ALBERT

Description: Key Points Clinical responsiveness to imexon represents the first demonstration of efficacy with modulating cellular redox in B-cell NHL. Antioxidant-related gene expression predicted for response to imexon.

Description: Abstract 1658 There is great interest in repurposing drugs currently available in the pharmacopeia to other indications like cancer as such an approach would significantly shorten the time needed and cost encumbered to provide new and effective cancer therapeutics. One such drug is the gold compound Auranofin (AF), which is FDA approved as a treatment for rheumatoid arthritis (RA). We demonstrate that AF induces MCL cell death with an LD50 of 1000 nM, 500 nM and 90 nM for Granta and Jeko cell lines, and cells derived from a patient biopsy, respectively. The increased sensitivity of primary MCL patient specimens to AF is confirmed on 4 additional patient samples tested. AF similarly induced DLBCL cell death with an LD50 of 500 nM, 500 nM and 1000 nM for OCI Ly-10, SUDHL-6 and −4 cell lines, respectively. Exposure of Granta cells to an AF concentration that induced cell death resulted in the generation of reactive oxygen species (ROS). Pre-treatment of these cells with N-acetyl-cysteine (NAC) or glutathione (GSH) abrogated both ROS generation and the induction of cell death supporting the notion that AF induces NHL cell death through a redox dependent pathway. Although AF does increase mitochondrial membrane permeability (although not through the classical permeability transition pore), the major mechanism through which AF induces NHL cell death is activation of the extrinsic apoptotic pathway. In this regard, AF induces the activation of caspases 7 and 8 and results in PARP cleavage, all of which are blocked by NAC. Despite the fact that AF is a known inhibitor of thioredoxin reductase (TR), its cytotoxic effect is independent of TR inhibition, as we observe no difference in the response to AF in U266 multiple myeloma cells transfected with an expression vector which results in the over-expression of TR. Given the redox dependence of AF-induced cytotoxicity we hypothesized that inhibition of the glutathione system with buthionine sulphoximine (BSO) would further augment AF induced cell death. To test this hypothesis, Granta cells were exposed to both AF and BSO. Significant synergistic interactions of these drugs were seen when tested in a Laska analysis of synergy. For example, whereas the LD50 for AF alone in Granta cells was 1000 nM, the LD50 for AF in combination with 5 μM BSO was 200nm; for Ly-10 cells, the LD50 for AF±BSO was 400 nm vs. 190 nM, respectively, and for SUDHL-6, 411 nM vs. 185 nM, respectively. Similar results were seen in MCL cells flow cytometrically sorted from patient biopsy specimens. As observed in studies using AF alone, the cytotoxic effects of the combination were blocked with both NAC and GSH. Similar to results with AF alone, the synergistic effects of AF and BSO on NHL cytotoxicity were independent of their effects on TR. Exposure of Granta cells to AF resulted in NF-κB inhibition. NF-κB was further inhibited with concomitant exposure to BSO over-expression of relA in Granta cells, however, had no effect on AF and BSO induced cell death, suggesting the synergistic effects of AF and BSO on NHL cell death may be NF-κB-independent. Finally, we demonstrate that AF and BSO modify free thiols on the plasma membrane. As we have recently shown that the redox agent parthenolide induces NHL death in part by modifying surface protein thiols, AF and BSO may induce NHL cell death through a similar mechanism. In summary, AF induces both MCL and DLCL cell death through a redox-dependent pathway that involves the extrinsic apoptotic pathway. Profound synergy is seen with concomitant depletion of glutathione by BSO. The data presented above, along with the fact that AF is a drug having a favorable safety profile in patients, and is an FDA-approved drug for the treatment of RA makes it an attractive candidate for further study as a single agent and in rational combination with other redox active drugs. Disclosures: No relevant conflicts of interest to declare.

Description: Backround: Anti CD-20 radioimmunotherapy (RIT) is effective therapy for indolent B-cell NHL, and under investigation in more aggressive histologies. Most data on safety and efficacy of RIT is from the pre-rituximab era, and the effect of rituximab exposure on RIT in pts with NHL is unknown. Gopal et al recently demonstrated that exposure to rituximab correlated with inferior tumor response and alteration in tumor: organ dosimetry ratio both in vitro and in mouse models following therapy with iodine-131 tositumomab (Blood 112:830). Two ongoing SWOG trials evaluating RIT consolidation therapy provide a unique opportunity to evaluate the impact of prior rituximab on pharmacodynamics of iodine-131 tositumomab in humans. S0016 enrolls previously untreated pts with follicular NHL, and iodine-131 tositumomab consolidation is administered after 6 cycles of CHOP. S0433 enrolls previously untreated pts with DLBCL, and iodine-131 tositumomab is administered after 6 cycles of CHOP with rituximab, and 2 additional cycles of CHOP alone. As rituximab leads to B-cell depletion for 6 months or more, we hypothesized the residence time of iodine-131 tositumomab would differ in pts exposed recently to rituximab compared to no prior rituximab. Methods: Prospective pts at the University of Rochester enrolled in S0016 and S0433 were analyzed. Residence times of iodine-131 tositumomab were calculated using serial imaging on a Picker XP 2000 gamma camera. Rituximab levels were performed within one week prior to dosimetric iodine-131 tositumomab administration using ELISA. Medians were used to summarize the data, and the 2-tailed Mann-Whitney-Wilcoxon test was used for hypothesis testing. Results: 16 pts (6 female) on S0016 and 12 pts (6 female) on S0433, were identified, with median ages of 54.5 and 69.5 respectively. All pts had advanced stage disease, and median BMI and creatinine were similar for both groups. Pts on S0433 had a median time from rituximab to RIT of 78.6 days (range 58–98 days). Despite this, rituximab levels were present at time of iodine-131 tositumomab in all pts measured (N=9; median rituximab level 37.2 ug/ml, range 15.6–61.69). Median absolute lymphocyte count appeared lower in the S0433 group compared to the S0016 group (600 vs 1050 /ul), but this difference was not significant (p=0.12). Pts on S0433 (all had received rituximab prior to iodine-131 tositumomab consolidation) had significantly longer RIT residence times when compared to those on S0016, (not treated with prior rituximab): 115 hours vs. 107 hours; p=0.02. Therapeutic doses of iodine-131 tositumomab were not significantly different between the two studies (S0433: 72 mCi vs. S00016: 78 mCi p=0.59). Conclusions: Our results indicate that prior therapy with rituximab results in a longer residence time of iodine-131 tositumomab when used as consolidation after chemotherapy. Measurable rituximab levels at time of RIT suggest that rituximab-induced B-cell depletion decreases clearance of RIT, possibly allowing for longer exposure times. The significance of this longer residence time is unknown but it could be associated with greater toxicity to normal organs, and could be indicative of decreased tumor binding. If confirmed in larger studies, these findings could have profound implications on RIT administration in the context of rituximab. Rituximab-induced B-cell depletion could obligate the need for unlabeled antibody dosing prior to RIT, and may affect dosimetry of RIT. Prospective studies of RIT in the rituximab era should evaluate the impact of prior rituximab and RIT residence time on toxicities and outcomes in pts treated with RIT.

Description: Abstract 3167 Background: Incidentally diagnosed venous thromboembolism (VTE) is a growing clinical problem. Although pancreatic cancer is well-known to be associated with VTE, contemporary rates of incidental and symptomatic VTE events and their association with mortality are incompletely understood. Methods: We conducted a retrospective cohort study of consecutive pancreatic adenocarcinoma patients seen at the University of Rochester from 2006–2009. Radiologic reports were reviewed for presence of pulmonary embolism (PE), deep venous thrombosis (DVT), and visceral vein thrombosis. Multiple clinical variables and mortality outcomes were collected. Data were analyzed using a multivariate Cox proportional hazards model. Results: A total of 1151 radiologic exams for 135 patients were included. Forty-seven patients (34.8%) experienced at least one VTE event. There were 12 PEs (n=12 patients, 8.9%), 34 DVTs (n=17 patients, 12.6%), 47 visceral vein (n=31 patients, 22.9%) and 2 arterial (n=2 patients, 1.5%) events. Twenty-one patients (15.5%) experienced more than one event. Incidental events comprised 33.3% (n=4) of PEs, 17.6% (n=6) of DVTs and 100% (n=47) of visceral VTE. Median survival for the study population was 237 (95% CI 199–277) days. Patients with VTE had significantly reduced survival (73 vs. 233 days at 3 months post-diagnosis; 66 vs. 245 days at 6 months post-diagnosis). There was no significant difference between asymptomatic and symptomatic events in terms of conditional median survival at 3 months-, 6 months- or 1 year-post diagnosis. In multivariate analysis, occurrence of either DVT (HR 7.4 95% CI 3.8–14.6, P

Description: Background: Indolent lymphomas, including follicular lymphoma (FL) and marginal zone lymphoma, are the most prevalent lymphomas in the United States. Though outcomes have improved substantially since the introduction of rituximab, with median overall survival now exceeding 20 yrs for FL, these diseases are characterized by an incurable clinical course (Tan et. al. Blood 2013). A subset of patients has substantially shorter survival and virtually all patients require intermittent or chronic, and often morbid therapy. Moreover, the financial cost of these therapies often exceeds $100,000 annually per patient. Therefore, lower intensity and better-tolerated, cost-effective treatments are needed. An analysis of newly diagnosed FL patients treated with standard chemotherapy plus anti-CD20 therapy in two clinical trials reported a significant association between low vitamin D levels and inferior clinical outcomes; this observation has been subsequently validated by other groups. The magnitude of this association is stronger than the individual clinical prognostic factors within the FL-IPI score and may be the strongest, and only modifiable, association reported to date of a pre-therapy prognostic factor in FL (Kelly et. al. J Clin Oncol 2015). Moreover, rituximab-mediated cytotoxicity is improved in vitro in the setting of sufficient vitamin D (Bittenbring et al. J Clin Oncol 2014). Based upon these observations, we designed a randomized trial to evaluate whether vitamin D supplementation improves outcomes in patients with indolent lymphoma treated with rituximab. Methods: This NIH-funded, multi-center, double-blind, randomized, placebo-controlled, phase III study is currently enrolling adults with biopsy-proven, indolent, low tumor burden FL, marginal zone, small lymphocytic lymphoma and mucosal associated lymphoid tissue histologies. Subjects are randomized, 2:1, to vitamin D, 2000IU daily or placebo daily for 3 years, or until disease progression. All patients receive standard treatment with rituximab in 4 weekly doses. The primary endpoint is 3-year event free survival (EFS), secondary endpoints include treatment response at week 13 and overall survival. Events are defined as lack of response at week 13, disease progression, initiation of new treatment and death. The study design provides 81% power to detect a HR of 0.55 at a 0.05 significance level, which corresponds to an increase in 3-year EFS from 40% to 60%. The primary analysis will include all treated subjects. Correlative studies of blood PTH and vitamin D will identify if baseline vitamin D levels can predict patients for whom supplementation is particularly effective or ineffective. Evidence suggests that response to vitamin D supplementation may be dependent upon specific genotypes of the vitamin D receptor and vitamin D binding protein, and we are performing whole exome sequencing of patients to determine whether vitamin D related germline variations are critical determinants of outcome in the context of supplementation. Implications: A positive finding of our study would have major implications on the treatment of follicular lymphoma and other indolent lymphomas, and given the low cost and low toxicity, the combination of vitamin D with rituximab would likely become standard of care for these patients. The cost of 3 years of vitamin D supplementation for all randomized patients in our trial is less than the commercial cost of two months of lenalidomide treatment for a single patient; lenalidomide has been approved in a similar patient population. Further implications of a positive study include support for subsequent studies of vitamin D supplementation in other lymphoma subtypes, and other malignancies treated with monoclonal antibodies relying on antibody dependent cellular cytotoxicity, such as the subset of breast cancer treated with trastuzumab, and solid tumors treated with cetuximab. Our study is currently open and accruing at 7 centers with an enrollment target of 210 subjects (NCT03078855). Disclosures Friedberg: Bayer: Honoraria, Other: Data & Safety Monitoring Committee; Acerta: Other: Data & Safety Monitoring Committee. Kahl:BeiGene: Consultancy; TG Therapeutics: Consultancy; ADC Therapeutics: Consultancy, Research Funding; Seattle Genetics: Consultancy. Leonard:Celgene: Consultancy; Celgene: Consultancy; Bayer Corporation: Consultancy; Nordic Nanovector: Consultancy; MorphoSys: Consultancy; Epizyme, Inc: Consultancy; Sutro Biopharma: Consultancy; Epizyme, Inc: Consultancy; Merck: Consultancy; Karyopharm Therapeutics: Consultancy; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy; Sandoz: Consultancy; Karyopharm Therapeutics: Consultancy; ADC Therapeutics: Consultancy; Akcea Therapeutics: Consultancy; Nordic Nanovector: Consultancy; Sandoz: Consultancy; Miltenyi: Consultancy; Akcea Therapeutics: Consultancy; AstraZeneca: Consultancy; Gilead: Consultancy; Miltenyi: Consultancy; BeiGene: Consultancy; MorphoSys: Consultancy; Bayer Corporation: Consultancy; Sutro Biopharma: Consultancy; ADC Therapeutics: Consultancy; Gilead: Consultancy; Merck: Consultancy; BeiGene: Consultancy; AstraZeneca: Consultancy. Lossos:NIH: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Janssen Scientific: Membership on an entity's Board of Directors or advisory committees. Flowers:Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Bayer: Consultancy; Acerta: Research Funding; Optimum Rx: Consultancy; Gilead: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Karyopharm: Consultancy; Eastern Cooperative Oncology Group: Research Funding; V Foundation: Research Funding; TG Therapeutics: Research Funding; National Cancer Institute: Research Funding; Burroughs Wellcome Fund: Research Funding; Celgene: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Denovo Biopharma: Consultancy; Spectrum: Consultancy; Millenium/Takeda: Research Funding; AstraZeneca: Consultancy; Pharmacyclics/Janssen: Consultancy, Research Funding. Cohen:Janssen Pharmaceuticals: Consultancy; Seattle Genetics, Inc.: Consultancy, Research Funding; Lymphoma Research Foundation: Research Funding; ASH: Research Funding; LAM Therapeutics: Research Funding; UNUM: Research Funding; Genentech, Inc.: Consultancy, Research Funding; Hutchison: Research Funding; Astra Zeneca: Research Funding; Bristol-Meyers Squibb Company: Research Funding; Takeda Pharmaceuticals North America, Inc.: Research Funding; Gilead/Kite: Consultancy. Nastoupil:TG Therapeutics: Honoraria, Research Funding; Bayer: Honoraria; Celgene: Honoraria, Research Funding; Genentech, Inc.: Honoraria, Research Funding; Gilead: Honoraria; Janssen: Honoraria, Research Funding; Novartis: Honoraria; Spectrum: Honoraria.

Description: Recent studies have described malignant stem cells as central to the initiation, growth, and potential relapse of acute and chronic myelogenous leukemia (AML and CML). Because of their important role in pathogenesis, rare and biologically distinct leukemia stem cells (LSCs) represent a critical target for therapeutic intervention. However, to date, very few agents have been shown to directly target the LSC population. The present studies demonstrate that parthenolide (PTL), a naturally occurring small molecule, induces robust apoptosis in primary human AML cells and blast crisis CML (bcCML) cells while sparing normal hematopoietic cells. Furthermore, analysis of progenitor cells using in vitro colony assays, as well as stem cells using the nonobese diabetic/severe combined immunodeficient (NOD/SCID) xenograft model, show that PTL also preferentially targets AML progenitor and stem cell populations. Notably, in comparison to the standard chemotherapy drug cytosine arabinoside (Ara-C), PTL is much more specific to leukemia cells. The molecular mechanism of PTL-mediated apoptosis is strongly associated with inhibition of nuclear factor κ B (NF-κB), proapoptotic activation of p53, and increased reactive oxygen species (ROS). On the basis of these findings, we propose that the activity of PTL triggers LSC-specific apoptosis and as such represents a potentially important new class of drugs for LSC-targeted therapy.

Description: Background Imexon is a 1-carboxamido-2-cyan-aziridine isomer investigated as an anti-cancer agent given its pro-oxidant properties. By binding reduced sulfhydryls leading to the accumulation of reactive oxygen species, imexon interferes with the endoplasmic reticulum and mitochondrial reduction-oxidation (redox) balance, inhibiting protein translation and cell growth and inducing apoptosis. Pre-clinical studies demonstrated activity across an array of tumor cells in vitro and increased activity amongst B-cell non-Hodgkin lymphomas (NHL). A partial response in a follicular lymphoma (FL) patient was observed in a previous phase I study. This phase II trial was initiated to further investigate the clinical activity of imexon in patients with relapsed or refractory NHL. Methods Histologically confirmed NHL, 〉 1 prior therapy, age≥18, ECOG performance status 0–2, measurable disease, signed informed consent, creatinine and bilirubin 〈 2.0 x IULN as well as G6PD 〉 IULN were required. Patients were treated with imexon 1000 mg/m2 IV daily on days 1-5 of a 21 day cycle for up to 1 year. Messenger RNA analysis was performed on pre-treatment tumor specimens, evaluating 22 genes important for antioxidant enzyme expression, 16 genes previously associated with outcome in NHL as well as 4 immune cell surface markers. Included were 13 genes used to generate a redox signature score, previously demonstrated to correlate with NHL prognosis (Tome, Blood 2005). Results Twenty-two NHL patients [9 FL, 5 diffuse large B cell (DLBCL), 3 mantle cell, 2 transformed follicular, 2 chronic lymphocytic leukemia and 1 Burkitt] with a median age of 64 (range 43-92) completed a median of 2.5 (range 1-13) cycles of therapy. With a median number of 4 prior therapies, 9 patients had undergone a prior stem cell transplant, 10 had stage IV disease and 6 were refractory to prior therapy. Twenty patients were evaluable for response, 2 pts discontinued therapy during cycle 1 due to progressive disease and grade 5 sepsis respectively. Of the 20 evaluable patients, the overall response rate was 30% (6/20) with another 35% achieving stable disease. Responses were observed in 4 FL and 2 DLBCL pts. After a median follow-up of 7 months, the median progression free survival (PFS) was 2.4 mos (range, 0.6 to 19.1 mos) with a median PFS of 6.7 mos (range, 1.2 to 9.0 mos) in FL patients. The median overall survival has not been reached. Grade 3 and 4 toxicities consisted of anemia (7 pts), thrombocytopenia (2 pts), neutropenia (2 pts), sepsis (2 pts), vomiting (2 pts), pneumonia (2 pts), fatigue (2 pts), dehydration (2 pts) as well as hypokalemia, hyperuricemia, transient ischemic attack, increased creatinine, rash and urinary tract infection in 1 pt each. 13 pts had available pre-treatment tumor biopsies, 2 of which attained a partial response with therapy. Patients with a higher redox score were more likely to achieve an objective response (p=0.03). Further, individual genes most predictive of response included CD68, GPX1 and SOD2. Conclusions This is the first trial to demonstrate that targeting the cellular redox environment is a viable therapeutic strategy in NHL and may be particularly effective in FL. The side effect profile may lend imexon to rational combination studies. Lymphomas reliant on antioxidant defense enzymes for proliferation and survival may be more susceptible to redox directed therapy. Evaluation of antioxidant related gene expression as a predictive biomarker is warranted in future investigations of imexon and similar targeted agents. (NCT01314014) Disclosures: Barr: Seattle Genetics: Consultancy; Celgene: Consultancy. Off Label Use: Imexon; being investigated for use in Non-Hodgkin lymphoma. Schwartz:HTG Molecular Diagnostics: Employment. Dorr:Amplimed Corporation: Employment.

Description: Abstract 3719 The clinical efficacy of mTOR inhibition in MCL is limited by known resistance pathways mediated through IRS-1 and mTORC2. Simultaneous inhibition of other molecules downstream of the B cell receptor, such as PI3Kδ, may abrogate such negative feedback mechanisms. PI3Kδ inhibition using GS-1101 has demonstrated early efficacy in MCL. Taken together, the combination of mTORC1 and PI3Kδ inhibition may represent a rationale combination to test in MCL. To this end, we utilized a panel of B cell lymphoma lines including established MCL cell lines (Granta, Jeko, Mino, Rec-1, HBL-2, Z-138), cytarabine resistant MCL lines (MinoAraCR, JekoAraCR, Rec-1AraCR, HBL-2AraCR) and primary MCL cells isolated from patients. In all cell lines, dose-finding experiments using GS-1101 and the mTOR inhibitors temsirolimus and everolimus were performed in triplicates. Cell viability was determined using an Alamar Blue reduction assay. Proteins downstream of PI3K – mTOR signaling were evaluated by western blot analysis. Synergy between the agents was evaluated using Laska et al's model–free test. For in vivo studies, severe combined immunodeficiency mice were injected with 10×106 Z-138 cells on day 0. GS-9820, a PI3Kδ inhibitor optimized for murine studies, was used in lieu of GS-1101. Upon detection of tumor engraftment, animals were divided into 6 groups, each containing 5 mice; Control, GS-9820 at 10 and 20mg/kg/dose, temsirolimus at 10 and 20mg/kg/dose, and GS-9820 plus temsirolimus at 10mg/kg/dose each. GS-9820 was administered by gastric lavage twice daily on days +15 to +19 and +22 to +26. Temsirolimus was administered via tail vein injection on days +15, +17, +19, +22, +24, and +26. Tumor measurements were used to determine therapeutic activity. The initial screen of lymphoma histologic subtypes demonstrated that cell viability was reduced across Burkitt, diffuse large B cell and MCL lines exposed to GS-1101. In MCL lines, the cell viabilities observed after 48 h treatments with GS-1101 (5uM) were 80% ± 6.9, 66% ± 2.2 and 68% ± 4.7 in Granta, Jeko and Rec-1 cells respectively. No difference was observed in cytarabine resistant cells suggesting non-cross resistance with cytarabine. The activity in primary MCL cells was similar using GS-1101 (5uM) [viability range 55%-65%] while peripheral blood mononuclear cells (PBMCs) appeared less sensitive to GS-1101 [78% ± 2.4]. Both mTOR inhibitors provided moderate reductions in viability after 48 h exposures. Compared to untreated controls, the viabilities of Granta, Jeko and Rec-1 cell lines after 48 h exposures to temsirolimus (5nM) were 73% ± 1.3, 53% % ± 6.9 and 54% ± 2.0 respectively as well as 68% ± 2.9, 50% ± 7.4 and 55% ± 2.0 respectively after everolimus (5nM). Similar results were observed in primary MCL cells using temsirolimus (5nM) [range 80%-85%] while PBMCs were largely unaffected [90% ± 2.2]. The combination of GS-1101 and either mTOR inhibitor produced largely additive reductions in cell viability. Synergistic interactions were observed in Rec-1 cells for 8 dose combinations of GS-1101 (0.1–5.0uM) and either temsirolimus (1–5nM) or everolimus (1–5nM) (unadjusted p 〈 0.05 for all 8 combinations). Evidence of synergy was insufficient at any combination after adjustment for multiple comparisons over the 3 cell lines. Sequential administration using 24 h pretreatment with each agent was evaluated; no benefit over simultaneous administration was demonstrated. Consistent with known mechanisms of action, immunoblotting revealed decreased 4EBP1 and S6K phosphorylation with mTOR inhibition while PI3K inhibition consistently decreased Akt phosphorylation. In vivo, GS-9820 appears active in the Z-138 xenografts at early time points. Tumor size was reduced to 60% ± 5.5 of control at day 18 and 23 using either 10 or 20 mg/kg of GS-9820. Testing of GS-9820 in combination with temsirolimus in this model is ongoing. Our findings indicate that PI3Kδ inhibition using GS-1101 and GS-9820 is active in vitro and also in a MCL murine xenograft. GS-1101 in combination with mTORC1 inhibition largely produced additive in vitro anti-lymphoma effects in MCL. Ongoing work is aimed at understanding the differences in molecular events downstream of PI3K and mTOR inhibition comparing Rec-1 cells, where synergy was demonstrated, with other cell lines to provide insight into optimal therapeutic combinations and to determine in which molecularly defined subsets of MCL they may be most active. Disclosures: Johnson: Gilead Sciences: Employment. Lannutti:Gilead Sciences Inc: Employment.