ALBERT

Description: Background: Talabostat (PT-100), an orally available inhibitor of dipeptidyl peptidases, is in Phase 2 studies for B-cell malignancies and solid tumors. Talabostat increases production of cytokines and chemokines in lymph nodes and spleen, stimulating both adaptive and innate immune responses (Adams S, Cancer Research, 2004;64:5471). Talabostat may thus enhance the antibody-dependent cytotoxicity of MAbs such as rituximab. Methods: This is a Phase 1 study to evaluate the safety and activity of talabostat and rituximab in patients with indolent NHL who did not respond or progressed following rituximab. Rituximab 375mg/m2 was administered weekly x 4. Total daily doses of talabostat 400μg (n=6), 600μg (n=3), or 800μg (n=6) were administered BID for 6 days following each dose of rituximab. Cytokines and chemokines were assessed pre-, 2, and 6 hours post-talabostat on Days 1, 6, 13, 20, and 27. Flow cytometry was performed at baseline and Day 28. Clinical and laboratory evaluations were performed at specified times. Adverse events (AEs) were graded per NCI-CTC and recorded throughout the study. Disease assessments were performed on Days 28 and 84. Results: 11 men and 4 women aged 48–82 with NHL (n=10) or SLL/CLL (n=5) have been treated. 9 patients completed the 28-day study: 4 at 400μg, 1 at 600μg, and 4 at 800μg. Enrollment continues at 400μg/day. The most frequent AEs have been edema (67%), nausea (47%), dizziness (40%), hypotension (33%), fatigue (33%), vomiting (33%), constipation (33%), thrombocytopenia (27%), and weight gain (27%). Grade 3 toxicities include: dizziness, myopathy (400μg/day), and 2 events of thrombocytopenia (600μg/day). Grade 3 peripheral edema, myalgia, dehydration, electrolyte imbalance, hypereosinophilia, elevated CPK (primarily CK-MM), and rhabdomyolysis were seen in 2/6 patients at 800μg/day; these events were DLTs. One partial response (PR) lasting 7 months was seen in one patient (800μg/day). A PR was seen in a second patient at 800μg/day but did not meet the strict NCI-WG criteria for response. Elevations in cytokines 〉ULN were reported across all doses following talabostat: G-CSF (13/15), IL-1β (10/15), IL-2 (7/15), IL-6 (8/15), IL-8 (8/15), IL-10 (11/15), TNF-α (11/15), and IFN-γ (3/15). At Day 28 or early termination, CD20 was decreased in most (12/15) patients. Increases were seen in the percentage of CD3 (12/15), CD3/4 (11/15) and CD3/8 (9/15). In all 5 patients with SLL/CLL, CD5+/CD20+ was

Description: Control of the intrinsic apoptotic pathway is exercised through the interplay of a 20-member family of proteins related to the founding member, Bcl-2. These proteins are confined to the vicinity of two membrane surfaces, the endoplasmic reticulum and the mitochondrial outer membrane. Like most of cellular biochemistry, reactions are confined in nano time and space which do not allow the approach to equilibrium seen at the macro level of the test tube, motivating us to initiate a dynamic systems biology approach to these non-equilibrium reactions. Our laboratory has investigated the mechanism of action of a non-peptide small molecule TW-37, which represents a new chemical class of compounds like ABT-737 (Abbott) useful in the induction of apoptosis in tumor cells. The drug induces apoptotic death in lymphoma cells with an observed EC50 of 290 nM (Mohammad et al., Clin. Cancer Res.13, 2226 (2007)). TW-37 binds with nanomolar affinity to the hydrophobic groove on the surface of the pro-survival Bcl-2 proteins which can accommodate the hydrophobic face of the amphipathic α-helix of BH3-only family members such as Bid. To model how the drug perturbs the Bcl-2 system, we have immobilized a highly-helical 24-mer Bid-derived BH3-domain peptide via its N-terminus to the surface of a CM5 chip (ligand) and analyzed peptide-protein interaction with flowing analyte proteins Bcl-2, Bcl-XL, Mcl-1, Bcl-w, and Bfl-1/A1 using the Biacore 3000. Using an analysis of Langmuir binding, we observe that the analyte proteins bind Bid with ON-rates (ka) varying modestly (3.01–7.72 E+05 1/Ms), but dissociate from Bid with 17-fold variation in OFF-rate (kd). The Bcl-w complex with Bid is the slowest to dissociate with a kd of 0.0015 (1/s); the most rapid dissociation is seen with Bcl-2 and Bid, where the kd is 0.0385. We find that Bid interacts with the multidomain proteins with dissociation constants (KD) between 4 and 82 nM, intermediate between earlier determinations (J. Med. Chem.49, 6139 (2006)); Molec. Cell17, 393 (2005)), but significantly lower than the KD for the drug TW-37 and the multidomain proteins (260–1100 nM reported previously; 79-333 nM measured here). Contrary to the suggestion of Shangary and Johnson (Biochemistry41, 9485 (2002)), our BH3-mimetic TW-37 is unable to disrupt already-formed Bid heterodimers with the mutidomain survival proteins; when heterodimers on the Biacore chip are challenged with doses of TW-37 up to 10000 nM. Consequently, the only opportunity the drug might have to perturb the Bcl-2 system is to locate the hydrophobic groove before Bid. Thus, a new picture emerges of drug action in which the dynamics of pre-apoptotic protein-protein interactions creates a temporal window for drug binding to the hydrophobic groove. In this light, attempts to develop small molecule inhibitors of protein-protein interaction (Current Topics in Medicinal Chem7(10), 922–927 (2007)) should incorporate surface plasmon resonance measurements of ka and kd into preclinical drug discovery programs in addition to KD estimates of drug binding to target. Figure Figure

Description: Abstract 4769 Introduction: Bone marrow biopsy is essential for the staging and monitoring of patients with non-Hodgkin's lymphoma (NHL). Based on a 1975 study, bilateral bone marrow biopsy is the standard practice in many institutions. Similar studies were conducted later, but with conflicting results, inadequate description and little statistical assessment of the value of conducting bilateral vs. unilateral bone marrow biopsies for the evaluation of patients with NHL. Objectives: To explore whether a unilateral bone marrow biopsy is comparable in yield to bilateral biopsies in staging of patients with NHL. Methods: We retrospectively reviewed electronic pathology reports for bone marrow biopsies done at our institution for staging NHL. We also collected data for age, gender, type of NHL, percentage of disease involvement, and size of biopsy. Patients were divided into those who had bilateral biopsies vs. unilateral biopsies. The bilateral group was further divided into bilaterally positive (matching), versus only one side (aspirate and/or biopsy) positive (non-matching). Results: Between 1995 and 2010, 256 patients were identified with the diagnosis of NHL, and found eligible for the study. 146 patients (57%) had low grade NHL and 83 patients (32.4%) had diffuse large B-cell NHL.107 patients had bilateral, and 149 had unilateral bone marrow biopsies. Overall positivity rate was 46.7% for bilateral (either side or both) and 41.4% unilateral (chi square, p = 0.884). For the low grade NHL group, the positivity rate was 56.2% for bilateral (either side or both) and 57.9% unilateral. For the Diffuse Large B-cell NHL group, the positivity rate was 26.7% for bilateral (either side or both) and 23.7% unilateral. Within the bilateral group, 97 patients had bilateral positive results (matching) and 10 had only one side positive (non-matching). Within the bilateral group (107 patients), the sensitivity of either side being positive, (i.e. left side vs. either, or right side vs. either) was 90% and the negative predictive value was 92%. Positivity rate of unilateral bone marrow biopsies size (≥2cm) was 10% more than that with size (

Description: Abstract 598 Recent evidence demonstrates that Non Hodgkin's Lymphoma (NHL) tumors have elevated expression of the XPO1 gene that codes for the nuclear exporter protein CRM-1 controlling the localization of critical tumor suppressors including p53 family members. For effective apoptosis and cell cycle regulation, p53/p73 nuclear localization and DNA binding are highly critical making CRM-1 an attractive therapeutic target for NHL that carries 〉90% wild type/functional p53 and only rare mutations in p73. We have identified novel small molecule inhibitors of CRM-1 (KPTs) that bind irreversibly and lock target proteins (including p53 and p73) in the nucleus leading to apoptosis of tumor cells. We demonstrate for the first time that KPTs can induce apoptosis in resistant NHL cell lines and corresponding xenograft models. The most potent CRM-1 inhibitor (KPT-185) induced growth inhibition and apoptosis in a panel of NHL cell lines with a median IC50 ∼25 nM. Western blot and confocal microscopy analyses demonstrated that KPT-185 treatment resulted in nuclear localization of p53 in wt-p53 and p73 in mut-p53 cell lines. Additionally, we observed KPT-185 mediated activation of p21 and Bax, known downstream executioners of p53/p73 cell cycle control and apoptosis, respectively. Most significantly, siRNA knockdown of p53 in WSU-FSCCL and p73 in WSU-DLCL2 abrogated the apoptotic potential of KPT-185, confirming that these were indeed p53/p73 dependent apoptotic events. KPT-185 showed a substantial enhancement in apoptosis when combined with genotoxic p53/p73 re-activating regimen CHOP. The KPTs selectively kill cancer cells with minimal toxicity to normal tissues, and possess clinically acceptable pharmacokinetic parameters. Using WSU-DLCL2 SCID models, we further show that oral administration of related CRM-1 inhibitor KPT-276 (75 and 150 mg/Kg p.o) resulted in 65 and 70% tumor reduction, respectively and subcutaneous injections of KPT-251 (25 and 75 mg/Kg) resulted in 70 and 74% suppression of tumor growth with no observed toxicity to the host. Remnant tumor tissue analysis (histology and protein markers) fell in line with our in vitro results with clear activation of p73 pathway. Our study verifies CRM-1 as a potential therapeutic target in NHL irrespective of the functional status of p53. These results build a strong case for the clinical use of our novel CRM-1 inhibitors either as single agents or in combination with CHOP. Disclosures: Kauffman: Karyopharm: Equity Ownership. McCauley:Karyopharm: Employment. Shacham:Karyopharm: Equity Ownership.

Description: Abstract 4991 Anti-CD20 RIT is the most effective single agent treatment for B-cell lymphoma. However, it is not widely used in community-based oncology because of perceived complexity of treatment and toxicity. We analyzed the first 48 patients treated with RIT at our institution between November 2003 and February 2011. A work sheet for referral, screening and treatment process was developed jointly between Oncology and Nuclear Medicine and a mid-level provider (MM) was designated as RIT coordinator. Pre-treatment evaluation of all patients was according to recent consensus conference report on RIT (Witzig T et al 2011) including pretreatment imaging and bone marrow (BM) aspiration and biopsy. Unless dictated by patient's renal function, bladder control status and ability to comply with post treatment radiation safety requirement, the type of RIT (Ibritumomab Tiuxetan [Zevalin®] or Iodine 131 Tositumomab [Bexxar®]) was up to the discretion of referring oncologist. For Bexxar-treated patients, dosimetry imaging was done at 48 and 120 hours; Therapy dose, on day 8, was calculated to deliver 75 cGy to the total body. Zevalin imaging (In-111) was done at 48h and therapeutic dose of 0.4mCi/kg or 0.3mCi/kg depending on platelet counts (≥150K/μL or 100–149K/ μL, respectively) on days 7, 8, or 9. Patients were monitored for toxicity (NCI-CTC v4.03) and evaluated for response after 12 weeks of therapy according to Revised Response Criteria for Malignant Lymphoma (Cheson BD et al 2007). The SPSS v19.0 program was used for statistical analysis. Median age for the whole group was 60 at diagnosis (range 34–88) and 68.5 (37-89) at RIT. There were 19 males and 29 females. All patients, except one, had relapsed or refractory disease. Twenty two patients had diffuse large B-cell lymphoma (DLBCL) and 25 had follicular lymphoma (FL) with or without transformation. Thirty four patients were treated with Bexxar and 14 were treated with Zevalin. Thirty three of 45 evaluable patients for response achieved complete (CR n=20, 44%) or partial (PR, n=13, 29%) response for overall response rate (ORR) of 73%. There was lower, but statistically not significant, ORR for DLBCL (65%) compared with FL (79%). Other factors that did not influence ORR were race, gender, type of RIT, stage of disease and BM involvement by lymphoma. There was a trend towards association between IPI score and response in DLBCL (100% ORR for scores 0–1 vs 40% for score 3). FLIPI, however, was not predictive due to the high ORR in FL. Prior therapy and response to last regimen prior to RIT (LRPtoRIT) was predictive of ORR. 100%, 79% and 47% of patients who received 1, 2, ≥3 prior regimens responded to RIT, respectively (P 0.01). 84% of patients who achieved CR or PR to LRPtoRIT responded to RIT compared with 44% of those who did not respond to LRPtoRIT (P 0.014). 87% of patients who did not receive prior external beam radiation (RT) responded to RIT compared with 43% in those who were previously treated with RT (P 0.002). Finally, patients ≤60 years of age had higher response (93%) compared with those 〉60 (65%) (P 0.047). Multivariate analysis showed that response to LRPtoRIT was still significant even after adjusting for age and RT prior to RIT. From Kaplan-Meier analysis, the median survival of the whole group was 48 months; 39 months for DLBCL and 82 months for FL (P 0.096). Patients who responded to RIT had a median survival of 81 months compared with those who did not (4.2 months) (P 0.000). Other factors that predicted for survival in univariate analysis were: number of prior regimens (P 0.017), response to LRPtoRIT (P 0.001), and prior external beam radiation (P 0.024). In multivariate analysis, response to RIT and response to LRPtoRIT were significant predictors of survival (P 0.048 and 0.026, respectively). There were no treatment-related deaths. Grade 3/4 toxicity was only hematologic including thrombocytopenia (38%), neutropenia (31%) and anemia (12.5%). Median time to nadir was 5, 6 and 8 weeks post therapy for platelets, neutrophils and hemoglobin, respectively. Two patients developed secondary AML and one patient developed MDS, all with complex chromosomal abnormalities. We conclude that with coordinated effort, RIT can be safely and effectively delivered in routine community setting with results comparable to those reported in clinical trials. Prior therapy and response to LRPtoRIT predict response to RIT whereas response to RIT and response to LRPtoRIT are predictive of overall survival. Disclosures: No relevant conflicts of interest to declare.

Description: A phase 1a/1b trial is planned for specific types of T-cell non-Hodgkin's lymphoma (T-NHL) using a new formulation of fenretinide for intravenous (IV) administration (ST-001, IND# 135475). Despite documented anticancer properties, clinical development of fenretinide has been hampered by poor bioavailability of the oral preparations and poor water solubility for IV infusion although some progress has been made. A phase 1 clinical trial of a 20% soy oil-in-water emulsion containing 2mg/mL of fenretinide, administered via 24-hour daily infusions x5 q3wks, demonstrated activity in mycosis fungoides /Sézary syndrome (MF/SS) and in angioimmunoblastic T-cell lymphoma (AITL) (Mohrbacher et al 2017). However, all doses exhibiting disease activity were associated with grade 4 hypertriglyceridemia. ST-001 from SciTech Development, LLC is a second generation, triglyceride-free, phospholipid-based nano-dispersion formulation of higher potency fenretinide (12.5mg/mL) suitable for IV infusion. The SciTech Delivery Vehicle (SDV) is composed of 4 clinically acceptable phospholipids (DPPC, DOPC, DMPC, and DMPG) which, in the presence of fenretinide, form lamellar bilayer structures

Description: Flavopiridol is a novel, potent inhibitor of cyclin-dependent kinases (CDK). This synthetic flavone has been reported to exhibit antitumor activity in murine and human tumor cell lines in vitro and in vivo and is currently undergoing clinical phase I evaluation. In the present study, 1 Epstein-Barr virus (EBV)-transformed B-prolymphocytic cell line (JVM-2), 1 EBV-transformed B-CLL cell line (I83CLL), and 1 non-EBV transformed B-CLL cell line (WSU-CLL) were used as targets. Treatment of the cells with flavopiridol (100 nmol/L to 400 nmol/L) led to a marked dose- and time-dependent inhibition of cell growth and survival as determined using trypan blue exclusion. Morphologic analysis showed characteristic apoptotic changes such as chromatin condensation and fragmentation, membrane blebbing, and formation of apoptotic bodies. Furthermore, quantitative assessment of apoptosis-associated DNA strand breaks by in situ TdT labeling showed that a significant number of flavopiridol-treated cells underwent apoptosis. These cellular effects were associated with a significant decrease in bcl-2 expression as observed by Northern and Western blotting. The results showed that flavopiridol downregulates bcl-2 mRNA and bcl-2 protein expression within 24 hours. Genistein and quercetin, two flavonoids that do not inhibit CDKs, did not affect bcl-2 expression. These data suggest an additional mechanism of action of this new flavone which might be useful as an agent in the treatment of chronic lymphoid malignancies.

Description: Background: PNT2258 is a 24-base, single-stranded liposome-encapsulated DNA oligonucleotide that hybridizes to regions of the BCL2 gene, modulating its function. As previously reported, PNT2258 demonstrates antitumor activity in patients with advanced-stage NHL. The updated clinical data that are provided in this abstract (as of 07/25/14) now include anti-tumor activity in patients with DLBCL and Richter’s syndrome. Methods: All patients had CT-measureable, FDG-PET positive disease with progression prior to study entry, and had previously received rituximab and combination cytotoxic chemotherapy. Refractory tumor status or concomitant medical issues precluded the use of additional cytotoxic therapy in all patients included in the study. PNT2258 induction cycles were administered as a 3-hour IV infusion at 120 mg/m2 days 1-5 of each 21-day cycle for up to 8 cycles. Response evaluation occurred at the end of cycles 2, 6 and 8. Following induction, patients could receive maintenance dosing at 100 mg/m2days 1-2 of each 28-day cycle until PD. Results: Thirteen patients (median age 65; 8 males, 5 females; ECOG PS 0/1/2: 3/9/1) received all scheduled doses of PNT2258. Regardless of attribution, there were no cycle 1, grade 3/4 AEs. The most common grade 1/2 AEs in cycle 1 included chills, low-grade fever, transient nausea, and tumor or back pain (n=5 for each). There was no evidence of tumor lysis syndrome, febrile neutropenia or significant GI toxicity. Of 4 patients with DLBCL, including 1 with Richter’s syndrome and 1 with Burkitt-like histology, PFS was 9.2 months, range 5.5-12.3 months as of 7/25/14. Best response data (CR/PR/SD/PD) for the DLBCL group was 2/1/1/0. Additionally, in 5 FL patients, best response was 1/1/3/0. Five patients, including 3 DLBCL and 2 FL remain on study as of 7/25/14 receiving maintenance treatment with PNT2258. Conclusions: PNT2258 is well tolerated and can be administered over prolonged periods of time. Durable single agent activity was previously reported in patients with FL, and has now been observed in patients with aggressive DLBCL. Studies in DLBCL and Richter’s syndrome are being implemented and updated information will be provided at the time of the meeting. Clinical trial information: NCT01733238. Disclosures Gaylor: ProNAi Therapeutics: Employment. Rodrigueza:ProNAi Therapeutics: Employment. Woolliscroft:ProNAi Therapeutics: Employment. Sooch:ProNAi Therapeutics: Employment. Messmann:ProNAi Therapeutics: Employment.

Description: Non-Hodgkin’s lymphoma (NHL) tumors include a group of heterogeneous diseases with varying natural histories and responsiveness to therapy; nonetheless, overexpression of Bcl-2 protein is seen in more than 80% of NHL. Throughout the years our laboratory succeeded in establishing a panel of B-cell lines representing various maturational stages of NHL. In this study, we have utilized a structure-based strategy to design a new class of potent nonpeptidic small-molecule inhibitor (SMI) of Bcl-2 family. TW-37, a lead compound that was designed to target the BH3 binding groove of antiapopototic Bcl-2 proteins. It binds to Bcl-2, Bcl-XL and Mcl-1 with Ki values of 290 nM, 1110 nM and 260 nM, respectively. TW-37 showed significant antiproliferative effect against Pre-B-Acute Lymphoblastic Leukemia (WSU-pre-B-ALL), Diffuse Large Cell Lymphoma (WSU-DLCL2), Follicular Small Cleaved Cell Lymphoma (WSU-FSCCL), Waldenstrom’s Macroglobulinemia (WSU-WM) and primary cells obtained from lymphoma patients, despite variations in their anti- and pro-apoptotic Bcl-2 proteins (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, Bim, Bad, BUMA and Bok). The IC50 for TW-37 varied from 165 nM in the WSU-FSCCL to 300 nM in WSU-DLCL2 cells. Apoptosis was independent of proliferative status or pathological classification of B-cell tumor. TW-37 was able to block Bim-Bcl-XL and Bim-Mcl-1 eterodimerization and induces apoptosis via activation of caspases -9, -3, PARP and DNA fragmentation. Although cell lines and patient samples expressed multiple Bcl-2 family proteins at various levels, TW-37 induced apoptosis was only strongly associated with Bax:Mcl-1 ratio. TW-37 administered to tumor-bearing SCID mice led to significant tumor growth inhibition (T/C), tumor growth delay (T-C) and Log10kill, when used at its maximum tolerated dose (40 mg/kg x 3days) via tail vein. failed to induce changes in the Bcl-2 proteins levels suggests that assessment of baseline Bcl-2 family proteins can be used to prognosticate the response to drug. These findings indicate activity of TW-37 across the spectrum of human B-cell tumors and support the concept of targeting the Bcl-2 system as a therapeutic strategy in the treatment of B-cell lymphoma.

Description: It is estimated that 63,000 cases of non-Hodgkin’s lymphoma (NHL) will be diagnosed in the United States alone in 2007, of which 19,000 will die of their disease. The selectively over-expressed, tumor-associated, lineage specific CD19 antigen is one of the hallmarks of B-cell NHL. Therefore, the design of high affinity-CD19-based modality epitomize an important step towards improving treatment outcome. In this report, we tested the efficacy of SAR3419, a novel humanized anti-CD19 antibody, i.e., huB4, conjugated to the cytotoxic maytansine derivative, DM4. The in vivo anti-tumor activity of SAR3419 was assessed as a single agent and in comparison with conventional therapies using our established CD19-positive human lymphoma xenograft models, the diffuse large cell lymphoma (WSU-DLCL2) and the transformed follicular small cleaved cell lymphoma (WSU-FSCCL), both EBV-negative. In all, we followed 112 tumor-engrafted SCID mice, 56 bearing bilateral WSU-DLCL2 tumors (subcutaneous) and 56 bearing the WSU-FSCCL (systemic disease). Starting 7 days after xenograft establishment (100 – 200 mg tumor size), SAR3419 (7.5, 15 and 30mg/kg), free DM4 (0.6mg/kg), naked huB4 (30mg/kg), CHOP (at maximum tolerated dose in SCID mice as previously defined) or rituximab (40mg/kg) were administered iv on Days 1 and 4 (except CHOP given on day 1 only). Animals were monitored 3 times weekly for tumor measurements and toxicity and were euthanized when tumor burden reached 10% of body weight (WSU-DLCL2) or became moribund (WSU-FSCCL). Our results showed that WSU-DLCL2-bearing animals treated with SAR3419 dosage of 15 mg/kg had no measurable tumor up to Day 155 (end of experiment) and considered cured (7/7 tumor-free survivors, TFS). In the higher dose of SAR3419 (30mg/kg), all animals (7/7) were tumor-free at time of death. However, 4/7 animals died of unrelated causes before the end of the experiment. At the dosage of 7.5 mg/kg, SAR3419 induced a T-C of 64 days and Log10 kill of 4.6 (2/7 TFS). Treatment with rituximab resulted in high anti-tumor activity (T-C of 72 days; Log10 kill of 5.19 and 4/7 TFS). Animals treated with huB4 or CHOP had T-C of 5 and 13 days, and Log10 kill 0.15 and 0.82, respectively. In the WSU-FSCCL model, by the end of the experiment (Day 150), SAR3419 induced 100%, 4/7 and 2/7 survivors, at 30, 15 and 7.5 mg/kg, respectively. The number of survivors in the other arms of the study was 2/7 for rituximab, 3/7 for CHOP and 2/7 for huB4. All animals in the no treatment (control arm) had died by Day 75. Necropsy and H&E analysis revealed that all deaths featured leptomeningeal lymphoma in the control and treated groups. Overall, SAR3419 is a very active immunoconjugate that produced 100% cures in the two models tested. SAR3419 was found better than either rituximab or CHOP at optimal dosages. We conclude that SAR3419 holds promise as novel and well tolerated immunoconjugate therapy in B-cell NHL.