Trudel, Suzanne

Abstract Abstract 1793 Thalidomide (THAL) and IMID® immunomodulary drugs lenalidomide (LEN) and (POM) have proven beneficial in the treatment of a variety of hematological malignancies. Pre-clinical studies demonstrate multiple direct and indirect anti-tumor activities including anti-angiogeneic, proapoptotic, anti-proliferative and immunomodulatory effects. Recent studies have identified cerebron (CRBN) as a potential direct physical target of THAL and IMiD compounds and CRBN expression is reportedly required for IMiD compound activity. However, the precise link between CRBN and IMiD compound mechanism of action (MOA) have not been clearly defined. We applied Drosophila as a drug discovery platform to assess the MOA of THAL and IMiD compounds in vivo. THAL or POM fed Drosophila demonstrate morphological phenotypes that replicate wingless (wg) mutants indicating that drugs are inhibitors of Wg/Wnt signaling. In this model system, THAL and IMiD compounds disrupt membrane localization of GSK-3 indicating that the bioactivity of IMiD compounds is achieved through spatial regulation and potentiation of Sgg/GSK-3 in Drosophila. Using epistasis analysis we show that Drosophila expressing genetic mutants lacking GSK-3 activity and myeloma cells in which GSK-3a and GSK-3b have been knocked down by siRNA fail to respond to POM. In both Drosophila and myeloma cells therefore it appears that GSK-3 activity is required for biological responses. To test the clinical validity of GSK-3 as a biomarker, we obtained patient tumor samples from a Phase II clinical trial of single agent LEN for previously untreated CLL (Chen CI et al., J Clin Oncol, 2011; 29:1175). Twenty five patients were enrolled onto this study and received LEN at a starting dose of 2.5 mg days 1–21 of a 28 day cycle with monthly escalation to a target dose of 10 mg. The primary clinical endpoint for the trial was objective response to lenalidomide (complete response (CR) and partial response (PR)) evaluated as defined in the revised 1996 NCI Working Group guidelines. Peripheral blood samples for correlative studies were collected on days 1 (pre-dosing) and 8 of cycles 1 and 2. CRBN expression was evaluated by gene expression profiling and Western blot and found to be uniformly expressed in all 19 evaluable day 1 patient samples regardless of LEN response. Thus CRBN expression does not appear to be a useful predictive biomarker of response in this population of previously untreated patients. However, GSK-3 localization was correlated with response. Paired analysis of CD19 selected CLL cells comparing day 1 vs day 8 revealed focal membrane localization of GSK-3 on day 1 and subcellular redistribution on day 8 in 11 out of 12 evaluable responders (PR or better). By contrast, in the CLL cells from all 6 evaluable non-responders, GSK-3 expression appeared diffusely distributed before and after treatment. In preliminary studies using confocal immunofluorescence microscopy we determined that CRBN and GSK-3 co-localize in day 1 CLL samples of responders but not in those of non-responders. In summary, our results indicate that THAL and the IMiD compounds target GSK-3 function by spatial regulation and identify GSK-3 localization as a potential clinical biomarker of IMiD response. Disclosures: Trudel: Celgene: Honoraria; GlaxoSmithKline: Research Funding; Janssen: Honoraria. Mercurio:Celgene: Equity Ownership, Research Funding. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Gaidarova:Celgene Corp: Employment, Equity Ownership. Webb:Celgene: Employment, Equity Ownership. Chen:Johnson & Johnson: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; GlaxoSmithKline: Research Funding; Lundbeck: Consultancy. Stewart:Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy. Manoukian:Celgene: Research Funding.

Abstract Abstract 1845 Poster Board I-871 Background: Proteasome inhibition is an effective strategy for the treatment of multiple myeloma. In patients, proteasome inhibition has primarily been measured in peripheral blood samples (whole blood or mononuclear cells). However, it is unknown whether myeloma cells in the bone marrow (BM) are equally sensitive to proteasome inhibitors such as bortezomib (BTZ) and carfilzomib (CFZ). Aim: To measure proteasome inhibition in purified tumor cells from BM samples taken from patients enrolled in two ongoing Phase 2 trials of single agent CFZ in relapsed or refractory myeloma: PX-171-003 (003) and PX-171-004 (004). Methods: CFZ was administered as an IV bolus of 20 mg/m2 on Days 1, 2, 8, 9, 15 and 16 of a 28-day cycle on both trials. Bone marrow samples, from an optional sub-study of both trials, were taken during screening and Day 2 (post-treatment) and sorted into CD138+ and CD138− cells. Proteasome activity was measured by an enzymatic assay using a fluorogenic substrate (LLVY-AMC) for the chymotrypsin-like (CT-L) activity and an active site ELISA (ProCISE) to quantitate levels of the CT-L subunits of the constitutive proteasome (Beta5) and immunoproteasome (LMP7) and the immunoproteasome subunit MECL1. Results: Whole blood samples from patients treated with CFZ showed inhibition of CT-L activity of ∼80+, similar to values obtained in Phase 1 studies. A total of 10 CD138+ screening samples, 6 from 004 and 4 from 003, and 9 post-dose samples, 5 from 004 and 4 from 003, were analyzed for proteasome levels and activity. In addition, 15 CD138−screening samples, 7 from 004 and 8 from 003, and 9 post-dose samples, 5 from 004 and 4 from 003, were analyzed. When compared to the average base-line activity, CFZ treatment resulted in 88% CT-L inhibition in CD-138+tumor cells from 004 patients (P = 0.0212 by unpaired t-test) and 59% CT-L inhibition in CD-138+ tumor cells from 003 patients (P = 0.25). Baseline CT-L activity in CD138+ tumor cells was 3-fold higher in 004 than 003, which includes a more heavily pre-treated patient population with greater prior exposure to BTZ. Higher specific enzymatic activity was due to increased levels of both constitutive and immunoproteasomes in tumor cells, where immunoproteasomes account for >75% of total cellular proteasomes. No differences between trials were seen in baseline CT-L activity from non-tumor (CD138−) cells. Inhibition in CD138− cells was 84% (P = 0.0380 and 42% (P = 0.38) in 004 and 003, respectively. Using ProCISE, we measured inhibition of LMP7 (66%), beta5 (48%) and MECL1 (64%) in CD138+ tumor cells from 004 patients. Three patients from 004 and one from 003 had both a screening and post-dose tumor cell samples available for analysis. Inhibition of CT-L activity was >80% in two of the 3 patients on 004; the third patient showed no proteasome inhibition by ProCISE and was unavailable for analysis by CT-L. CT-L activity in the CD138+ tumor cells in the 003 patient was not inhibited, however, inhibition was seen in non-tumor cells. Conclusions: CFZ inhibits the proteasome activity of myeloma cells in the bone marrow of relapsed and refractory myeloma patients. The levels of inhibition were similar to those measured in whole blood samples, supporting the use of the blood-based assay as a surrogate marker for proteasome inhibition in tumor cells. CFZ treatment resulted in inhibition of both CT-L subunits as well as additional subunits of the immunoproteasome in tumor cells. Reduced baseline activity in the more heavily pretreated 003 patients may reflect reduced tumor-dependency on the proteasome and may be related to prior treatment with BTZ in these patients. More samples are needed in order to make correlations between levels of proteasome inhibition in bone marrow tumor cells and prior therapies or response. These observations support further evaluation of proteasome activity and the effects of this promising new agent in primary tumors cells from myeloma patients. Disclosures: Trudel: Celgene: Honoraria, Speakers Bureau; Ortho Biotech: Honoraria. Lee:Proteolix, Inc.: Employment. Kirk:Proteolix, Inc.: Employment. Lonial:Celgene: Consultancy; Millennium: Consultancy, Research Funding; BMS: Consultancy; Novartis: Consultancy; Gloucester: Research Funding. Wang:Proteolix, Inc.: Research Funding. Kukreti:Celgene: Honoraria. Stewart:Genzyme, Celgene, Millenium, Proteolix: Honoraria; Takeda, Millenium: Research Funding; Takeda-Millenium, Celgene, Novartis, Amgen: Consultancy. Jagannath:Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. McDonagh:Proteolix: Research Funding. Zonder:Celgene: Speakers Bureau; Pfizer: Consultancy; Seattle Genetics, Inc.: Research Funding; Amgen: Consultancy; Millennium: Research Funding. Bennett:Proteolix: Employment.

Abstract Abstract 576 Stat3 appears to play a critical role in sustaining and generating memory B-cells and long-lived plasma cells (PCs) the normal counterparts of myeloma cells. In contrast to the transient nature of Stat3 activation in normal cells, many hematological tumors including multiple myeloma (MM) harbor constitutive Stat3 activity. Aberrant STAT signaling is recognized as a master regulator of direct and indirect tumor processes including proliferation, apoptosis, invasion, angiogenesis and cancer inflammation and thus Stat3 represents an important therapeutic target. Using structure-based design, targeting the phosphotyrosine (pY)-SH2 domain interactions that stabilizes active Stat3 dimers, phosphopeptides have been identified that block Stat3 dimerization and DNA binding activity. Peptidic agents however suffer from limited cell permeability and metabolic lability, properties that have restricted their practical application in vivo at therapeutic doses. Here we describe the dependence of myeloma tumors on Stat3 and characterize the anti-tumor activity of BP-2-047, a novel, non-phosphorylated small molecule with reduced peptidic character. We analyzed the activity of Stat3 using a Stat3 gene expression index based on Stat3 target genes in 60 human myeloma cell lines (HMCLs). Several available myeloma cell lines (XG6, XG7, JJN3 and UTMC2) demonstrated elevated STAT3 gene expression signatures (2.3 to 14.8) with confirmed constitutive Stat3 phosphorylation by Western blot analysis. RNA interference revealed that Stat3 is essential for XG6, JJN3, and UTMC2 survival. We similarly analyzed the activity of Stat3 in publicly available gene expression profile data sets derived from newly diagnosed MM patients. Approximately a third of patients demonstrated a functionally significant level of Stat3 signature, comparable to those in the Stat3-dependent cell lines. We next evaluated the anti-myeloma activity of the BP-2-047 in cell-based assays against a panel of HMCLs with high and low STAT3 activity. BP-2-047 delivered a desirable activity profile for targeted therapy. Consistent with its predicted mechanism of action, we observed dose-dependent inhibition of Tyr705Stat3 phosphorylation in XG6, XG7, JJN3 and UTMC2 cells, presumably through the blockade of Stat3 binding to the pTyr motifs of gp130 and the prevention of de novo phosphorylation by JAKs. Using a luciferase reporter assay we confirmed inhibition of Stat3 transcriptional activity in MM cells stably expressing the Stat3-dependent luciferase reporter (pLucTKS3). Inhibition of transcription was associated with decreased expression of short half-life proteins, c-Myc and Mcl-1. BP-2-047 inhibited viability of myeloma tumors with IC50 values of 3.1–7.4 microM, in cell lines with constitutive Stat3 activity. Inhibition of Stat3 phosphorylation resulted in induction of dose-dependent apoptosis as determined by PI/annexin V staining and PARP cleavage. However, cells were resistant to BP-2-047-induced apoptosis when co-cultured with bone marrow stroma cells. Identification of factors that confer survival advantage in the presence of stroma is in progress. Exposure of MM patient derived bone marrow mononuclear cells to < 15 microM BP-2-047 preferentially induced apoptosis of CD138 + MM cells. In contrast, BP-2-047 at concentrations of 30 microM failed to inhibit normal bone marrow-derived CD34 colony formation. In vitro combination studies in cell lines demonstrate synergy with dexamethasone, bortezomib and lenalidomide. Studies assessing in vivo activity of BP-2-047 as a single agent and in combination with bortezomib against Stat3 activated myeloma tumors in xenograft mice are ongoing and will be reported. These data suggest that Stat3 may represent an important therapeutic target in a subset of myeloma patients and supports innovative drug development with a focus on Stat3. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 4029 Introduction: Chromosomal translocation of the FGFR3 oncogene in t(4;14)-positive multiple myeloma (MM) causes FGFR3 overexpression in plasma cells, chemoresistance, and poor prognosis leading to shorter overall survival in MM patients (pts). MFGR1877S is a human monoclonal antibody that targets FGFR3 to prevent ligand binding, receptor-receptor association, and FGFR3 signaling. In preclinical studies, anti-FGFR3 antibody suppresses FGFR3-mediated cell proliferation, and exerts strong anti-tumor activity in mouse xenograft models of both t(4;14)-positive MM and bladder carcinoma. Methods: This Phase I study assessed the safety, tolerability, and biologic activity of MFGR1877S given intravenously, weekly for 3 weeks, followed by every 28-day dosing, to eligible patients with t(4;14)-positive MM. Dose escalation started with single-pt cohorts at 1 and 2 mg/kg, followed by a standard 3+3 dose-escalation scheme at doses ranging from 4–15 mg/kg. Dose escalation decisions were made based on monitoring pts for dose-limiting toxicities (DLT) during the first 22 days on study. FGFR3 expression, safety, pharmacokinetics (PK), and response (EBMT/IMWG criteria) were assessed. Main efficacy outcome measures include serum/urine M protein and free light chain (FLC) quantities. Results: A total of 14 pts (median age 66, range 45–78; 43% female) with a baseline ECOG status of 0–2, and a median number of 5 prior therapies (range 1–10), received a median of 3.5 doses (range 1–7) of MFGR1877S. Adverse events (AEs) deemed related to MFGR1877S were diarrhea, fatigue, and nausea (14% each), and anemia, increased creatinine, bone pain, confusion, decreased appetite, myalgia, and fever (7% each). The only Grade ≥ 3 related AE was fatigue (7%). Six pts experienced 9 serious AEs (SAE), one of which was Grade 2 pyrexia attributed to MFGR1877S that occurred within 24 hours of infusion and required hospitalization. This pt later discontinued due to a second SAE of Grade 2 pyrexia requiring hospitalization not attributed to MFGR1877S. One pt died of intracranial hemorrhage not attributed to MFGR1877S. No Grade 4 SAEs were reported. Other reported SAEs included Grade 3 hypercalcemia, neutropenia, pain in extremity, and musculoskeletal chest pain (1 pt each), Grade 2 ankle fracture, pneumonia, and pyrexia × 2 (1 pt each). No maximum tolerated dose was identified, as no DLTs were observed through the highest dose tested (15 mg/kg). Preliminary PK analysis for limited number of multiple myeloma patients (n=14) by non-linear mixed effect modeling demonstrated a trend of dose proportional increase of exposure (area under the concentration-time curve and maximal concentration) from 2 to 15 mg/kg. Population clearance of MFGR1877S was estimated to be ∼0.62 L/day, suggesting that MFGR1877S appeared to have a slightly faster clearance in MM patients, compared to the typical IgG1 monoclonal antibody clearance in human. The central volume of distribution of MFGR1877S was ∼3.4 L, which approximated human serum volume and appeared similar to the central volume of distribution of other typical IgG1 monoclonal antibodies. While some degree of FGFR3 expression was detected by immunohistochemistry in 10/14 patient bone marrow samples taken at screening, FACS analysis of FGFR3 surface expression on myeloma cells, although largely consistent with IHC measurements, also revealed heterogeneity in the levels of detectable surface FGFR3 expression. Six pts had stable disease as their best response: 2 pts up to 4 cycles, 1 pt up to 3 cycles, and 3 pts up to 1 cycle. Conclusions: MFGR1877S was well-tolerated overall in these patients with multiple relapsed or refractory MM. Although no objective responses were observed, stable disease was observed in 3 pts for 3–4 cycles. Disclosures: Off Label Use: MFGR1877S is a human monoclonal antibody that targets FGFR3 to prevent ligand binding, receptor-receptor association, and FGFR3 signaling. Singhal:Millennium and Celgene: Speakers Bureau. Niesvizky:Onyx, Millemium, Celgene. Speakers bureau: Millenium and Celgene: Consultancy, Research Funding. Comenzo:Millenium, Neotope; Onyx, Osiris, Millenium: Consultancy, Research Funding. Lebovic:Genentech: Speakers Bureau. Choi:Genentech: Employment. Lu:Genentech: Employment. French:Genentech: Employment. Penuel:Genentech: Employment. Ho:Genentech: Employment.

Abstract Abstract 718 Introduction: In a previously reported phase 2 study of single agent lenalidomide in 25 untreated CLL patients (pts), we reported an overall response rate (ORR) of 56% (14 pts), 40% SD (10 pts) and no CR at a median follow-up of 20.7 months (Chen et al. JCO 2010;29:1175). Although an amended protocol with conservative lenalidomide dosing was used to mitigate tumor lysis and severe myelosuppression observed in the first 2 accrued pts, we continued to observe frequent toxicities of grade 3–4 neutropenia (72%) and tumor flare (TF 88%). We now report long-term efficacy and toxicity from this study at a median follow-up of 47 months (mos). Methods: Patients were eligible if previously untreated and symptomatic (cytopenias, symptomatic adenopathy/organomegaly, constitutional symptoms, lymphocyte doubling count <12 mos). The amended starting dose for lenalidomide was 2.5mg daily on days 1–21 of a 28 day cycle, with slow monthly dose escalations (2.5mg cycle 1, 5mg cycle 2, 10mg cycle 3 and if required for response, further 5mg increments to a maximum of 25mg daily were allowed). Results: Longterm toxicities: Hematologic toxicities were common: grade 3–4 neutropenia (76%), thrombocytopenia (28%), anemia (20%). With longer term use, neutropenia tended to recur (12% of all cycles) and 10 pts required GCSF support (5 routinely during each cycle). Most common non-hematologic toxicities (all grades) were TF (88%), fatigue (76%), rash (60%), muscle cramping (40%), diarrhea (40%). All non-hematologic toxicities were mild (grade 1–2), except for 1 pt each with grade 3 rash and diarrhea. Although TF was most common during cycle 1, repeat flare symptoms upon resuming lenalidomide after the 7 day rest period of each cycle were noted in 16% of all 898 cycles administered, and as late as at cycle 28. Infections were mild (most respiratory, skin) with only 2 grade 3 events (disseminated zoster, S.pneumoniae bacteremia). Other malignancies: 2 pts developed transformed large cell lymphoma 7 and 18 mos after study discontinuation, 1 pt developed squamous cell carcinoma of skin at cycle 51, and 1 pt developed recurrence of remote non-small cell lung cancer at cycle 34. Dose modifications/discontinuation: The median highest dose achieved for all 25 pts was 15 mg (range 2.5–25 mgs); 8 pts were able to escalate to the maximal 25mg dose. Ten pts (40%) required dose reductions for grade 3 cytopenias [neutropenia (2), thrombocytopenia (2), both (2)], febrile neutropenia (2), and diarrhea (2). Of all 25 pts, the median duration on therapy was 31.1 mos (range 28 days – 60.6 mos). Twelve pts (52%) currently remain on study, receiving a median of 59 cycles of therapy (range 48–66). Causes of discontinuation for 13 pts included: treatment-related toxicity (8), lack of response/progressive disease (4), and recurrence of remote lung cancer (1). Toxicities leading to discontinuation included: prolonged cytopenias (3), recurrent infections (1), atrial fibrillation (1), disseminated herpes zoster (1), persistent grade 2 diarrhea (1), and grade 3 skin rash (1). Efficacy: With extended median follow-up from 20.7 to 47 mos, the ORR improved from 56% (14 pts) to 72% (18 pts), with 3 pts in PR upgrading to CR, and 1 SD to PR. Although the median time to response was 7.7 mos, responses occurred as quickly as 1.8 mos to as late as 27.0 mos of therapy. For the 3 CR pts, prolonged therapy with an additional 14.9, 28.3 and 40.6 mos beyond the time of first response was required to achieve CR. To date, 7 pts have progressed with 3-year PFS 68.8% (95% CI:52–91%) and OS 85.3% (95% CI:71.1–100%). Correlatives: Cereblon (CRBN), recently identified as a direct protein target of lenalidomide, was evaluated by gene expression profiling and Western blot and found to be uniformly expressed in all 19 evaluable day 1 pt samples regardless of lenalidomide response. Thus, baseline CRBN expression does not appear to be a useful predictive biomarker of response in this population. The mechanism by which CRBN is linked to response is reported by Trudel et al, ASH 2012. Conclusions: Long-term followup of this study demonstrates that when using low doses of single agent lenalidomide in CLL, prolonged therapy is feasible and may be required for the achievement of durable, high quality responses. Maximal daily doses of 25mg can be reached and may also be needed for optimal response, though recurrent myelosuppression remains limiting. Disclosures: Chen: Celgene: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; Lundbeck: Consultancy; Janssen: Consultancy, Research Funding. Off Label Use: Lenalidomide is not approved for use in chronic lymphocytic leukemia. Kukreti:Roche: Honoraria; Celgene: Honoraria; Janssen Ortho: Honoraria. Trudel:Celgene: Honoraria, Research Funding, Speakers Bureau.

SummaryThis single institution, open label Phase I‐II dose escalation trial evaluated the safety and efficacy of the combination of lenalidomide (Revlimid®), cyclophosphamide and prednisone (CPR) in patients with relapsed/refractory multiple myeloma. The maximal administered dose of CPR consisted of cyclophosphamide 300 mg/m2 on day 1, 8, and 15, lenalidomide 25 mg on d 1–21 and prednisone 100 mg every other day in a 28‐d cycle. Between November 2007 and June 2009, 32 patients were entered in cohorts of three at three dose levels. The median age was 64 years, 59% were male, with a median two prior regimens. Responding patients could stay on treatment until progression. The full‐dose CPR regimen produced no dose‐limiting toxicity and was delivered for a median of 16 months (3·5–65 months) with acceptable safety and tolerance. The overall response rate (≥ partial response) was 94% at a median follow up of 28 months. The median progression‐free survival was 16·1 months [95% confidence interval (CI); 10·9–22·5 months], while the median overall survival was 27·6 months (95% CI; 16·8–36·6 months). Only the beta‐2 microglobulin level at protocol entry correlated with a better survival (P = 0·047). These observations compare favourably with other 2‐ and 3‐ drug combinations for relapsed/refractory myeloma, and suggest that CPR should be evaluated further in the setting of relapsed/refractory disease, or in newly diagnosed patients.

SummaryAutologous stem‐cell transplant has been widely used to treat patients with AL amyloidosis. However, transplant‐related mortality rates are high, and a recent randomized trial suggested that non‐transplant regimens produced comparable results with less toxicity. In order to define the role of patient selection in stem cell transplantation, we evaluated 78 consecutive AL amyloidosis patients transplanted at our centre. Transplant‐related mortality occurred in 11·5%. Complete haematological response and organ response were achieved in 56% and 60%. Median overall survival was significantly lower for patients with brain‐type natriuretic peptide (BNP) >300 pg/ml (17·5 months vs. not‐reached) (P = 0·0004), troponin‐I >0·07 ng/ml (13·5 months vs. not‐reached) (P = 0·00001) and those not achieving a complete haematological response (88 months vs. not‐reached) (P = 0·0345); high BNP and troponin‐I were the most important predictive factors in a multivariate analysis. Based on this study, patients with BNP <300 pg/ml and/or normal levels of troponin‐I should be considered transplant candidates.