapeutics. We are investigating the role of interstitial fluid pressure in the anti-tumor effect of Apo2L/TRAIL alone and in combination with gemcitabine. We first carried out a histological analysis of Colo205 tumors treated with a single 1000 g dose of Apo2L/TRAIL. Apoptosis (ck18+ cells) and infiltration of macrophages (F4/80+ cells) peaks by 8 hrs; by 24 and 48 hours, apoptotic debris has been cleared and the lumen of tumor vessels is clearly apparent. This is in sharp contrast to untreated tumors in which cells are densely packed and vessels with an open lumen are seldom seen. Correspondingly, measurements of tumor interstitial fluid pressure (IFP) taken 48 hrs after a single treatment of Apo2L/TRAIL revealed a significant reduction in IFP compared to tumors in the untreated mice (n=5; 0.3 cm H2O vs. 3.4 cm H2O, P= 0.002). We have characterized the Apo2L/TRAIL sensitivity of a panel of patient pancreatic tumor xenografts and found a range of responses to Apo2L/TRAIL from actual regression (tumor growth inhibition, TGI, of 108%) to complete resistance (TGI of 0%). Histological analysis of a sensitive tumor showed that Apo2L/TRAIL induced both the physical changes seen in Colo205 as well as a similarly significant reduction in IFP (n=7; P= 0.003). Importantly, in these patient xenografts, combination therapy with Apo2L/TRAIL and chemotherapy has been shown to enhance the antitumor effect of gemcitabine. Therefore, we investigated the effect of Apo2L/TRAIL induced lowering of IFP on gemcitabine uptake. In these studies, a single dose of gemcitabine was administered 48 hrs after Apo2L/TRAIL administration. Pharmacological analysis reveals a 33% increase in gemcitabine uptake in tumors in the 6 hours following Apo2L/TRAIL administration; no such increase was observed in normal liver. These results support the idea that pretreatment of a tumor with Apo2L/TRAIL leads to “decompression” of the tumor and will selectively improve tumor uptake of chemotherapeutics. We are currently investigating different schedules of administration in order to maximize the uptake of gemcitabine and optimize the anti-tumor efficacy of this combination therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-380.

e15540 Background: Currently, advanced AGC has no clear standard regimen. The loss of transforming growth factor-β (TGFβ) response contributes to oncogenesis and has been described in gastric cancer. V, an inhibitor of histone deacetylase (HDAC) can restore TGFβ activity. We hypothesize that the addition of V to a standard chemotherapy regimen, FOLFIRI will result in improved therapeutic efficacy. Methods: AGC (esophagus, gastric, hepatocellular) patients (pts) with adequate organ function, performance status (ECOG 0–1), and 0–1 prior chemotherapy regimens are eligible for this phase 1 study to determine the MTD of V. Treatment consists of standard FOLFIRI (I 180mg/m2, leucovorin 400mg/m2, 5FU 400mg/m2 followed by 46-hr infusion 5FU 2400 mg/m2 q2w) with escalating doses of V given orally daily starting on day 2 (doses 200mg, 300mg, 400mg) in part 1 of the study. Per pre-specified design, initial intra- and inter-patient dose escalation was permitted, but then changed to a standard 3+3 design after observed toxicities. Tumor biopsy pre-Rx and at D13 are being done for TGFβ and survivin expression. PKs for I, SN-38 & SN-38G are being evaluated in part 1. Part 2 examines PK of V at the highest V dose tolerated. Results: 10 pts (7 M, 3 F), with a median age of 52 yr have been treated at 3 dose levels of V in part 1 (2 at 200mg, 5 at 300mg, 3 at 400mg). 2 pts have been given 400mg V in part 2. Major toxicities included neutropenia, leukopenia, fatigue, diarrhea, anemia, and hypoalbunemia. No DLT was noted at any dose level. Of the 8 pts evaluable for response, 2 pts experienced a partial response and 5 pts had stable disease. SN-38 exposure was similar prior to (316–395 ng-hr/mL) and in combination with escalating doses of V (426–469 ng-hr/mL), with a terminal half-life ranging from 7.5–14 hr. Conclusions: V continuously at 400mg/d with FOLFIRI was tolerable in AGC pts. Drug exposure of SN-38 is not affected by V coadministration. The expansion cohort will determine the recommended phase 2 dose. Correlative studies of survivin and TGFβ expression are ongoing. Part 3 will examine intermittent V dosing. This study is supported by an IISP research grant from Merck & Co., Inc. [Table: see text]

2574 Background: SLM reduces Iri toxicity in xenograft models at associated selenium (Se) concentrations (Con) = 15μM. We conducted a phase I clinical trial of a fixed dose Iri with escalating doses of SLM in order to identify the highest safe dose of SLM that achieves and maintains Se Conc > 15μM. Methods: A standard 3–3 escalation was conducted. Iri was given at 125mg/m2/week x 4 weeks every 6 weeks. SLM was started 1 week prior to 1st dose of Iri. A loading BID dose of SLM was given for 1 week, followed by a daily maintenance dose. Selenium trough levels were obtained on days 8 and 29 of the study (days 1 and 22 irinotecan) and once every 6 week-cycle. Seven dose-levels of SLM were investigated (loading/maintenance, in mcg): 3,200/2,800, 3,200/3,200, 4,000/3,200, 4,000/4,000, 4,800/4,800, 5,600/5,600, and 7,200/7,200. Results: 31 patients enrolled on this study: age (median 57, range 21–80), Male/Female 21/10, ECOG 0/1 (15/16), 31 with prior chemotherapy, 12 with prior radiation, 22 colorectal and 9 mixed solids. Dose escalation was successful up to dose level 7 (7,200mcg SLM PO BID × 1 week followed by 7,200mcg SLM PO QD), which was declared the recommended dose. 2 Dose limiting toxicities occurred on study: one DLT of Grade (G) 3 febrile neutropenia, G3 sepsis, G3 dehydration occurred on dose-level 1; one DLT of G3 febrile neutropenia, dehydration occurred on dose-level 7. Both DLT occurred in a setting of partial small bowel obstruction related to peritoneal carcinomatosis. Non-DLT = G3 treatment-related toxicities included: 3 G3 neutropenia (< 1 week) at DL2, 3 & 7; 2 G3 diarrhea lasting < 48 hours on DL5 & 7. A lower incidence of G2+ toxicities was noted in patients with higher Se Conc (86% for Se < 15; 67% for Se 15–20; 57% for Se> 20 μM). Toxicities related to SLM were limited to garlic-like smell to breath, sweat, and urine in few patients. Responses included 7 confirmed stable diseases and 2 confirmed partial responses. A Se Con > 15 μM was achieved at all SLM dose levels of 4,800 mcg and above. Conclusions: Doses of SLM up to 7,200 mcg BID × 7 days followed by 7,200 mcg QD can be administered safely with standard doses of Iri. Formal phase II and III studies are needed to determine if SLM reduces Iri toxicity. No significant financial relationships to disclose.

AIM We previously showed SLM reduces CPT-11 toxicity and improves activity at plasma levels ≥ 1200 ng/mL Se in animals. Our aim was to characterize SLM PK and design a dose regimen to achieve this target in pts. METHOD 13 refractory solid tumor pts enrolled in a Phase I trial of SLM (2200ug Se as SLM PO QD) started 1 wk before dose escalated CPT-11. Intensive plasma PK of Se on d8 of cycle 1 and troughs during therapy. CPT-11 and metabolite (SN38, SN38G) PK determined wks 1 and 4. PK analyzed by compartmental modeling. RESULTS Se accumulation was slow: 4–6 wks to achieve steady state. Mean (SD) CLt/F and T1/2 of Se after SLM was 0.12 (0.05) L/h and 183 (94)h. CPT-11 biliary index, BI=AUC(CPT11.SN38/SN38G), associated with GI toxicity, was 85% lower after 4 wks SLM vs. wk 1. Simulations were used to design regimens to achieve the 1200 ng/mL target within 7d, before CPT-11. These candidate SLM strategies are being formally tested in an ongoing Phase I trial of fixed dose CPT-11 and dose escalated SLM based on toxicity and[Se]. 16 pts have enrolled: 4/6 achieved the 1200 ng/mL target within 7d after a loading dose of 4000ug SLM BID x7d; 2/6 subjects were within 23 and 14% of the target. Maintenance doses of 3200ug QD maintained 5/6 patients > 1200 ng/mL for 3 – 4 months. CONCLUSION SLM demonstrated a long T1/2, significant and slow accumulation, and may reduce CPT-11 GI toxicity by altering BI. The use of modeling, simulation, and a ‘learn and confirm’ approach to the development of SLM as a therapeutic has improved the ability to evaluate this novel compound rapidly and efficiently. Clinical Pharmacology & Therapeutics (2005) 79, P31–P31; doi: 10.1016/j.clpt.2005.12.114