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Table S2. Baseline Characteristics in the Tumor SPARC-Evaluable Population

Purpose: nab-Paclitaxel plus gemcitabine was superior to gemcitabine alone for patients with metastatic pancreatic cancer (MPC) in the phase III MPACT trial. This study evaluated the association of secreted protein acidic and rich in cysteine (SPARC) levels with efficacy as an exploratory endpoint.Experimental Design: Patients with previously untreated MPC (N = 861) received nab-paclitaxel plus gemcitabine or gemcitabine alone. Baseline SPARC level was measured in the tumor stroma and epithelia (archival biopsies) and plasma. Experiments were performed in pancreatic cancer mouse models in which SPARC was intact or deleted.Results: SPARC was measured in the tumor stroma of 256 patients (30%), the tumor epithelia of 301 patients (35%), and plasma of 343 patients (40%). Stroma-evaluable samples were from metastases (71%), from the pancreas (11%), or of unidentifiable origin (insufficient tissue to determine; 17%). For all patients, stromal SPARC level [high (n = 71) vs. low (n = 185)] was not associated with overall survival (OS; HR, 1.019; P = 0.903); multivariate analysis confirmed this lack of association. There was no association between stromal SPARC level and OS in either treatment arm. Neither tumor epithelial SPARC nor plasma SPARC was associated with OS. Results from a SPARC knockout mouse model treated with nab-paclitaxel plus gemcitabine revealed no correlation between SPARC expression and tumor progression or treatment efficacy.Conclusions: SPARC levels were not associated with efficacy in patients with MPC. This exploratory analysis does not support making treatment decisions regarding nab-paclitaxel plus gemcitabine or gemcitabine alone in MPC based on SPARC expression. Clin Cancer Res; 21(21); 4811–8. ©2015 AACR.

Figure S1 . Representative SPARC staining. (A) Representative SPARC staining in stroma. (B) Representative SPARC staining in tumor epithelia.

supplemental figure legend

Background: Albumin-bound paclitaxel (nab-paclitaxel, nab-PTX) plus gemcitabine (GEM) combination has demonstrated efficient antitumour activity and statistically significant overall survival of patients with metastatic pancreatic ductal adenocarcinoma (PDAC) compared with GEM monotherapy. This regimen is currently approved as a standard of care treatment option for patients with metastatic PDAC. It is unclear whether cremophor-based PTX combined with GEM provide a similar level of therapeutic efficacy in PDAC. Methods: We comprehensively explored the antitumour efficacy, effect on metastatic dissemination, tumour stroma and survival advantage following GEM, PTX and nab-PTX as monotherapy or in combination with GEM in a locally advanced, and a highly metastatic orthotopic model of human PDAC. Results: Nab-PTX treatment resulted in significantly higher paclitaxel tumour plasma ratio (1.98-fold), robust stromal depletion, antitumour efficacy (3.79-fold) and survival benefit compared with PTX treatment. PTX plus GEM treatment showed no survival gain over GEM monotherapy. However, nab-PTX in combination with GEM decreased primary tumour burden, metastatic dissemination and significantly increased median survival of animals compared with either agents alone. These therapeutic effects were accompanied by depletion of dense fibrotic tumour stroma and decreased proliferation of carcinoma cells. Notably, nab-PTX monotherapy was equivalent to nab-PTX plus GEM in providing survival advantage to mice in a highly aggressive metastatic PDAC model, indicating that nab-PTX could potentially stop the progression of late-stage pancreatic cancer. Conclusions: Our data confirmed that therapeutic efficacy of PTX and nab-PTX vary widely, and the contention that these agents elicit similar antitumour response was not supported. The addition of PTX to GEM showed no survival advantage, concluding that a clinical combination of PTX and GEM may unlikely to provide significant survival advantage over GEM monotherapy and may not be a viable alternative to the current standard-of-care nab-PTX plus GEM regimen for the treatment of PDAC patients.

Purpose: nab-Paclitaxel plus gemcitabine was superior to gemcitabine alone for patients with metastatic pancreatic cancer (MPC) in the phase III MPACT trial. This study evaluated the association of secreted protein acidic and rich in cysteine (SPARC) levels with efficacy as an exploratory endpoint. Experimental Design: Patients with previously untreated MPC (N = 861) received nab-paclitaxel plus gemcitabine or gemcitabine alone. Baseline SPARC level was measured in the tumor stroma and epithelia (archival biopsies) and plasma. Experiments were performed in pancreatic cancer mouse models in which SPARC was intact or deleted. Results: SPARC was measured in the tumor stroma of 256 patients (30%), the tumor epithelia of 301 patients (35%), and plasma of 343 patients (40%). Stroma-evaluable samples were from metastases (71%), from the pancreas (11%), or of unidentifiable origin (insufficient tissue to determine; 17%). For all patients, stromal SPARC level [high (n = 71) vs. low (n = 185)] was not associated with overall survival (OS; HR, 1.019; P = 0.903); multivariate analysis confirmed this lack of association. There was no association between stromal SPARC level and OS in either treatment arm. Neither tumor epithelial SPARC nor plasma SPARC was associated with OS. Results from a SPARC knockout mouse model treated with nab-paclitaxel plus gemcitabine revealed no correlation between SPARC expression and tumor progression or treatment efficacy. Conclusions: SPARC levels were not associated with efficacy in patients with MPC. This exploratory analysis does not support making treatment decisions regarding nab-paclitaxel plus gemcitabine or gemcitabine alone in MPC based on SPARC expression. Clin Cancer Res; 21(21); 4811–8. ©2015 AACR.

Abstract 3287 Background: Durable responses with lenalidomide monotherapy have been reported in patients with non-Hodgkin lymphoma. In relapsed/refractory diffuse large B-cell lymphoma (DLBCL), higher responses were observed in the activated B-cell-like (ABC) subtype than in the germinal centre B-cell (GCB)-like subtype (Czuczman, et al. British Journal of Haematology, 2011, 154, 477–481). Herein, the molecular mechanisms involved in the differential efficacy of lenalidomide in DLBCL subtypes were investigated. Methods: A panel of DLBCL cell lines, with 5 of ABC-subtype and 11 of non-ABC subtype, was collected and cell of origin subtype was confirmed based on literature, molecular and genetic analysis. The direct antiproliferative effect of lenalidomide on DLBCL cells was assessed using the 3H-thymidine incorporation assay and apoptosis analysis. The molecular mechanisms involved in the antiproliferative efficacy of lenalidomide in DLBCL subtypes were investigated by western blot, immunohistochemistry (IHC) and qRT-PCR analysis of key signaling events during B-cell receptor (BCR)-dependent NF-κB activation. The critical roles of interferon regulatory factor 4 (IRF4), and cereblon (CRBN) in lenalidomide efficacy were established by knock-in or knock-down of these proteins in sensitive ABC cells. Finally, a mouse xenograft model was used to confirm the antitumor effect of lenalidomide and the relevance of the molecular mechanism involved. Results: Using DLBCL cell lines, lenalidomide treatment was found to preferentially suppress proliferation of ABC-DLBCL cells in vitro at a concentration range of 0.01–100 μM (the median plasma concentration at Cmax for patients receiving 25 mg lenalidomide is 2.2 μM) and delay tumor growth in a human tumor xenograft model of OCI-Ly10 cells (lenalidomide 3–30 mg/kg, p.o. qdX28), with minimal effect on non-ABC-DLBCL cells. This tumoricidal effect of lenalidomide was associated with downregulation of IRF4, a survival factor in ABC-DLBCL cells. Treatment with lenalidomide for 1–3 days, similar to the inhibitors of PKCb and MALT1 (LY-333,531 and z-VRPR-fmk, respectively), was found to significantly (p Furthermore, knockdown of CRBN in OCI-Ly10 (p Conclusions: These data may provide a mechanism for the preferential efficacy of lenalidomide in ABC-DLBCL observed in clinical studies. These findings suggest that lenalidomide has direct antitumor activity against DLBCL cells, preferentially ABC-DLBCL cells, by blocking IRF4 expression and the BCR-NF-κB signaling pathway in a cereblon-dependent manner (also see Figure below). Disclosures: Zhang: Celgene Corp: Employment, Equity Ownership. Kosek:Celgene Corp: Employment, Equity Ownership. Wang:Celgene Corporation: Employment, Equity Ownership. Heise:Celgene Corporation: Employment, Equity Ownership. Schafer:Celgene: Employment, Equity Ownership. Chopra:Celgene Corporation: Employment, Equity Ownership.

Multi-drug resistance and cumulative cardiotoxicity are major limitations for the clinical use of anthracyclines. Here, we evaluated and compared the cross-resistance of amrubicin, a third-generation synthetic anthracycline and potent topoisomerase (topo)-II inhibitor with little or no observed cardiotoxicity to other anthracyclines and the topo-II inhibitor etoposide in drug-resistant tumor models in order to elucidate its potential mechanisms of action.Amrubicin activity was assessed in multi-drug-resistant cell lines and human tumor explants using cytotoxicity assays, confocal microscopy, fluorescence time-lapse imaging, flow cytometry, immunoblotting, and gene expression profiling techniques.We demonstrate that both doxorubicin-resistant tumor cell lines and several drug-resistant human ovarian and breast tumor explants retain sensitivity to amrubicin. In addition, we observed similar levels of amrubicin uptake and accumulation in doxorubicin-sensitive versus doxorubicin-resistant cell lines. Although amrubicin is a weak P-glycoprotein substrate, transport and retention of amrubicin were not solely modulated by P-glycoprotein in the resistant cell lines overexpressing drug efflux pumps. The cellular retention of amrubicin is likely to be a result of rapid influx due to its high intrinsic permeability and lipophilic properties, and this may explain why amrubicin overcomes pleiotropic drug resistance. Consistent with drug accumulation studies, amrubicin induced DNA damage, G(2)-M cell cycle arrest, and apoptosis in both doxorubicin-sensitive and doxorubicin-resistant lines. Using gene expression profiling studies, several classes of genes were significantly and uniquely regulated following amrubicin, but not doxorubicin or etoposide, treatment.Amrubicin appears to have a distinct mode of action that overcomes typical anthracycline resistance mechanisms. Therefore, amrubicin may be useful in the treatment of anthracycline-refractory or anthracycline-resistant tumors.

Lenalidomide has demonstrated clinical activity in myelodysplastic syndromes, particularly in patients with a deletion in the long arm of chromosome 5 (del[5q] abnormality). It has a direct effect on the del(5q) clone, which may contribute to its ability to induce cytogenetic responses. Lenalidomide also stimulates erythropoiesis, leading to erythroid responses in certain patients. Other effects include immunomodulation, anti-inflammatory effects and angiogenesis inhibition. New findings indicate that lenalidomide modulates key genes located within the del(5q) region, including tumor suppressor genes, and regulators of the actin cytoskeleton and cell membrane. Its effects on cytoskeleton regulation may explain its direct antitumor and immunomodulatory effects. This article provides an overview of the known effects of lenalidomide and new insights into its activity in del(5q) myelodysplastic syndromes.

Inhibition of histone deacetylase 6 (HDAC6)-dependent aggresome function by pan HDAC inhibitors was recently reported to be a key mechanism underlying the synergistic activity between proteasome inhibitors and HDAC inhibitors in a variety of tumour types. Because these combinations induce significant thrombocytopenia in vivo, we examined whether less toxic, isotype-selective HDAC inhibitors may still synergize with proteasome inhibitors, and if so, by what mechanisms. Here, we showed that the class I HDAC inhibitor, MGCD0103, has a potent antiproliferative activity in Hodgkin lymphoma (HL) cell lines. Furthermore, MGCD0103 induced tumour necrosis factor α (TNF-α) expression and secretion, which was associated with nuclear factor (NF)-κB activation. Selective inhibition of TNF-α expression by short interfering mRNA, or inhibition of MGCD0103-induced NF-kB activation by proteasome inhibitors enhanced MGCD0103-induced cell death. Thus, our results demonstrate that MGCD0103 may synergize with proteasome inhibitors by HDAC6-independent mechanisms, providing mechanistic rationale for exploring this potentially less toxic combination for the treatment of lymphoma.

The inhibition of key receptor tyrosine kinases (RTKs) that are implicated in tumor vasculature formation and maintenance, as well as tumor progression and metastasis, has been a major focus in oncology research over the last several years. Many potent small molecule inhibitors of vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) kinases have been evaluated. More recently, compounds that act through the complex inhibition of multiple kinase targets have been reported and may exhibit improved clinical efficacy. We report herein a series of potent, orally efficacious 4-amino-3-benzimidazol-2-ylhydroquinolin-2-one analogues as inhibitors of VEGF, PDGF, and fibroblast growth factor (FGF) receptor tyrosine kinases. Compounds in this class, such as 5 (TKI258), are reversible ATP-competitive inhibitors of VEGFR-2, FGFR-1, and PDGFRbeta with IC(50) values

Purpose: To determine the maximum tolerated dose (MTD) dose-limiting toxicity, and pharmacokinetic and pharmacodynamic profile of TKI258 (formerly CHIR-258). Experimental Design: A phase I dose escalating trial in patients with advanced solid tumors was performed. Treatment was initially as single daily doses on an intermittent 7-day on/7-day off schedule. Following a protocol amendment, a second schedule comprised, during cycle 1, 7-day on/7-day off treatment followed by 14 days of continuous daily dosing; subsequent cycles comprised 28 days of daily dosing. Pharmacokinetics and evaluation of phosphorylated extracellular signal-regulated kinase (ERK) in peripheral blood mononuclear cells were done during the first 28 days of each schedule. Results: Thirty-five patients were treated in four intermittent (25-100 mg/d) and three continuous (100-175 mg/d) dosing cohorts. Observed drug-related toxicities were nausea and vomiting, fatigue, headache, anorexia, and diarrhea. Dose-limiting toxicities were grade 3 hypertension in one patient at 100 mg continuous dosing, grade 3 anorexia in a second patient at 175 mg, and grade 3 alkaline phosphatase elevation in a third patient at 175 mg. One patient had a partial response (melanoma) and two patients had stable disease >6 months. TKI258 pharmacokinetics were linear over the dose range of 25 to 175 mg. Five of 14 evaluable patients had modulation of phosphorylated ERK levels. Conclusions: The MTD was defined as 125 mg/d. Evidence of antitumor activity in melanoma and gastrointestinal stromal tumors warrants further investigation, and other phase I studies are ongoing. Further pharmacodynamic evaluation is required in these studies to evaluate the biological effects of TKI258.

Purpose: To evaluate the therapeutic and biological effects of CHIR-258, an orally bioavailable, potent inhibitor of class III-V receptor tyrosine kinases, in colon cancer models. Experimental Design: The pharmacologic activity of CHIR-258 was characterized by monitoring target modulation as well as by evaluating the antitumor and antiangiogenic effects in human colon xenograft models. Results: CHIR-258 inhibits vascular endothelial growth factor receptor 1/2, fibroblast growth factor receptor 1/3, and platelet-derived growth factor receptor β (PDGFRβ) and shows both antitumor and antiangiogenic activities in vivo. Treatment of KM12L4a human colon cancer cells with CHIR-258 resulted in a dose-dependent inhibition of vascular endothelial growth factor receptor 1 and PDGFRβ phosphorylation and reduction of phosphorylated extracellular signal-regulated kinase (ERK) levels, indicating modulation of target receptors and downstream signaling. In vivo administration of CHIR-258 resulted in significant tumor growth inhibition and tumor regressions, including large, established tumors (500-1,000 mm3). Immunohistochemical analysis showed a reduction of phosphorylated PDGFRβ and phosphorylated ERK in tumor cells after oral dosing with CHIR-258 compared with control tumors. These changes were accompanied by decreased tumor cell proliferation rate and reduced intratumoral microvessel density. CHIR-258 inhibited the phosphorylation of PDGFRβ and ERK phosphorylation in tumors within 2 hours following dosing and the inhibitory activity was sustained for >24 hours. Significant antitumor activity was observed with intermittent dosing schedules, indicating a sustained biological activity. Conclusion: These studies provide evidence that biological activity of CHIR-258 in tumors correlates with efficacy and aids in the identification of potential biomarkers of this multitargeted receptor tyrosine kinase inhibitor. CHIR-258 exhibits properties that make it a promising candidate for clinical development in a variety of solid and hematologic malignancies.

The ONYX-015 virus is a mutated adenovirus that in theory selectively replicates and induces cytolysis in tumor cells lacking functional p53. The present study investigated whether ONYX-015 viral infection alone or in combination with conventional chemotherapeutic agents could significantly increase apoptosis in human colon cancer cell lines, regardless of p53 status, compared to untreated cells. A pair of colon cancer cell lines that differ only in their p53 status (RKO with wild-type p53 and RKOp53 with deficient p53) was tested. Two chemotherapeutic agents, 5-fluorouracil (5-FU) and CPT-11, were tested in combination with ONYX-015. Final concentrations of these agents corresponded to peak plasma levels achievable in patients. ONYX-015 concentration was 10 p.f.u./cell. In RKO and RKOp53 cell lines, ONYX-015 viral infection alone or in combination with 5-FU or CPT-11 induced a significant increase in apoptosis compared to chemotherapeutic agents alone, regardless of p53 status. Moreover, the combination of ONYX-015 and chemotherapeutics induced more apoptosis than chemotherapeutics alone in the two colon cancer cell lines independently of their p53 status. We conclude that ONYX-015 virus infection alone or in combination with 5-FU or CPT-11 induced apoptosis in human colon cancer cell lines, independently of p53 status.

Inflammatory breast cancer (IBC) is the most metastatic variant of locally advanced breast cancer. IBC has distinctive characteristics including invasion of tumor emboli into the skin and rapid disease progression. Given our previous studies suggesting that HDAC inhibitors have promise in targeting IBC, the present study revealed that the class I HDAC inhibitor Romidepsin (FK-288, Istodax; Celgene Corporation, Summit, NJ) potently induced destruction of IBC tumor emboli and lymphatic vascular architecture. associated with inhibition of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1alpha, (HIF1alpha) proteins in the Mary-X pre-clinical model of IBC. Romidepsin treatment induced clinically relevant biomarkers in including induction of acetylated Histone 3 (Ac-H3) proteins, apoptosis, and increased p21WAF1/CIP1. Romidepsin, alone and synergistically when combined with Paclitaxel, effectively eliminated both primary tumors and metastatic lesions at multiple sites formed by the SUM149 IBC cell line. This is the first report of the ability of an HDAC inhibitor to eradicate IBC tumor emboli, to destroy the integrity of lymphatic vessel architecture and to target metastasis. Furthermore, Romidepsin, in combination with a taxane, warrants evaluation as a therapeutic strategy that may effectively target the skin involvement and rapid metastasis that are hallmarks of IBC.

Novel therapies are needed for locally advanced pancreatic carcinoma. ONYX-015 (dl1520) is an E1B-55 kDa region-deleted adenovirus that selectively replicates in and lyses tumor cells with abnormalities in p53 function (eg gene mutation). We carried out a phase I dose escalation study of ONYX-015 in patients with unresectable pancreatic cancer. ONYX-015 was administered via CT-guided injection (n = 22 patients) or intraoperative injection (n = 1) into pancreatic primary tumors every 4 weeks until tumor progression. Interpatient dose escalation was carried out with at least three patients per dose level from 10(8) p.f.u. up to the 10(11) p.f.u. dose level (two patients treated at this dose). The majority of patients had abnormally low cellular immunity (CD4 counts and hypersensitivity skin testing). Injection of ONYX-015 into pancreatic carcinomas was well-tolerated. Mild, transient pancreatitis was noted in only one patient. Dose-escalation proceeded to the highest dose level. Neutralizing antibodies rose post-treatment in all patients. After injection, ONYX-015 was detectable in the blood 15 min later, but not between 1 and 15 days later. Viral replication was not documented, however, in contrast to trials in other tumor types. No objective responses were demonstrated. Intratumoral injection of an E1B-55 kDa region-deleted adenovirus into primary pancreatic tumors was feasible and well-tolerated at doses up to 10(11) p.f.u. (2 x 10(12) particles), but viral replication was not detectable.

Sotatercept (ACE-011), a recombinant human fusion protein containing the extracellular domain of the human Activin receptor IIA, binds to and inhibits activin and other members of the transforming growth factor -β (TGF-β) superfamily. Administration of sotatercept led to a rapid and sustained increase in red blood cell (RBC) count and haemoglobin (Hb) in healthy volunteers (phase I clinical trials), but the mechanism is not fully understood. Mice treated with RAP-011 (murine ortholog of ACE-011) respond with a rapid (within 24 h) increase in haematocrit, Hb, and RBC count. These effects are accompanied by an equally rapid stimulation of late-stage erythroid precursors in the bone marrow (BM). RAP-011 also induces a significant increase in erythroid burst-forming units and erythropoietin, which could contribute to additional, sustained effects on RBC production. Further in vitro co-culture studies demonstrate that BM accessory cells are required for RAP-011 effects. To better understand which TGF-β family ligand(s) mediate RAP-011 effects, we evaluated the impact of several of these ligands on erythroid differentiation. Our data suggest that RAP-011 may act to rescue growth differentiation factor 11/Activin A-induced inhibition of late-stage erythropoiesis. These data define the mechanism of action of a novel agent that regulates RBC differentiation and provide the rationale to develop sotatercept for the treatment of anaemia and ineffective erythropoiesis.

ONYX-015 is an adenovirus with the E1B 55-kDa gene deleted, engineered to selectively replicate in and lyse p53-deficient cancer cells while sparing normal cells. Although ONYX-015 and chemotherapy have demonstrated anti-tumoral activity in patients with recurrent head and neck cancer, disease recurs rapidly with either therapy alone. We undertook a phase II trial of a combination of intratumoral ONYX-015 injection with cisplatin and 5-fluorouracil in patients with recurrent squamous cell cancer of the head and neck. There were substantial objective responses, including a high proportion of complete responses. By 6 months, none of the responding tumors had progressed, whereas all non-injected tumors treated with chemotherapy alone had progressed. The toxic effects that occurred were acceptable. Tumor biopsies obtained after treatment showed tumor-selective viral replication and necrosis induction.

Adenovirus biology and cancer biology have progressed in a complementary fashion. For example, proteins encoded at the virus’ E1 region were found to bind to the tumor suppressor proteins p53 and pRB, and these interactions played an important role in characterizing the role of these tumor suppressors in virus-dependent and -independent transformation (1, 2). Perhaps, then, it is a natural extension of this history that human adenoviruses are now being developed as anticancer agents. Ironically, although advances in genetic engineering have permitted the development of replication-disabled adenoviruses as simple delivery vehicles for therapeutic genes (3–5), replication-competent wildtype strains were actually the first to be administered safely to patients (6). Now, nearly a half-century later, replication-competent adenoviruses are once again being studied as therapeutic agents (7–9). However, genetic engineering has now been used with the goal of maximizing tumor-selective replication.

ONYX-015 is an E1B-deleted adenovirus that replicates in and causes lysis of p53-deficient cancer cells selectively. To study the efficiency of intratumoral (i.t.) spread by ONYX-015, we infected specific fractions of tumor cells (two p53-deficient tumor lines and one p53 functional line) in vitro before subcutaneous inoculation into nude mice. Infection of as few as 5% of p53- tumor cells prevented tumor development in all cases; infection of 1% of p53- tumor cells resulted in significant growth inhibition but did not prevent tumor formation. In contrast, infection with ONYX-015 had no significant effect on p53+ tumor formation. These data suggested that replication-dependent tumor cell lysis and spread was occurring, but that tumor destruction might be improved by increasing i.t. virus distribution. Two treatment parameters were then varied to determine whether virus distribution, and consequently efficacy, could be improved. Divided i.t. injections of virus were more efficacious than a single injection of the same total dose. Likewise, increasing the volume of the viral suspension for i.t. injection allowed better distribution within the tumor mass and increased efficacy. These results have implications for the treatment of cancer patients with viral agents.

The 55-kilodalton (kDa) protein from the E1B-region of adenovirus binds to and inactivates the p53 gene, which is mutated in half of human cancers. We have previously shown that the replication and cytopathogenicity of an E1B, 55-kDa gene-attenuated adenovirus, ONYX-015, is blocked by functional p53 in RKO and U20S carcinoma lines. We now report that normal human cells were highly resistant to ONYX-015-mediated, replication-dependent cytolysis. In contrast, a wide range of human tumor cells, including numerous carcinoma lines with either mutant or normal p53 gene sequences (exons 5-9), were efficiently destroyed. Antitumoral efficacy was documented following intratumoral or intravenous administration of ONYX-015 to nude mouse-human tumor xenografts; efficacy with ONYX-015 plus chemotherapy (cisplatin, 5-fluorouracil) was significantly greater than with either agent alone.

The human adenovirus E1B gene encodes a 55-kilodalton protein that inactivates the cellular tumor suppressor protein p53. Here it is shown that a mutant adenovirus that does not express this viral protein can replicate in and lyse p53-deficient human tumor cells but not cells with functional p53. Ectopic expression of the 55-kilodalton EIB protein in the latter cells rendered them sensitive to infection with the mutant virus. Injection of the mutant virus into p53-deficient human cervical carcinomas grown in nude mice caused a significant reduction in tumor size and caused complete regression of 60 percent of the tumors. These data raise the possibility that mutant adenoviruses can be used to treat certain human tumors.

An Adenovirus Mutant That Replicates Selectively in p53- Deficient Human Tumor Cells The human adenovirus E1B gene encodes a 55-kilodalton protein that inactivates the cellular tumor suppressor protein p53. Here it is shown that a mutant adenovirus that does not express this viral protein can replicate in and lyse p53-deficient human tumor cells but not cells with functional p53. Ectopic expression of the 55-kilodalton EIB protein in the latter cells rendered them sensitive to infection with the mutant virus. Injection of the mutant virus into p53-deficient human cervical carcinomas grown in nude mice caused a significant reduction in tumor size and caused complete regression of 60 percent of the tumors. These data raise the possibility that mutant adenoviruses can be used to treat certain human tumors.

We investigated SIV infection and expression of adhesion molecules in the small intestine of rhesus macaques infected with pathogenic SIV (SIVmac) or nonpathogenic clone (SIV1A11). There was a wider dissemination and marked difference in tissue localization of SIVmac relative to SIV1A11. Our results also indicate that viral pathogenicity is associated with increased migration of inflammatory cells expressing VLA-alpha 4, LFA-1 alpha, Mac-1 alpha, ICAM-1, and beta 2 integrin into the intestinal mucosa.

We present data from a phase II study investigating a novel treatment strategy for relapsed/refractory mantle cell lymphoma (MCL). Twenty-six patients received lenalidomide 25�mg/d (days 1-21 of a 28-d cycle) for up to 6 cycles followed by low-dose maintenance lenalidomide (15�mg) in responding patients. Eight patients achieved complete or partial response to give an overall response rate of 31% with median response duration of 22�2�months [95% confidence interval (CI) 0�0-53�6] and median progression-free survival (PFS) of 3�9�months (95% CI 0�0-11�1). An additional six patients (23%) achieved stable disease. Eleven patients received maintenance with median PFS of 14�6�months (95% CI 7�3-21�9). Correlative studies showed that peripheral T and Natural Killer (NK) cells increased in responding patients by 40-60% over the first 6 cycles with an initial dip in NK cells suggestive of tumour infiltration. Peripheral regulatory T cells were increased in MCL patients (P�=�0�001) and expanded further following lenalidomide. Sequential plasma analysis showed increased IL12 p40 and IL7 alongside decreased MMP9, IL10, and adiponectin. Finally, a significant correlation (P�=�0�02) between gender and response suggested that female MCL patients were more sensitive to lenalidomide than males. In summary, we confirm the activity, safety and immunomodulatory properties of lenalidomide in MCL and highlight its potential as a low-dose maintenance agent.

Introduction: Lenalidomide (Len) is indicated for the treatment of relapsed/refractory (R/R) Mantle Cell Lymphoma (MCL) in the United States and Switzerland. Len binds to the cullin 4 ring E3 ubiquitin ligase complex resulting in ubiquitination and subsequent proteasomal degradation of lymphoid transcription factors Aiolos and Ikaros leading to stimulation of immune cells, such as T-cells. Clinical trial CC-5013-MCL-002 (NCT00875667) is a randomized open-label phase II study in R/R MCL patients in which Len was given orally at 25 mg/day on days 1-21 of each 28-day cycle until progression (N=170). The control arm consisted of investigator choice of single-agent rituximab, gemcitabine, fludarabine, chlorambucil, or cytarabine (N=84). We explored the immune effects of Len treatment in MCL patients enrolled in CC-5013-MCL-002 and further investigated our findings in in vitro MCL co-culture models. Methods: Peripheral blood samples for exploratory analysis were collected at Cycle 1 Day 1 (C1D1, pre-treatment), Cycle 1 Day 4 (C1D4), Cycle 2 Day 15 (C2D15) and at treatment discontinuation. Flow cytometric profiling of T, B and natural killer (NK) cell subsets was performed and differences were analyzed for correlation with clinical outcomes (response rate and progression free survival [PFS]). Cell dependent cytotoxicity was measured in 1) anti-CD3 stimulated peripheral blood mononuclear cells (PBMC) treated with vehicle or 1-10000 nM Len for 3 days and incubated with target tumor cells for an additional 4 hours followed by an apoptosis assay as measured by Annexin V/ToPro-3 flow cytometry and 2) negatively selected CD56+ NK cells stimulated with IL-2 and treated with Len (1 nM to 10 μM) for 18 hrs and incubated with target tumor cells for an additional 4 hours followed by apoptosis assay. Results: At baseline, no significant differences were observed in the absolute levels of immune subsets when comparing non-responders (NR) and responders (R) in either Len (NR=11, R=23) or control (NR=4, R=5) arms. However, in the Len arm, significantly elevated (adj. p < 0.05) proportions of CD3-CD56+CD16+ NK cells (difference of means = 8.73; 95%CI [4.48, 12.98]) were observed at C1D4 compared to baseline in the R (N=19) outcome sub-group compared to NR (N=11). A similar trend in levels of NK subsets was observed at C2D15, however the difference was not significant. In addition, elevated proportions of CD3-CD56+CD16+ NK cells (p≤0.016) at C1D4 relative to total lymphocytes correlated significantly to longer PFS in the Len arm. Immune subset analysis in the control arm did not show any correlation to response or PFS at any visit. The mechanism whereby NK cell modulation contributes to clinical benefit demonstrated by Len in patients was further explored in in vitro co-culture systems with MCL cell lines. Len treated PBMC co-cultured with Jeko-1, Granta-519, and Mino MCL cell lines resulted in 38-47.5% more apoptosis compared to DMSO (p≤0.001). We examined the effect of Len on Aiolos and Ikaros protein expression in CD56+ NK and CD3+ T cells within anti-CD3 antibody stimulated PBMCs treated with DMSO or various concentrations of Len (1 nM to 10 μM) for 72 hours. Degradation of both Aiolos (40%) and Ikaros (95%) was observed after drug treatment in CD56+ NK cells. Aiolos and Ikaros levels were also monitored in CD3+ T cells and showed decreased levels after Len treatment, consistent with previous reports (Gandhi, 2014; Kronke, 2014). Furthermore, purified CD56+ NK cell mediated cytotoxicity produced a similar pro-apoptotic effect as the PBMC assay in all MCL cell lines versus DMSO (p≤0.01). Supernatants from co-cultures of NK cells with MCL cell lines showed significantly elevated granzyme B levels as compared to DMSO controls (p≤0.0001), suggesting that the apoptotic effects observed are induced by granzyme B. Conclusions: Lenalidomide is an immune modulating agent and NK cell modulation in particular may play a role in its clinical activity in MCL. A significant increase in proportions of NK cell subsets (vs total lymphocytes) at C1D4 versus baseline was observed and is a potential response indicator of favorable clinical outcome in R/R MCL patients treated with Len. In vitro, Len enhances cell mediated cytotoxicity of MCL cell lines in two co-culture model systems. Understanding NK cell mediated mechanism(s) has potential to enhance guiding patient selection strategies and rational combination therapies of lenalidomide in MCL. Disclosures Hagner: Celgene: Employment, Equity Ownership. Chiu:Celgene: Employment, Equity Ownership. Ortiz-Estevez:Celgene: Employment, Equity Ownership. Biyukov:Celgene: Employment, Equity Ownership. Brachman:Celgene: Employment, Equity Ownership. Trneny:Celgene: Consultancy, Honoraria, Other: Travel, accommodations, expenses, Research Funding. Morschhauser:Genentech Inc./Roche: Other: Advisory boards. Stilgenbauer:AbbVie, Amgen, Boehringer-Ingelheim, Celgene, Genentech, Genzyme, Gilead, GSK, Janssen, Mundipharma, Novartis, Pharmacyclics, Roche: Consultancy, Honoraria, Research Funding. Milpied:Celgene: Honoraria, Research Funding. Musto:Sandoz: Consultancy; Celgene: Honoraria; Roche: Honoraria; Sanofi: Consultancy; Genzyme: Consultancy; Novartis: Honoraria; Janssen: Honoraria; Mundipharma: Honoraria. Martinelli:AMGEN: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; ROCHE: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; MSD: Consultancy. Heise:Celgene: Employment, Equity Ownership. Daniel:Celgene: Employment, Equity Ownership. Chopra:Celgene: Employment, Equity Ownership. Carmichael:Celgene: Employment, Equity Ownership. Trotter:Celgene Corporation: Employment. Gandhi:Celgene: Employment, Equity Ownership. Thakurta:Celgene Corporation: Employment, Equity Ownership.

Histone deacetylase inhibitors are a group of recently developed compounds that modulate cell growth and survival. We evaluated the effects of the histone deacetylase inhibitor MGCD0103 on growth of pancreatic carcinoma models following single agent treatment and in combination with gemcitabine. MGCD0103 inhibited tumor cell growth and acted synergistically with gemcitabine to enhance its cytotoxic effects. Gene expression analysis identified the cell cycle pathway as one of the most highly modulated gene groups. Our data suggest that MGCD0103 + gemcitabine might be an effective treatment for gemcitabine-refractory pancreatic cancer.

Aaron N Nguyen1, Paul W Hollenbach1, Normand Richard2, Antonio Luna-Moran1, Helen Brady2, Carla Heise1, Kyle J MacBeth11Celgene Corporation, San Francisco, CA, USA; 2Celgene Corporation, San Diego, CA, USAAbstract: Azacitidine (AZA) and decitabine (DAC) are cytidine azanucleoside analogs with clinical activity in myelodysplastic syndromes (MDS) and potential activity in solid tumors. To better understand the mechanism of action of these drugs, we examined the effects of AZA and DAC in a panel of non-small cell lung cancer (NSCLC) cell lines. Of 5 NSCLC lines tested in a cell viability assay, all were sensitive to AZA (EC50 of 1.8–10.5 µM), while only H1299 cells were equally sensitive to DAC (EC50 of 5.1 µM). In the relatively DAC-insensitive cell line A549, both AZA and DAC caused DNA methyltransferase I depletion and DNA hypomethylation; however, only AZA significantly induced markers of DNA damage and apoptosis, suggesting that mechanisms in addition to, or other than, DNA hypomethylation are important for AZA-induced cell death. Cell cycle analysis indicated that AZA induced an accumulation of cells in sub-G1 phase, whereas DAC mainly caused an increase of cells in G2/M. Gene expression analysis of AZA- and DAC-treated cells revealed strikingly different profiles, with many genes distinctly regulated by each drug. In summary, while both AZA and DAC caused DNA hypomethylation, distinct effects were demonstrated on regulation of gene expression, cell cycle, DNA damage, and apoptosis.Keywords: apoptosis, azacitidine, decitabine, gene expression, non-small cell lung cancer

BACKGROUND: The cytidine nucleoside analogs azacitidine (AZA) and decitabine (DAC) are used for the treatment of patients with myelodysplastic syndromes and acute myeloid leukemia (AML). Few non-clinical studies have directly compared the mechanisms of action of these agents in a head-to-head fashion, and the agents are often viewed as mechanistically similar DNA hypomethylating agents. To better understand the similarities and differences in mechanisms of these drugs, we compared their in vitro effects on several end points in human AML cell lines. METHODOLOGY/PRINCIPAL FINDINGS: Both drugs effected DNA methyltransferase 1 depletion, DNA hypomethylation, and DNA damage induction, with DAC showing equivalent activity at concentrations 2- to 10-fold lower than AZA. At concentrations above 1 microM, AZA had a greater effect than DAC on reducing cell viability. Both drugs increased the sub-G1 fraction and apoptosis markers, with AZA decreasing all cell cycle phases and DAC causing an increase in G2-M. Total protein synthesis was reduced only by AZA, and drug-modulated gene expression profiles were largely non-overlapping. CONCLUSIONS/SIGNIFICANCE: These data demonstrate shared mechanisms of action of AZA and DAC on DNA-mediated markers of activity, but distinctly different effects in their actions on cell viability, protein synthesis, cell cycle, and gene expression. The differential effects of AZA may be mediated by RNA incorporation, as the distribution of AZA in nucleic acid of KG-1a cells was 65:35, RNA:DNA.

Recombinant interleukin-2 (rIL-2) is a pleiotropic cytokine that activates select immune effector cell responses associated with antitumor activity, including antibody-dependent cellular cytotoxicity (ADCC). Rituximab is an anti-CD20 monoclonal antibody that activates ADCC in non-Hodgkin lymphoma (NHL). The ability of rIL-2 to augment rituximab-dependent tumor responses was investigated. The efficacy of rIL-2 in combination with rituximab was evaluated in 2 NHL tumor xenograft models: the CD20hi, rituximab-sensitive, low-grade Daudi model and the CD20lo, aggressive, rituximab-resistant Namalwa model. Combination of rIL-2 plus rituximab was synergistic in a rituximab-sensitive Daudi tumor model, as evidenced by significant tumor regressions and increased time to tumor progression, compared with rIL-2 and rituximab single agents. In contrast, rituximab-resistant Namalwa tumors were responsive to single-agent rIL-2 and showed an increased response when combined with rituximab. Using in vitro killing assays, rIL-2 was shown to enhance activity of rituximab by activating ADCC and lymphokine-activated killer activity. Additionally, the activity of rIL-2 plus rituximab F(ab')2 was similar to that of rIL-2 alone, indicating a critical role for immunoglobulin G1 Fc-FcgammaR-effector responses in mediating ADCC. Antiproliferative and apoptotic tumor responses, along with an influx of immune effector cells, were observed by immunohistochemistry. Collectively, the data suggest that rIL-2 mediates potent tumoricidal activity against NHL tumors, in part, through activation and trafficking of monocytes and natural killer cells to tumors. These data support the mechanistic and therapeutic rationale for combination of rIL-2 with rituximab in NHL clinical trials and for single-agent rIL-2 in rituximab-resistant NHL patients.

Purpose: The ectopically expressed and deregulated fibroblast growth factor receptor 3 (FGFR3) results from a t(4;14) chromosomal translocation that occurs in ∼15% of multiple myeloma (MM) patients and confers a particularly poor prognosis. This study assesses the antimyeloma activity of CHIR-258, a small-molecule inhibitor of multiple receptor tyrosine kinases that is currently in phase I trials, in a newly developed FGFR3-driven preclinical MM animal model. Experimental Design: We developed an orthotopic MM model in mice using a luciferase-expressing human KMS-11-luc line that expresses mutant FGFR3 (Y373C). The antimyeloma activity of CHIR-258 was evaluated at doses that inhibited FGFR3 signaling in vivo in this FGFR3-driven animal model. Results: Noninvasive bioluminescence imaging detected MM lesions in nearly all mice injected with KMS-11-luc cells, which were mainly localized in the spine, skull, and pelvis, resulting in frequent development of paralysis. Daily oral administration of CHIR-258 at doses that inhibited FGFR3 signaling in KMS-11-luc tumors in vivo resulted in a significant inhibition of KMS-11-luc tumor growth, which translated into a significant improvement in animal survival. Conclusions: Our data provide a relevant preclinical basis for clinical trials of CHIR-258 in FGFR3-positive MM patients.

Overexpression of fibroblast growth factor receptor 3 (FGFR3) is a hallmark of t(4;14) multiple myeloma (MM). To dissect the mechanism of FGFR3 oncogenesis in MM, we used 3 FGFR selective kinase inhibitors—CHIR258, PD173074, and SU5402—and FGFR3-specific siRNA to modulate FGFR3 activity. Conversely, the ligand FGF was used to stimulate FGFR3 function in human MM cells. The transcriptional response to FGFR3 modification was recorded, and gene expression changes common to all 5 modifiers were documented. Ten genes were commonly regulated. Macrophage inflammatory protein-1 alpha (MIP-1α) was the single most differentially altered gene. MIP-1 α promoter function, gene expression, and protein secretion were each down-regulated following inhibition of FGFR3 signaling. Down-regulation of MIP-1 α was not, however, observed following FGFR3 inhibition in MM cells with RAS mutations implicating RAS-MAPK in MIP-1 α regulation. As confirmation, inhibition of ERK1 also down-regulated MIP-1 α in FGFR3 inhibitor-resistant cells harboring RAS mutations. MIP-1 α is implicated in the survival and proliferation of MM cells and the pathogenesis of MM bone disease. Our observation is the first to directly link an initiating IgH translocation not only to MM-cell growth and survival but also to the disease-associated bone disease.

3-Benzimidazol-2-yl-1H-indazole analogs were developed as inhibitors of receptor tyrosine kinases (RTK). The synthesis and SAR of this series is reported.

Background and Aim: Advanced pancreatic cancer is both deadly and difficult to treat with success. Here, we disseminate the results of comprehensive preclinical studies which laid a strong foundation for the rapid bench to bedside development of nab-paclitaxel, an albumin-bound formulation of paclitaxel, in combination with gemcitabine, a regimen recently approved by U.S. Food and Drug Administration as a first-line treatment for patients with metastatic pancreatic cancer. Materials and Methods: We enrolled a total of 650 mice with established pancreatic tumors originated from a collection of patient-derived pancreatic cancer xenografts. The present study investigated the anti-tumor activity, survival advantage and mechanism of action of nab-paclitaxel or Cremophor EL™ (CreEL)-based paclitaxel monotherapy and in combination with gemcitabine. Results: When tested in mice with subcutaneous tumors originating from 11 separate individual patient xenografts, nab-paclitaxel plus gemcitabine treatment demonstrated superior tumor regression response, robustly depleted the tumor desmoplatic stroma, leading to enhanced gemcitabine uptake (2.8-fold) in the tumor compared to tumors in the gemcitabine alone treated mice. In orthotopic models, nab-paclitaxel treatment leads to an average of 3.64-fold decrease in primary tumor volumes compared to CreEL-based paclitaxel. Intra-tumor stromal collapse combined with decreased tumor cell proliferation was clearly evident in the primary tumors of nab-paclitaxel treated mice compared to CreEL-based paclitaxel, when the mice were sacrificed immediately after five consecutive day's treatment or three weeks after the final dose of the agents. In a highly aggressive orthotopic model, nab-paclitaxel plus gemcitabine treatment prevented primary tumor progression, and metastatic spread to liver, lymph nodes and diaphragm. While CreEL-based paclitaxel plus gemcitabine treatment failed to enhance mouse survival compared to gemcitabine monotherapy, the nab-paclitaxel plus gemcitabine combination proved statistically significant (p=0.0133) in enhancing survival. nab-paclitaxel monotherapy demonstrated statistically significant survival advantage compared to CreEL-based paclitaxel monotherapy (p=0.0072). Remarkably, nab-paclitaxel monotherapy was equivalent to nab-paclitaxel plus gemcitabine in providing survival advantage in a highly aggressive metastatic model of pancreatic cancer. Conclusion: Our results demonstrated that co-treatment with nab-paclitaxel and gemcitabine resulted in superior tumor regression response, stromal depletion and enhanced intra-tumoral gemcitabine uptake compared with either single agent alone. nab-paclitaxel demonstrated superior anti-tumor activity and provided a statistically significant survival advantage compared to CreEL-based paclitaxel. Our results provide further rationale for future preclinical and clinical trials in pancreatic cancer using nab-paclitaxel as a backbone therapy in combination with novel experimental and targeted agents. Acknowledgements: The study was supported by funding from Celgene Corporation and AACR-Stand Up To Cancer Dream Team Translational Cancer Research Grant (SU2C-AACR-DT0509). Citation Format: N.V Rajeshkumar, Shinichi Yabuuchi, Shweta G. Pai, Scott Bateman, Ellen Filvaroff, Daniel W. Pierce, Carla Heise, Daniel D. Von Hoff, Anirban Maitra, Manuel Hidalgo. Highlights of innovative preclinical studies which guided the rapid bench to bedside development of nab-paclitaxel plus gemcitabine combination for the treatment of pancreatic 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 LB-94. doi:10.1158/1538-7445.AM2014-LB-94

The t(4;14) translocation that occurs uniquely in a subset (15%) of patients with multiple myeloma (MM) results in the ectopic expression of the receptor tyrosine kinase (RTK), fibroblast growth factor receptor 3 (FGFR3). Inhibition of activated FGFR3 in MM cells induces apoptosis, validating FGFR3 as a therapeutic target in t(4;14) MM and encouraging the clinical development of FGFR3 inhibitors for the treatment of these patients, who have a poor prognosis. We describe here the characterization of a novel, small-molecule inhibitor of class III, IV, and V RTKs, CHIR-258, as an inhibitor of FGFR3. CHIR-258 potently inhibits FGFR3 with an inhibitory concentration of 50% (IC50) of 5 nM in in vitro kinase assays and selectively inhibited the growth of B9 cells and human myeloma cell lines expressing wild-type (WT) or activated mutant FGFR3. In responsive cell lines, CHIR-258 induced cytostatic and cytotoxic effects. Importantly, addition of interleukin 6 (IL-6) or insulin growth factor 1 (IGF-1) or coculture on stroma did not confer resistance to CHIR-258. In primary myeloma cells from t(4;14) patients, CHIR-258 inhibited downstream extracellular signal-regulated kinase (ERK) 1/2 phosphorylation with an associated cytotoxic response. Finally, therapeutic efficacy of CHIR-258 was demonstrated in a xenograft mouse model of FGFR3 MM. These studies support the clinical evaluation of CHIR-258 in MM.

Replication-selective oncolytic adenoviruses are being developed for the treatment of cancer, but the safety and feasibility of repeated adenovirus delivery to tumors via the bloodstream was unknown, particularly in light of a patient death after hepatic artery infusion of a replication-defective adenovirus vector. We performed a Phase II trial of an oncolytic replication-selective adenovirus (dl1520, also known as Onyx-015) administered by hepatic artery infusion in patients with gastrointestinal carcinoma metastatic to the liver (n = 27). dl1520 was infused into the hepatic artery (2 x 10(12) particles) on days 1 and 8 as a single agent, and thereafter starting on day 22 in combination with i.v. 5-fluorouracil and leucovorin every 28 days. Repeated viral infusions were feasible, and no deaths occurred on study; reversible grade 3/4 hyperbilirubinemia occurred in 2 patients. Systemic inflammatory cytokine responses varied greatly between patients and even between cycles within a given patient. Proinflammatory cytokines [e.g., tumor necrosis factor, IFN-gamma, and interleukin (IL) 6] typically rose within 3 h and were followed at 18 h by a rise in IL-10. However, in the single patient who suffered a severe but reversible systemic inflammatory response, a unique cytokine profile was detected: marked acute increases of IL-6 (20-fold higher than average for all of the patients) and inhibition of IL-10 production. Delayed secondary peaks of viremia were reproducibly detected 3-6 days after treatment, even in the presence of high level neutralizing antibody titers and antiviral cytokines. Mathematical modeling was used to calculate the number of virus particles produced and shed into the blood with each replication cycle. The combination of virotherapy and chemotherapy had antitumoral activity in some chemotherapy-resistant colorectal tumors. The intra-arterial infusion of oncolytic adenoviruses warrants additional study.

Background CNS manifestations of aggressive non-Hodgkin Lymphoma are associated with serious morbidity and adverse prognosis. Primary CNS lymphomas (PCNSL) exhibit a dichomatous growth pattern, either dissemination within brain, typical at presentation, and/or leptomeningeal spread, common at relapse. Elucidation of the mechanistic basis of CNS lymphoma progression as well as drug resistance requires preclinical models that recapitulate their pathogenesis. Methods We developed a novel method to derive cell lines of CNS lymphoma that recapitulate disease phenotypes upon intracranial implantation into mice. We are applying genomics, in vitrochemotaxis, preclinical testing of targeted therapies and neuroimaging to evaluate mechanisms of invasion and resistance. Results We developed 7 CNS lymphoma cell lines; 6 DLBCL (all ABC-type), 1 Burkitt; 5 from secondary CNS lymphoma (SCNSL), and 2 from PCNSL, of which 1 was treatment naïve. Intracranial implantation of lymphoma cells from these tumours within NSG mice provides a reproducible model to dissect the pathogenesis of CNS lymphomas. PCNSL specimens were 10X more efficient in CNS dissemination than SCNSL. High resolution array-CGH demonstrated that intracranial tumour growth was associated with retention of genomic aberrations of the original tumours (e.g. del 6q, gains on 12, etc) and that these were maintained with serial passage in vivo. CNS-infiltrative lymphomas expressed significantly increased levels of MMP-7 and RGS-13 transcripts compared to lymphomas that did not infiltrate brain, while osteopontin and cathepsin D expression by lymphoma cells did not correlate with CNS invasion. Targeted shRNA-mediated knockdown of RGS-13 was performed using lentiviral infection and resulted in significant delay of CNS lymphoma growth in vivo in a xenograft model but had no effect on lymphoma proliferation in culture. Therapeutic response to lenalidomide, minus and plus rituximab, was recapitulated in RAG-/- mice, despite deficient T-cell function and correlated with baseline relative cereblon expression, as quantified using a highly specific immunohistochemical assay. The emergence of resistance to lenalidomide in human CNS lymphoma xenografts also correlated with loss of cereblon protein expression, supporting a role for cereblon in the efficacy of lenalidomide in CNS lymphomas. Notably, significant cereblon protein expression by lymphoma cells was detected by immunohistochemistry in 12/22 diagnostic specimens of aggressive CNS lymphoma. Metabolic imaging of model CNS lymphomas using magnetic resonance spectroscopy demonstrated significant intratumoural lactate production in the microenvironment, detectable before evidence of aberrant T2 signal and reduced diffusion. Lenalidomide reduced tumour expression of lactate dehydrogenase and lactate, as well as RGS-13, consistent with anti-proliferative as well as anti-invasive effects. Conclusions To the best of our knowledge we have developed the first panel of patient-derived CNS lymphoma cell lines. We have used these to generate intracranial xenografts that provide a highly reproducible model system to dissect key elements of CNS lymphoma pathogenesis, leading to the elucidation of mechanisms of CNS lymphoma growth and invasion as well as resistance. Our results support a direct, T-cell independent effect of lenalidomide on CNS lymphoma growth and invasion which may be cereblon-dependent. Additional studies are needed to define the role of cereblon as a biomarker and mediator of lenalidomide efficacy in CNS lymphomas. In addition, we are using these models to identify novel genomic and metabolic aberrations predictive of early resistance to lenalidomide and other targeted therapies. Supported by the Lymphoma Research Foundation, Leukemia and Lymphoma Society, and by NIH R01CA139-83-01A1. Disclosures: Heise: Celgene: Employment, Equity Ownership. Rubenstein:Celgene: Research Funding; Genentech: Research Funding. Off Label Use: use of lenalidomide in CNS lymphoma.

Introduction The B-cell receptor (BCR) and its downstream effectors have emerged as important therapeutic targets in B-cell malignancies. CC-292 is a novel, potent, covalent, and highly selective inhibitor of Btk (IC50apparent of 0.5 nM, kinact/KI ratio of 7.69 × 104 M-1s-1), that does not appreciably inhibit other kinases involved in BCR signaling (eg, IC50 Lyn kinase, 4401 nM) (Evans et al., J Pharmacol Exp Ther. 2013). Here, we report preclinical characterization and clinical data in CLL from a single-agent phase 1 dose-escalation trial of CC-292 in B-cell malignancies, with a focus on how target engagement and downstream events correlate with clinical activity. Results Pharmacodynamic effects of Btk inhibition by CC-292 can be monitored by occupancy of the Btk catalytic site, Btk autophosphorylation on Y223, and downstream phosphorylation of Plc-γ2 and Erk. We developed a sensitive (10 pg/mL lower limit of quantification) and quantitative assay to measure covalent binding of CC-292 to Btk (Evans et al., J Pharmacol Exp Ther. 2013), as well as Western and novel phos-flow assays to probe downstream signal transduction. These methods showed that CC-292 treatment blocks Btk autophosphorylation and downstream pathway activation in both tumor cells and human peripheral blood mononuclear cells (PBMCs). The extent of CC-292 binding to Btk correlated with its in vitro and in vivo effects. The occupancy assay demonstrated that CC-292 effectively targets Btk in tumor cell lines, PBMCs, spleen, and lymph nodes (LNs) in animal models, and in PBMC and lymph node samples from clinical trial subjects. In rats and non-human primates treated with CC-292, Btk occupancy in spleen and LNs was dose-dependent. Measured occupancy in rat spleen and axillary, mesenteric, and superficial cervical LNs was 94%, 92%, 90%, and 76% respectively, 4 hours (hrs) after a single 30-mg/kg dose. Interim data from the phase 1 CLL trial showed that PBMC Btk was completely occupied in the majority of subjects 4 hrs post-dose with both QD and BID dosing. Twenty-four hrs post-dose at 750 and 1000 mg QD, CC-292 exhibited 83% ± 17% Btk occupancy, whereas with BID dosing at 375 and 500 mg, occupancy was 94% ± 16% at the corresponding time point (12 hrs after the second dose). Thus, while both schedules achieved extensive and sustained Btk occupancy, residual free Btk levels were lower with the BID schedule, offering a rationale for an early trend towards more rapid nodal responses, lymphocytosis, and partial responses on the BID schedule observed to date in the phase 1 study. In the 10 clinical LN biopsies tested to date, no measurable levels of unoccupied Btk have been detected, although Btk protein was present as determined by Western blotting, showing that CC-292 was able to penetrate LNs and inhibit Btk in human subjects as it did in preclinical models. For monitoring downstream signal transduction, we developed reagents and assays including a phos-flow assay based on a novel rabbit monoclonal antibody to detect Btk pY223 levels in PBMC subsets. CC-292 effectively inhibited constitutive and induced phosphorylation of Btk and Plc-γ2 at low nanomolar concentrations. CC-292 also inhibited BCR activation and nurse-like cell–supported survival of CLL cells. Furthermore, CC-292 reduced CLL cell migration and actin polymerization in response to chemokines (CXCL12, CXCL13) and inhibited secretion of the chemokines CCL3 and CCL4 by CLL cells. These chemokines are essential for migration and retention of normal and neoplastic B cells in the marrow and secondary lymphatic tissues. Consistent with this preclinical data, CC-292 treatment resulted in rapid reductions in circulating CCL3 and CCL4 levels. In subjects treated at the 750 mg QD, 1000 mg QD, 375 mg BID, and 500 mg BID dose levels, plasma CCL3 was reduced from 99 ± 16 pg/ml before treatment to 28 ± 5 pg/ml (N = 48, mean ± SEM) at 24 hrs after the first dose, while CCL4 was reduced from 235 ± 59 pg/ml to 74 ± 16 pg/ml (N = 51). Conclusions These data demonstrate that CC-292 achieves significant and durable occupancy of Btk in vitro and in vivo, inhibits Btk-mediated downstream signaling events and chemokine production, and that these preclinical activities have translated into the clinic. Taken together, these results argue that Btk inhibition is necessary and sufficient for clinical activity in CLL. These emerging data support continued development of CC-292 for the treatment of B-cell malignancies. Disclosures: Pierce: Celgene: Employment, Equity Ownership. O'Brien:Genentech: Consultancy, Research Funding; Emergent: Consultancy, Research Funding; CLL Global Research Foundation: Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy; Gilead Sciences: Consultancy, Research Funding; Infinity: Consultancy, Research Funding; MorphoSys: Research Funding; Pharmacyclics: Consultancy, Research Funding; Talon: Consultancy, Research Funding; Teva/Cephalon: Consultancy. Heise:Celgene: Employment, Equity Ownership. Nacht:Celgene: Employment, Equity Ownership. Aslanian:Celgene: Employment, Equity Ownership. Liu:Celgene: Employment, Equity Ownership. Hong:Celgene: Employment, Equity Ownership. Wu:Celgene: Employment, Equity Ownership. Zavodovskaya:Celgene: Employment, Equity Ownership. Marine:Celgene: Employment, Equity Ownership. Barnett:Celgene: Employment, Equity Ownership. Nava-Parada:Celgene: Employment, Equity Ownership. Mei:Celgene: Employment, Equity Ownership. Chopra:Celgene: Employment, Equity Ownership. Burger:Pharmacyclics: Research Funding; Gilead: Research Funding. Singh:Celgene: Employment, Equity Ownership.

Introduction Patients with mantle cell lymphoma (MCL) typically respond to initial therapy and almost inevitably relapse with frequent chemoresistance over time and poor outcome. Multiple phase II studies have established the efficacy and safety of lenalidomide, an immunomodulatory agent with tumoricidal and antiproliferative properties, in relapsed/refractory MCL. The prospective phase II multicenter MCL-001 “EMERGE” study led to FDA approval of lenalidomide for patients with relapsed/refractory MCL after 2 prior treatments, that included bortezomib. The activity of lenalidomide was seen regardless of MIPI, number of prior therapies, prior high dose therapy, bulky disease or high tumor burden. One of the most established prognostic factors in MCL is the proliferation index Ki67 (MIB1), now confirmed both in standard and dose-intensive high-dose therapy strategies. We present here longer follow-up of efficacy and safety from the MCL-001 study in patients relapsed/refractory to bortezomib and the potential relationship between Ki-67 and efficacy outcomes. Methods Patients with heavily pretreated MCL, that included prior bortezomib, received lenalidomide 25 mg/day PO, days 1-21 in 28-day cycles until disease progression or intolerability. Primary study endpoints were overall response rate (ORR) and duration of response (DOR); secondary endpoints included complete response (CR), time to response (TTR), progression-free survival (PFS), overall survival (OS), and safety. Response rates and time-to-event data were analyzed by independent central reviewers per modified IWG criteria and Kaplan-Meier estimates respectively (data cut-off March 20, 2013). Ki-67 was examined as an exploratory endpoint by immunohistochemistry for 81/134 patients (60%) either performed on biopsy samples for 24 patients, or based on the Ki-67 scores reported in local pathology reports for 57 patients. Results Median age for the enrolled intent-to-treat patient population (N=134) was 67 years (range, 43-83; 63% ≥65 years). The median number of previous therapies was 4 (range, 2-10; 78% received ≥3), 93% stage III/IV, and 72% were Conclusions Single-agent lenalidomide in heavily pretreated patients with relapsed/refractory MCL post-bortezomib showed durable long-term efficacy with a consistent safety profile. Consistent with what is reported in the literature, high Ki-67 is associated with poor outcome in our cohort with shorter OS. Though based on retrospective evaluation and subsets of patients, the ORR to lenalidomide was similar in both low and high Ki-67 groups, suggesting lenalidomide can be active in patients expressing high levels of Ki67. Prospective studies are needed to confirm these findings. Disclosures: Goy: Celgene: Consultancy, Research Funding, Speakers Bureau. Off Label Use: This is a phase 2 clinical study of safety and efficacy for lenalidomide in patients with MCL. Williams:Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Drach:Celgene: Honoraria. Ramchandren:Celgene: Research Funding. Zhang:Celgene: Employment. Cicero:Celgene: Employment. Fu:Celgene: Employment. Heise:Celgene: Employment, Equity Ownership. Witzig:Celgene: Research Funding.

Sotatercept, a recombinant human fusion protein containing the extracellular domain of ActRIIA binds to and inhibits activin and other members of the TGFß superfamily to induce a rapid increase in red cell number and hemoglobin. We sought to investigate if RAP-011, the murine ortholog of sotatercept, might regulate iron availability, which is important for erythropoiesis, through modulating hepcidin levels. Hepcidin is a circulating peptide hormone that negatively regulates iron transport into the plasma membrane by binding the iron transporter, ferroportin, and causing degradation of the complex. Hepcidin is primarily secreted by hepatocytes and also by hematopoietic cells, macrophages, kidney, heart, pancreas, and adipose cells (Gantz, T., Blood, 2003). The hepcidin promoter can be activated by both SMAD-4 and STAT-3, downstream targets of BMP-6 and IL-6 respectively (Andriopoulos Jr, B., Nature Genetics, 2009). BMP-6 binds its receptor, triggering downstream SMAD-1/5/8 signaling leading to SMAD-4 activation and initiation of HAMP (the gene encoding hepcidin) transcription. SMAD-4 signaling is also implicated in STAT-3 activation of HAMP transcription downstream of inflammatory signals such as IL-6 (Wang, R.H., Cell Metabolism, 2005). In the current study, we evaluated the effect of RAP-011 on hepcidin expression and regulation in primary human hepatocytes. Consistent with its role as an activin receptor ligand trap, RAP-011 reduced baseline p-SMAD 1/5/8 expression and subsequently decreased HAMP expression. Next, we asked whether RAP-011 could also decrease HAMP expression under conditions of exogenous BMP-6 stimulation. When hepatocytes were treated with 50 ng BMP-6, an approximately 50-fold induction of HAMP was observed; interestingly, RAP-011 treatment decreased this induction by half. Similarly, treatment of the hepatocytes with the inflammatory cytokine IL-6 led to nearly a 7-fold induction of HAMP which was reduced to baseline levels following the addition of RAP-011. Hepcidin protein levels, although much more variable, mirrored that of HAMP expression following BMP-6 and combined BMP-6 + RAP-011 treatment. Protein changes following IL-6 stimulation were less apparent; this may be due to the fact that the peak of IL-6-stimulated HAMP expression occurred earlier than the 48 hours post-treatment at which the protein was assayed. Our data demonstrate that RAP-011 can modulate SMAD signaling and HAMP expression in primary human hepatocytes and suggests that RAP-011 may allow for increased iron availability for erythropoiesis. Importantly, RAP-011 was able to rescue IL-6-induced HAMP expression, suggesting that the drug might be able to restore iron availability in antinflammatory environment. Anemia of inflammation is associated with diseases such as renal, Castleman’s, and Rheumatoid arthritis. Hepcidin has been shown to play a role in restricting iron and promoting anemia in these pathologies (Steinbicker, A.U., Blood, 2011). In summary, RAP-011, through a yet unknown mechanism, may help to regulate iron availability by regulating hepcidin in pathological situations such as inflammation, iron restriction, or stress erythropoiesis. Disclosures: Malek: Celgene Corp.: Employment. Heise:Celgene: Employment, Equity Ownership. Chopra:Celgene Corp.: Employment, Equity Ownership. Daniel:Celgene Corp.: Employment, Equity Ownership. Sung:Celgene Corp.: Employment, Equity Ownership.

Erythropoiesis, the process of cell proliferation and differentiation that produces erythrocytes, is a tightly regulated process, but apart from early progenitor development and the EPO-dependent response, very little is known about other molecular signals which control cellular fate during RBC production. Members of the transforming growth factor beta (TGFβ) superfamily have been studied as potential regulators of erythropoiesis, iron regulation and globin expression. Sotatercept, an ActRIIA ligand trap, binds to and inhibits activin and other members of the TGFβ superfamily to induce a rapid increase in red cell number and hemoglobin in healthy volunteers. Pharmacological findings demonstrate that RAP-011, a murine ortholog of sotatercept, stimulates RBC parameters in mice through a mechanism distinct from EPO. We conducted the current study to evaluate if RAP-011 may stimulate expansion of a late-stage erythroblast population that is not normally expanded and/or may induce faster differentiation of erythroid precursors. In order to determine if RAP-011 promotes proliferation or differentiation during erythropoiesis, the number of cell divisions was quantified by CFSE staining. During in vitro erythroid differentiation, RAP-011 did not appear to alter the number of cell divisions; however, the percentage of cells that underwent the last division was higher in cultures treated with RAP-011, suggesting that the drug induced faster cellular maturation/differentiation. We also analyzed cell viability of GPA+ cells at the end of the differentiation process and observed that the percentage of apoptotic death was higher in control vs. RAP-011-treated cells. This suggests that RAP-011 may promote survival of late-stage precursors. To assess potential candidates which may mediate the erythropoietic effects of RAP-011, we selected three high affinity RAP-011 ligands, Activin A, Activin B and GDF-11, and proceeded to evaluate their effects on Smad signaling and on erythroid differentiation of human bone marrow progenitors. First, we observed that RAP-011 blocked ligand-induced Smad2/3 phosphorylation in the bone marrow-derived cells. Secondly, RAP-011 rescued activin A-induced inhibition of BFU-E colony formation. Finally, when mature CD36+ cells were differentiated in liquid media containing each of the three ligands, RAP-011 was able to reverse GDF-11- and Activin A-induced inhibition of of erythroid cell proliferation. GDF-11 and Activin A also significantly decreased the percentage of GPA-positive cells in culture, while significantly increasing the percentage of CD45-positive cells. Consistent with proliferation results, RAP-011 blocked these ligand effects. Treatment of CD36+ cells with Activin B did not alter growth or differentiation. These data suggest that GDF-11 and Activin A may contribute, in part, to the erythropoietic stimulatory effects of RAP-011. Several members of the TGFβ superfamily of ligands have been implicated as negative growth regulators, or “chalones”, functioning in homeostasis to maintain specific, mature tissue size. The results from our studies using the ActRIIA-Fc ligand trap, RAP-011, suggest that GDF-11 and Activin A, as well as other sotatercept ligands, may also be “chalones” for the blood, specifically regulating homeostasis of mature RBCs. We suggest that sotatercept increases red blood cell maturation and survival by blocking the negative growth regulation by TGFβ members. In pathologic states such as ineffective erythropoiesis, sotatercept may have an even greater impact than in the healthy, homeostatically-balanced environment. Disclosures: Carrancio: Celgene Corp.: Employment. Markovics:Celgene Corp.: Employment. Wong:Celgene Corp.: Employment. Heise:Celgene: Employment, Equity Ownership. Daniel:Celgene Corp.: Employment, Equity Ownership. Chopra:Celgene: Employment, Equity Ownership. Sung:Celgene Corp.: Employment.

Intraperitoneal (i.p.) recurrence of cisplatin-refractory and p53 mutant ovarian cancer is a major clinical problem, despite surgery and chemotherapy. dl1520 (ONYX-015) is an E1B–55 kDa gene-deleted adenovirus engineered selectively to replicate in and destroy cancer cells lacking functional p53. However, a correlation between efficacy and p53 function has not been definitively studied in vivo to date, and efficacy following i.p. administration had not been previously described. We therefore carried out experiments to address these issues in three nude mouse–human ovarian carcinomatosis xenograft models. Intraperitoneal treatment with dl1520 led to complete tumor eradication and/or significantly improved survival in two p53(−) nude mouse–human ovarian tumor xenograft models. OVCAR3 i.p. xenografts underwent complete regressions in 11 of 12 mice (versus one of seven controls; P = 0.001), while mice bearing cisplatin-resistant A2780 tumors had significantly improved survival versus controls (P = 0.05). In contrast, the A2780 p53(+) ovarian cancer xenograft was resistant to dl1520. The efficacy of i.p. dl1520 in the p53(−) models correlated strongly with tumor burden present at the time of treatment initiation, and no efficacy was seen with non-replicating/UV-inactivated dl1520. Selectively replicating viruses such as dl1520 hold promise as i.p. therapies for p53-deficient and chemotherapy-resistant ovarian carcinomas. A phase I clinical trial of i.p. dl1520 (ONYX-015) is underway in patients with cisplatin-resistant ovarian carcinoma.

Nab®-paclitaxel is an albumin-bound nanoparticle formulation of paclitaxel (ptx) that does not contain Cremophor EL (CrEL), and results in higher drug levels in xenografts and increased clinical activity in breast and lung cancers compared to ptx formulated with CrEL (Taxol®). Above the critical micellar concentration of 0.009%, CrEL forms long-lived micelles in circulation that can sequester ptx (peak plasma concentration in clinical use is 0.3%). Previous studies have shown CrEL reduces ptx binding to albumin, and Taxol® has reduced association with, and transport across endothelial cells compared to nab®-ptx (Desai, CCR 2006). Endothelial cells take up albumin, which is trafficked into recycling or transcytosis pathways or into lysosomes for degradation. Here, we explore mechanisms of uptake and trafficking of albumin and ptx in endothelial cells and the effect of CrEL on these events. Using fluorescence microscopy, we visualized the uptake of rhodamine-albumin and fluorescent ptx (Flutax). Albumin was present in EEA1-positive early endosomes and LAMP1-positive lysosomes. Notably, ptx was also present in vesicular structures and was often co-localized with albumin. The uptake of albumin was blocked by increasing concentrations (0.003%-0.3%) of CrEL, and also by inhibitors of caveolin-mediated endocytosis including indomethacin (blocks internalization of caveolae) and methyl-β-cyclodextrin (prevents formation of lipid rafts). The effect of CrEL on paclitaxel and albumin cellular uptake was confirmed by flow cytometry studies. 0.3% CrEL reduced the uptake of Flutax in DMSO, Flutax-modified nab®-ptx, and FITC-labeled albumin to close to background levels in both HUVEC and PC3 cells. Thus, in addition to its drug sequestration activity, CrEL directly affects endocytosis. We further evaluated CrEL effects on Flutax and ptx transport across endothelial monolayers in transwell chambers using a fluorescence detection assay. Two-fold more ptx crossed monolayers exposed to Flutax-containing nab®-ptx as compared to Taxol®. The effects of 0.001% to 0.3% CrEL on ptx transport at varying timepoints were investigated by mass spectrometry. Dose-dependent inhibition was observed, with a 3-fold reduction in transported ptx at 24 hrs. In summary, we have demonstrated that ptx co-localizes with albumin in endothelial cells, suggesting that the nab-ptx complex can remain intact within cells. Furthermore, CrEL interferes with albumin uptake at clinically relevant concentrations, thereby affecting paclitaxel cellular uptake and transport. These mechanistic studies further elucidate the basis of increased delivery of drug into the target cells by the nab®-ptx formulation as compared to Taxol®. Citation Format: Xiping Liu, Carrie Brachmann, Sean Hong, Shijuan Wu, Zeen Tong, Tapas De, Willard Foss, Gondi Kumar, Sekhar Surapaneni, Rajesh Chopra, Daniel Pierce, Carla Heise. Albumin and paclitaxel co-localize in endocytic vesicles in HUVEC cells, and uptake is blocked by Cremophor EL. [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 5666. doi:10.1158/1538-7445.AM2013-5666

Abstract 372 The regulation of erythropoiesis requires stem cell factor and erythropoietin (EPO) for the proliferation and survival of erythroid progenitor and early precursor cells. While recombinant EPO is widely used for treating various types of anemia, it often lacks efficacy in cases of anemia due to ineffective erythropoiesis in which immature erythroid precursors undergo apoptosis. Thus, there is an need for new therapies to treat the later stages of erythropoiesis. Members of the transforming growth factor beta (TGFβ) superfamily have been studied as potential regulators of erythropoiesis, iron regulation and globin expression. Sotatercept (ACE-011), a recombinant fusion protein consisting of the extracellular domain of the human activin receptor IIA (ActRIIA) linked to the human immunoglobulin G1 (IgG1) Fc domain, is a ligand trap which binds a number of TGFβ superfamily ligands including activin A, activin B, growth differentiation factor-11 (GDF-11) and bone morphogenetic protein-10 (BMP-10). Administration of sotatercept led to substantial increases in red cell number and hemoglobin in human subjects, but the mechanism is not fully understood. We utilized both mouse in vivo and human in vitro models to investigate the mechanism of sotatercept in promoting erythropoiesis. In order to compare the effects of RAP-011 (the murine version of sotatercept) to EPO on red blood cell (RBC) parameters, C57/Bl mice were dosed with RAP-011, EPO or control vehicle. RAP-011-treated mice had a rapid and statistically significant increase in hematocrit, hemoglobin, and RBC number in less than 72-hours. As rapidly as 24 hours after treatment, RAP-011 induced a significant increase in RNA-negative, enucleated cells in the bone marrow (BM). RAP-011 also rapidly increased BM BFU-e and CFU-e erythroid progenitors, while EPO was more effective on spleen-derived progenitors. These data suggest that RAP-011 acts primarily on both bone marrow progenitor cells and late erythroblasts to promote erythropoiesis. In order to investigate the cellular mechanism by which RAP-011 increases red blood cell parameters, we conducted a series of in vitro experiments and found no evidence to support direct effects of RAP-011 on human CD34+ cells assessed in colony formation assays and in erythroid differentiation in liquid culture. As both clinical and pharmacological findings point to a clear role for RAP-011 in stimulating RBC parameters, we hypothesized that RAP-011 effects may be mediated by accessory cells in the BM microenvironment. Human CD36+ cells, which are highly enriched for erythroid progenitors, were co-cultured with long-term BM cultures and erythroid differentiation was assessed following 6 days of culture in EPO (2U/mL)-supplemented media. At day 6 the output of these cultures was predominantly characterized as EryA (∼basophilic erythroblast) but with the addition of RAP-011 (50μM), a significant fraction of CD36+ cells matured into EryB/C cells (polychromatic/orthochromatic erythroblasts), suggesting that factors produced by BM accessory cells mediate RAP-011 erythropoietic effects and that, in contrast to EPO, RAP-011 may play a role in the latter stages of erythroblast maturation. To identify cytokines that may mediate RAP-011 effects, CD36+ cells were treated with several activin receptor IIA ligands. GDF-11 treatment significantly decreased proliferation of GPA+ cells during the differentiation process and RAP-011 effectively reversed this effect, but had no consequence on untreated cells. These data suggest that GDF-11 may mediate the erythroipoietic stimulatory effects of RAP-011. In summary, RAP-011 induced a rapid increase in RBC parameters in mice (reflected in the number of enucleated cells found in the bone marrow), likely mediated by BM accessory cells. Our data also suggest that effects of sotatercept may be mediated at least partly by GDF-11, acting as a potential negative regulator of the terminal stages of erythropoiesis. The ability of sotatercept to reverse this inhibition would lead to a rapid release of terminal erythroid cells into the circulation. These data support the rationale to develop sotatercept for the treatment of anemia and ineffective erythropoiesis, especially in patients who may not respond to EPO. Disclosures: Carrancio: Celgene Corporation: Employment. Markovics:Celgene Corporation: Employment. Wong:Celgene Corporation: Employment. Leisten:Celgene Corporation: Employment. Groza:Celgene Corporation: Employment. Raymon:Celgene Corporation: Employment. Heise:Celgene Corporation: Employment. Chopra:Celgene Corp: Employment, Equity Ownership. Daniel:Celgene Corporation: Employment. Sung:Celgene Corporation: Employment.

Abstract 2766 Background: Lenalidomide plus rituximab therapy is a highly effective and well-tolerated therapy in patients (pts) with follicular lymphoma (FL). In a Phase II trial, this combination induced a complete remission rate of 87% in pts with advanced stage untreated FL (Fowler et al, Ann Oncol, 2011; 22; suppl 4:137). A randomized Phase III trial was recently initiated to compare this combination with current standard of care therapies in pts with FL. Although lenalidomide is known to be an immunomodulatory drug with effects on a variety of immune cells in vitro, its effects have not been well studied in vivo in humans. Understanding the in vivo effects of lenalidomide could lead to novel combination strategies to enhance the efficacy and improve clinical outcome in FL and other malignancies. Methods: Pts received lenalidomide 20 mg/day on days 1–21 of each 28-day cycle and rituximab was given at 375 mg/m2on day 1 of each cycle. Peripheral blood mononuclear cells (PBMC) were phenotyped by multiparametric flow cytometry at baseline, on cycle 2 day 15 (C2D15), and at the end of cycle 6. In addition, peripheral blood (PB) samples were collected in PAXgene Blood RNA tubes at baseline and on C2D15 for whole genome gene expression profiling (GEP). Results: Immunophenotyping of baseline and end of cycle 6 PBMC (n=17) showed that the percentages and absolute numbers of CD3+, CD4+, CD8+, TCRgd, and Foxp3+ regulatory T cells; and NK, NKT, and myeloid dendritic cells were not significantly different between the two time points. However, a significant increase in CD4+CD45RO+ (p To understand lenalidomide-induced changes on a molecular level, we compared GEP data at C2D15 vs. baseline for 7 pairs of PB samples. The paired significance analysis of microarrays method, based on Student's t test, identified 1,748 differentially expressed genes (DEG; 713 up, 1035 down), without a fold-change threshold, in C2D15 samples vs. baseline. Results were influenced by rituximab-induced depletion of B cells in C2D15 samples, but there were many changes that suggested altered PBMC physiology. Noteworthy up-regulated genes (>1.5 fold) included genes associated with T and NK cell activation including BATF, CCR2, CD1B, CD2, CD160, CTLA4, CXCR3, ICOS, and LAG3; and CD163 and CD209, phagocytic receptors expressed on monocytes/macrophages. Down-regulated genes (>1.5 fold) included CXCR5, which mediates B cell migration into follicles; and IL1B and TNFSF13B (BAFF), which are produced by activated macrophages and induce B cell proliferation. Gene set enrichment analysis of all GEP results, and Ingenuity Pathway Analysis of DEGs, indicated up regulation of multiple pathways and processes including ribosomal and mitochondrial components involved in translation and oxidative phosphorylation, CTLA4 signaling in cytotoxic T cells, and differentiation and signaling by ICOS and CD28 in T helper cells. We confirmed up regulation of CTLA4, ICOS, and LAG3 at the protein level in C2D15 PBMC by flow cytometry. Furthermore, treatment of PBMC derived from untreated FL pts with lenalidomide in vitro resulted in up regulation of these molecules in T and/or NK cells consistent with our in vivo results. Conclusions: In FL pts, lenalidomide induced multiple changes in the immune system including increases in PDC and memory T cell subsets, activation of T and NK cells, and down-regulation of certain genes mediating B cell migration and proliferation. These results provide insights into the mechanism of action of lenalidomide and suggest that it can be combined with other immunostimulatory agents such as therapeutic vaccines, adoptive T cell therapy strategies, and immune checkpoint inhibitors to further enhance its efficacy in FL and other malignancies. Disclosures: Fowler: Celgene: Research Funding. Heise:Celgene Corporation: Employment, Equity Ownership. Lacerte:Celgene: Honoraria. Samaniego:Celgene: Research Funding. Neelapu:Celgene Corporation: Research Funding.

Human immunodeficiency virus (HIV)-associated intestinal abnormalities can occur before immunodeficiency or infection with opportunistic enteropathogens. Rhesus macaques infected with simian immunodeficiency virus (SIV) develop an AIDS-like illness that frequently includes enteropathy. The development of enteropathy and its association with SIV infection in the intestinal tract was examined. By 1 week after infection, SIV-infected macrophages and T lymphocytes were detected in gut-associated lymphoid tissue. In contrast to findings in the asymptomatic stage, SIV-infected macrophages were numerous in primary and terminal stages of infection. An acute enteropathy syndrome was observed in the primary acute stage of infection. Functional abnormalities of absorptive epithelium, indicated by D-xylose malabsorption and decreased sucrase activity, occurred before the onset of diarrhea or opportunistic enteric infections. These findings indicate that macrophages and T cells in the intestinal tract are early targets of SIV infection and may play a critical role in the development of SIV-associated intestinal dysfunction.

Intestinal tissues from SIV-infected rhesus macaques with subclinical as well as with terminal SIV disease were evaluated for SIV infection. SIV-infected mononuclear cells were detected throughout the gastrointestinal tract in all animals. In early infection, SIV-infected cells were diffusely distributed in lymphoid tissue and lamina propria, whereas in late stages of the disease, the viral infection was more severe and widely disseminated. Lymphoid cells of the lamina propria and gut-associated lymphoid tissue were the primary targets of SIV.