scholarly journals The Novel PI3K/mTOR Dual Inhibitor PQR309 in Pre-Clinical Lymphoma Models: Demonstration of Anti-Tumor Activity As Single Agent and in Combination and Identification of Gene Expression Signatures Associated with Response

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1782-1782
Author(s):  
Chiara Tarantelli ◽  
Eugenio Gaudio ◽  
Ivo Kwee ◽  
Andrea Rinaldi ◽  
Matteo Stifanelli ◽  
...  
Keyword(s):  
Gene Expression ◽  
Phase I ◽  
B Cell ◽  
Cell Lines ◽  
Proliferative Activity ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Dual Inhibitor

Abstract Dual PI3K/mTOR inhibitors represent a promising class of anti/cancer compounds, of potential interest in lymphoid neoplasms which present activation of both targeted pathways. PQR309 is a novel, oral, member of this class of compounds and, as single agent, is currently being evaluated in a phase I for patients with solid tumors (NCT01940133). Here, we present the activity of the compound in pre-clinical models of mature lymphoid tumors, also integrating response data with genomic features. Methods. 48 cell lines [27 derived from diffuse large B-cell lymphoma (DLBCL), 10 from mantle cell lymphoma (MCL), 3 from splenic marginal zone lymphoma (SMZL), 8 from anaplastic large cell lymphoma (ALCL)] were treated with increasing doses of PQR309 and MTT assays were performed after 72 hrs exposure. A second dual PI3K/mTOR inhibitor, GDC0980, and the PI3Kdelta inhibitor Idelalisib were also used on all the cell lines. IC50, GI50, LC50, and TGI values were used to estimate the cytotoxic and cytostatic effects. PQR309-induced cytotoxic activity was tested by AnnexinV assay. Synergy was assessed by the Chou-Talalay combination index (CI) on 2 DLBCL cell lines (TMD8, U2932) exposed to increasing doses of PQR309 alone or in combination with increasing doses of other drugs for 72 hrs. Baseline gene expression profiling (GEP) was obtained on the cell lines with the Illumina HumanHT-12 Expression BeadChips and integrated with the anti-proliferative effect. Results. PQR309 showed potent anti-proliferative activity in most of the cell lines tested. The median IC50 was 242 nM (18nM-3.6 mcM), GI50 141 nM (25 nM-1.7 mcM), LC50 2.7 mcM (306 nM->10 mcM), TGI 711 nM (69 nM - >10 mcM). DLBCL (median IC50=166 nM), MCL (234 nM) and SMZL (214 nM) were all more sensitive than ALCL (664 nM) (P=0.005). Activated B-cell like (ABC) and germinal center B-cell like (GCB) DLBCL subtypes were equally sensitive. Across the 48 cell lines, PQR309 and GDC0980 presented a highly correlated pattern of anti-proliferative activity (R=0.95). Idelalisib appeared significantly less active and its pattern of sensitive cell lines was less correlated with PQR309 (R=0.67) or GDC0980 (R=0.71). In DLBCL cell lines, PQR309 (1 mcM) was able to inhibit IgM-stimulation induced p-AKT(Ser 473) in 2/2 cells and the baseline p-AKT(Ser 473) levels in 1/1. PQR309 (500 nM, 72 hrs) caused apoptosis in 1/7 cell lines. Synergism or additive effected were observed in 2/2 cells combining PQR309 with the BCL2 inhibitor ABT199 (CI = 0.1 and 0.5), the immunomodulatory drug lenalidomide (0.5 and 0.4), the BTK-inhibitor ibrutinib (0.6 and 0.57) or the proteasome inhibitor bortezomib (0.9 and 0.9), and in 1/2 with the anti-CD20 monoclonal antibody rituximab (0.6), the BET inhibitor JQ1 (0.7) and the chemotherapy agent bendamustine (0.7). We then looked for baseline GEP features associated with sensitivity to PQR309, by comparing very sensitive (IC50 < 200 nM) versus less sensitive DLBCL cell lines (IC50 > 400 nM). Transcripts more expressed in sensitive cells were significantly enriched of genes involved in B-cell receptor pathway/signaling, kinases regulation, immune system. Transcripts associated with less sensitive cells were enriched of members of proteasome pathway, oxidative phosphorylation, translation initiation. Genes coding for individual proteins involved in PI3K signaling cascade were differentially expressed between the two groups of cells. Conclusions. PQR309 showed promising activity as single agent and in combination providing the basis for phase I/II studied dedicated for lymphoma patients. Baseline features associated with response were identified and are worth of being validated in the context of the next clinical trials. (CT and EG contributed equally to this work) Disclosures Hillmann: Piqur Therapeutics AG: Employment. Fabbro:Piqur Therapeutics AG: Employment. Hebeisen:Piqur Therapeutics AG: Employment. Betts:Piqur Therapeutics AG: Consultancy. Wicki:Piqur Therapeutics AG: Research Funding; University Hospital Basel: Employment. Cmiljanovic:Piqur Therapeutics AG: Employment, Membership on an entity's Board of Directors or advisory committees. Bertoni:Piqur Therapeutics AG: Research Funding.

scholarly journals Combination of Ibrutinib and BCL-2 or SYK Inhibitors in Ibrutinib Resistant ABC-Subtype of Diffuse Large B-Cell Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 505-505 ◽  
Author(s):  
Hsu-Ping Kuo ◽  
Richard Crowley ◽  
Ling Xue ◽  
Karl J. Schweighofer ◽  
Leo WK. Cheung ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tumor Growth ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Large B Cell Lymphoma ◽  
B Lymphoma Cells ◽  
Bcr Signaling

Abstract Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin's lymphoma (NHL), accounting for roughly 30% of newly diagnosed cases in the US. DLBCL can progress quickly, and in advanced cases is inconsistently cured with current therapies. Ibrutinib, a first-in-class Bruton's tyrosine kinase (BTK) inhibitor, is approved as a treatment for patients (pts) with mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) who have had one prior treatment. The ABC subtype of DLBCL is considered especially high risk and characterized by chronic active B-cell receptor (BCR) signaling, which is blocked by ibrutinib. Recent phase II clinical trial results of ibrutinib as a single agent in DLBCL pts show an overall response rate of 41% in the ABC subtype (Wilson et al. ASH 2012). Responses of various cancers to single kinase targeted therapies are often limited by the cell's ability to bypass the target via alternative pathways or acquired mutations in the target or its pathway. It has been shown that a small number of CLL pts acquire resistance to ibrutinib through mutations in BTK and its substrate phospholipase C gamma 2 (PLCG2) in the B lymphoma cells following prolonged treatments (Woyach et al. NEJM 2014). Such mechanisms may be overcome by combinations of targeted agents. Through screening of wild-type and acquired ibrutinib-resistant ABC-DLBCL cell lines (e.g. expressing BTK C481S), we identify and report herein B-cell lymphoma-2 (BCL-2) and spleen tyrosine kinase (SYK) inhibitors that synergize with ibrutinib in vitro and in vivo. Methods: Gene expression was analyzed by RT-qPCR using TaqMan Gene Expression Master Mix. Human DLBCL cell lines were treated with drugs for 3 days and the effect on cell growth was determined by CellTiter-Glo luminescent cell viability assay. SCID mice were treated when the TMD8 tumors reached 100-150 mm3. Annexin-V-positive and PI-negative population was detected as apoptotic cells in tumor cells at sacrifice. Cell adhesion and migration assays were performed as previously described (Chang et al. Blood 2013). Analysis of clinical samples used for BCL-2 gene expression profiling was performed using Affymetrix microarrays on FFPE specimens from the phase 2 PCYC-1106 trial (NCT01325701) and a rank based statistic (RankProd) was used to determine the significance of gene expression changes. Results: DLBCL cell lines with higher BCL-2 expression were more sensitive to single agent ABT-199 than those with lower expression. Treatment of DLBCL cells with ibrutinib alone increased BCL-2 expression as well as their sensitivity to BCL-2 inhibitors. Combination treatment with BCL-2 inhibitors and ibrutinib completely inhibited tumor growth in murine models of ABC-DLBCL (Figure). Increased apoptotic cell populations were detected in the combination treated tumors compared to either treatment alone. Clinically, pretreatment tissue samples (n=28) from ABC-DLBCL pts who experienced objective responses to ibrutinib (CR+PR) had lower BCL-2 gene expression. A high BCL-2 mutation rate was observed in pts with poor response to ibrutinib (SD+PD). However, none of these mutations occurred in the BH3 or BH1 domains, both of which appear to interact with ABT-199 based on a 3-dimensional co-crystal structure of the inhibitor with BCL-2 (PDB code 4MAN) and further molecular simulation results. These findings suggest the potential benefit from combination therapy. SYK is another downstream mediator of BCR signaling. Pretreatment of DLBCL cells with SYK inhibitors (e.g. R406) increased their sensitivity to ibrutinib. Ibrutinib resistant B-lymphoma cells with either C481S BTK or R665W PLCG2 mutations were re-sensitized to ibrutinib in combination with BCL-2 or SYK inhibitors, inhibiting cell growth, IgM-induced calcium flux, cell adhesion or migration in mutant containing cells. Conclusions: Consistent with previous results from high-throughput combinatorial screenings of drugs interact favorably with ibrutinib (Mathews Griner, et al. PNAS 2013), we found BCL-2 and SYK may function in alternative survival pathways in DLBCL cells upon BTK inhibition. Human B lymphomas harboring ibrutinib-resistant C481S BTK or R665W PLCG2 may be re-sensitized by BCL-2 or SYK inhibitors, both of which provide a rationale for the design of combination therapies. Figure 1 Combination of ibrutinib and ABT-199 on the effect of TMD-8 tumor growth. Figure 1. Combination of ibrutinib and ABT-199 on the effect of TMD-8 tumor growth. Disclosures Kuo: Pharmacyclics: Employment. Crowley:Pharmacyclics: Employment. Xue:Pharmacyclics: Employment. Schweighofer:Pharmacyclics: Employment. Cheung:Pharmacyclics: Employment. Hsieh:Pharmacyclics: Employment. Eckert:Pharmacyclics: Employment. Versele:Janssen Pharmaceutica: Employment. Chang:Pharmacyclics, Inc: Employment, Equity Ownership.

scholarly journals A Phase I Trial of Brentuximab Vedotin in Combination with Lenalidomide in Relapsed or Refractory Diffuse Large B-Cell Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3988-3988 ◽  
Author(s):  
Jeffrey P. Ward ◽  
Jessica Thein ◽  
Jingqin Luo ◽  
Nina D. Wagner-Johnston ◽  
Amanda F. Cashen ◽  
...  
Keyword(s):  
Phase I ◽  
B Cell ◽  
Research Funding ◽  
De Novo ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Brentuximab Vedotin ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Background: The addition of rituximab to CHOP has improved the overall survival of patients with diffuse large B-cell lymphoma (DLBCL); however, approximately 30% of patients will relapse. Stem cell transplantation (SCT) provides a second chance at cure, but the prognosis for patients who are not candidates for SCT or who have refractory disease is poor, and new treatments with novel agents are needed. Brentuximab vedotin (BV), an antibody-drug conjugate that combines an anti-CD30 monoclonal antibody and the microtubule disrupting agent MMAE, has a single agent response rate (RR) of 44% (CR 17%) in CD30 (+) (Jacobsen, Blood 2015) and 27% (CR 3.7%) in CD30 (-) relapsed/refractory (rel/ref) DLBCL (Bartlett, ASH 2014). Lenalidomide (Len), an immunomodulator with multiple described mechanisms of action, has a single agent RR of 28% (CR 7%) in rel/ref DLBCL (Witzig, Ann Oncol 2011). Notably, the Len RR was 52.9% in the subset of patients with non-germinal center-like (non-GCB) DLBCL, compared to 8.7% in GCB DLBCL (Hernandez-Ilizaliturri, Cancer 2011). Given that both compounds have single agent activity in DLBCL and favorable, non-overlapping toxicity profiles, we hypothesized that the combination of BV and Len would be an effective and tolerable regimen in rel/ref DLBCL. Methods: This investigator initiated, phase I/dose expansion trial is ongoing to identify the safety and maximum tolerated dose (MTD) of the combination of BV and Len (Clinical Trials.gov NCT02086604). Eligible patients have rel/ref de novo or transformed DLBCL after at least one prior systemic therapy and have previously received or are ineligible for SCT. Response assessments are performed after cycles 2, 4, 6, 9 and then every six months for two years by PET/CT scan and response determined per the Revised International Working Group Response Criteria for Malignant Lymphoma 2007. The study is in two parts, a dose-escalation portion using a 3+3 design to determine the MTD, followed by a dose-expansion cohort enrolling patients with either CD30 (+) or CD30 (-) DLBCL assessed by visual assessment using routine IHC per local laboratory. BV is administered every 21 days and Len is dosed continuously for a maximum of 16 cycles until disease progression or unacceptable toxicity. Results: Eighteen patients have been enrolled to date. The median age is 61 years (range 51-79), with 83% having an ECOG performance status of 0-1. Median number of prior therapies is 2 (range 1-6), with 39% undergoing a previous autoSCT, and one patient a previous alloSCT. 72% of patients were refractory to their last regimen. 13 patients have CD30 (-) and 5 CD30 (+) DLBCL. Treatment-related adverse events (AEs) occurring in >20% of patients include anemia (50%), elevated ALT (28%), hypocalcemia (22%), peripheral neuropathy (22%), neutropenia (28%), thrombocytopenia (33%), and hypokalemia (28%). Anemia, febrile neutropenia, thrombocytopenia, and hypokalemia were the only grade 3/4 related AEs observed in >10% of patients. Growth factors were not given during cycle 1 but were administered in 11 patients with subsequent cycles. One patient came off study for thrombocytopenia after completing 8 cycles, while in a CR. 47% have required at least one dose reduction. The DLTs per dose cohort are summarized in the table. The MTD of the combination is 1.2 mg/kg of BV Q21d with 20 mg Len given continuously. At the time of this analysis, 17 patients (1 too early) have had restaging evaluations; 7 CR (41%), 2 PR, 3 SD, and 5 PD, for an overall RR of 53%. Five CRs occurred after C2, 1 after C6 and 1 after C8. All responses are ongoing with a range of 5 to 35 wks. Among the 7 CRs, 2 patients have CD30 (+) and 5 patients CD30 (-) DLBCL, 4 pts were GCB and 3 non-GCB. Of the four patients with CD30 (-) disease categorized as GCB, two achieved a CR. Conclusions: This Phase I study of BV plus Len identified the MTD of the combination at BV 1.2 mg/kg Q21d with Len 20 mg/d continuously. Dose expansion cohorts of 15 patients each for CD30 (-) and CD30(+) DLBCL are currently accruing. The predominant toxicity of the study regimen is related to cytopenias, consistent with prior experience. Although patient numbers are small, the high CR rate is intriguing. Updated results will be presented at the meeting. Table 1. # Patients Assigned BV Dose Assigned Len Dose # of DLT DLT Toxicity 9 1.2mg/kg 20mg 1 Neutropenia 6 1.2mg/kg 25mg 2 Neutropenia, DVT 3 1.8mg/kg 25mg 2 Fatigue, Neutropenia Disclosures Ward: Boehringer Ingelheim: Consultancy. Wagner-Johnston:Celgene: Research Funding; Gilead: Consultancy. Fehniger:Celgene: Research Funding. Bartlett:Gilead: Consultancy, Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Millennium: Research Funding; Colgene: Research Funding; Medimmune: Research Funding; Kite: Research Funding; Insight: Research Funding; Seattle Genetics: Consultancy, Research Funding; MERC: Research Funding; Dynavax: Research Funding; Idera: Research Funding; Portola: Research Funding; Bristol Meyers Squibb: Research Funding; Infinity: Research Funding; LAM Theapeutics: Research Funding.

PIM as a Rational Target for B-Cell Lymphomas.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3946-3946
Author(s):  
Cristina Gomez-Abad ◽  
Helena Pisonero ◽  
Juan F Leal ◽  
Giovanna Roncador ◽  
Jose A. Martinez-Climent ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Specific Inhibitor ◽  
B Cell Lymphoma ◽  
Large B Cell Lymphoma ◽  
Micromolar Range ◽  
Large B Cell ◽  
Stat Pathway

Abstract Abstract 3946 Poster Board III-882 INTRODUCTION The Pim kinases are a family of serine/threonine kinases composed by three members: Pim1, Pim2 and Pim3, involved in the phosphorylation and regulation of several proteins that are essential for cell cycle progression, metabolism or apoptosis (BAD, p21, p27KIP, AKT, Mdm2 and cMyc, among them). Overexpression, translocation or amplification of Pim family have been described in many human cancers, including B-cell Non Hodgkin's Lymphoma, Multiple Myeloma, Prostate cancer and Pancreatic cancer. In addition, 50% of patients diagnosed with diffuse large B-cell Lymphoma (DLBCL) present somatic mutations in Pim1. Despite of its important role in cancer progression, very few chemical inhibitors have been described in the literature, being effective all of them in the high micromolar range. PURPOSE Validating PIM as a rational therapeutic target in B-cell lymphoma, developing tools for patient stratification and pharmacodynamic studies on PIM inhibition. MATERIAL AND METHODS Gene expression profiling and Copy Number data were obtained from a series of 94 B-cell Non-Hodgkin Lymphoma patients (DLBCL, FL, MALT, MCL and NMZL). The effect of Pim inhibition was checked on cell lines by using a novel specific inhibitor for the Pim family (ETP-39010). Newly produced antibodies and RT-PCR primers and protocols were standarized. RESULTS Gene expression data revealed high Pim isoforms expression in a subset of patients with Mantle cell lymphoma (MCL), and Diffuse Large B-cell lymphoma (DLBLC)-ABC type. CGH analysis focused on chromosomal regions containing Pim family and its main regulatory upstream pathway (JAK/STAT) was performed. Heterozygous gains of Pim1 (6p21.2) and Pim3 (22q13.33) were identified in 13.6% of DLBCL patients and in 4.2% of MCL. Alterations in JAK/STAT pathway were also detected in 59.1% of DLBCL patients, and 37.5% of MCL patients presented any alteration in JAK/STAT pathway, being frequent losses of JAK2 chromosomal region. Analysis of additional pathways involved in the up-stream regulation of Pim family disclosed heterozygous gains of PIK3C3 in 40.9% of DLBCL patients, and gains of PIK3CA in 45.9% of MCL patients. Lymphoma cell lines (15) derived from both MCL (9) and ABC-DLBLC (6) subtype, have been analyzed by qRT-PCR and Western-blot, showing variable expression levels of Pim1, Pim2 and Pim3. IC50 obtained for the ETP-39010 compound is in the low micromolar range for the MCL (0.7-8.7 micromolar) and DLBCL-ABC (0.8-10.3 micromolar) cell lines. Since Pim kinase family phosphorilate multiple sites of Bad and AKT, we have checked the inhibition of its phosphorilation as molecular biomarkers for the ETP-39010 effect. Our data show an inhibition of at least 20% of pBad (S112) and almost a complete inhibition of pAKT (S473) 4h after treatment. In addition, cell cycle arrest at G1 and induction of apoptosis were observed 24h after the treatment. CONCLUSION Pim family genes are a rational therapeutic target in MCL and DLBCL-ABC lymphoma subtypes. Stratification and pharmacodynamic markers have been developed for PIM inhibition using a novel specific inhibitor compound -ETP-39010-. Disclosures: No relevant conflicts of interest to declare.

Differential Patterns of Gene Expression and CD20 Localisation by the Type II Anti-CD20 Antibody Obinutuzumab (GA101) Compared with the Type I Antibody Rituximab Suggests Binding to Functionally Distinct CD20 Subpopulations on B-Cell Lymphoma Cell Lines,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3721-3721
Author(s):  
Gerhard Niederfellner ◽  
Olaf Mundigl ◽  
Alexander Lifke ◽  
Andreas Franke ◽  
Ute Baer ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Mechanisms Of Action ◽  
Type I ◽  
Type Ii ◽  
Cd20 Antibody ◽  
Anti Cd20

Abstract Abstract 3721 The anti-CD20 antibody rituximab has become central to the treatment of B-cell malignancies over the last decade. Recently, it has been shown that anti-CD20 antibodies can be divided into two types based on their mechanisms of action on B cells. Rituximab is a type I antibody that redistributes CD20 into lipid rafts and promotes complement-dependent cytotoxicity (CDC), while the type II, glycoengineered antibody GA101 has lower CDC activity but higher antibody-dependent cellular cytotoxicity and direct cell death activity. In preclinical studies GA101 was superior to rituximab in B-cell killing in vitro, depletion of B cells from whole blood, and inhibition of tumour cell growth in lymphoma xenograft models. GA101 is currently being evaluated in Phase II/III trials, including comparative studies with rituximab. To investigate the differences in direct effects of GA101 and rituximab on B-cell lymphoma signaling, we have analysed the effects of antibody binding on gene expression in different B-cell lines using a GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix). Rituximab and GA101 rapidly induced gene expression changes in SUDHL4 and Z138 cells, including regulation of genes associated with B-cell-receptor activation such as EGR2, BCL2A1, RGS1 and NAB2. The effects on gene expression differed markedly between different cell lines and between the two antibodies. SUDHL4 cells showed pronounced changes in the gene expression pattern to rituximab treatment, while Z138 cells, which represent a different B-cell stage, showed less pronounced changes in gene expression. The reverse was true for GA101, suggesting not only that the signaling mediated by CD20 differs in different cell lines, but also that in a given cell line the two types of antibodies bind CD20 molecules with different signaling capacity. For each cell line, gene expression induced by other type I antibodies (LT20, 2H7, MEM97) was more like rituximab and that induced by other type II antibodies (H299/B1, BH20) was more like GA101 in terms of the number of genes regulated and the magnitude of changes in expression. Unbiased hierarchical clustering analysis of gene expression in SUDHL4 could discriminate type I from type II antibodies, confirming that the two classes of antibody recognised CD20 complexes with inherently different signalling capacities. By confocal and time-lapse microscopy using different fluorophores, rituximab and GA101 localised to different compartments on the membrane of lymphoma cells. GA101/CD20 complexes were relatively static and predominantly associated with sites of cell–cell contact, while rituximab/CD20 complexes were highly dynamic and predominantly outside areas of contact. These findings suggest that type II antibodies such as GA101 bind distinct subpopulations of CD20 compared with type I antibodies such as rituximab, accounting for the differences in mechanisms of action and anti-tumour activity between these antibodies. Disclosures: Niederfellner: Roche: Employment. Mundigl:Roche: Employment. Lifke:Roche: Employment. Franke:Roche: Employment. Baer:Roche: Employment. Burtscher:Roche: Employment. Maisel:Roche: Employment. Belousov:Roche: Employment. Weidner:Roche: Employment. Umana:Roche: Employment, Patents & Royalties. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

A Phase I Study of Myeloablative I-131-Anti CD-20 (Tositumomab) Radioimmunotherapy with Escalating Doses of Fludarabine Followed by Autologous Hematopoietic Stem Cell Transplantation (ASCT) for Adults ≥ 60 Years of Age with High-Risk or Relapsed/Refractory B-Cell Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 663-663
Author(s):  
Ajay K. Gopal ◽  
Ted Gooley ◽  
Joseph Rajendran ◽  
John M. Pagel ◽  
Darrell R. Fisher ◽  
...  
Keyword(s):  
High Risk ◽  
Phase I ◽  
B Cell ◽  
Radiation Exposure ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Nucleoside Analogs ◽  
B Cell Lymphoma ◽  
High Dose ◽  
Hematopoietic Stem

Abstract Abstract 663 Background: The majority of patients with relapsed or refractory B-cell, non-Hodgkin's lymphoma (NHL) are over 60 years of age, yet many are denied potentially curative high-dose regimens due to concerns of excessive toxicity. We have shown that myeloablative anti-CD20 radioimmunotherapy (RIT) can safely deliver effective radiation doses to tumor sites while limiting exposure to normal organs in older adults requiring high-dose therapy, however, not all patients in this series remained progression-free (Gopal, JCO 2007). Preclinical data suggest that improved anti-tumor activity may be attained by the concurrent administration of nucleoside analogs (fludarabine, cytarabine) which synergize with RIT (Johnson, Int J Cancer; Gopal, BBMT, 2006). We hypothesized that a prolonged regimen of fludarabine could be administered concurrently with myeloablative RIT and ASCT to safely augment the efficacy of this approach. We present the phase I data evaluating this strategy. Methods: Patients were eligible if they were 60 years of age or older, had mantle cell lymphoma (MCL) in first remission or relapsed/refractory B-NHL, an ECOG PS of 0–1, acceptable organ function, >2×106 autologous CD34+ peripheral blood stem cells/kg collected, <20 Gy prior radiation to critical organs, and no human-anti-mouse-antibodies (HAMA). All patients underwent outpatient biodistribution studies for dosimetry using tositumomab (1.7 mg/kg, n=3 or 485mg flat dose, n=33) labeled with ∼10mCi I-131 followed by serial quantitative gamma camera imaging to calculate individualized organ-specific absorbed dose estimates. Patients then received therapeutic infusions of I-131-tositumomab to deliver 27Gy to the critical normal organ receiving the highest radiation exposure. Forty-eight hours later fludarabine was administered in escalating doses to patients (Table) to define a regimen associated with a dose limiting toxicity (DLT = grade III/IV Bearman toxicity) rate of <25%. ASCT occurred when radiation exposure was estimated to be <2mR/hr at 1meter. Filgrastim at 5μg/kg/day or pegfilgrastim at 6mg × 1 was started on day 1. Response was scored using standard criteria. Results: Between July 2005 and May 2011 36 patients were treated. Baseline characteristics included: median age 65 yrs (range 60–76), stage III/IV = 34 (94%), median number of prior regimens = 2 (range 1–9), chemoresistant disease (defined as < a partial response [PR] to the most recent regimen) = 12 (33%), >1 extranodal site = 14 (39%), elevated LDH at treatment = 13 (36%), IPI score at transplant 3–5 = 53%. Histology: MCL = 23, diffuse large B-cell = 8 (with 5 transformed from follicular lymphoma [FL]), FL=3, and marginal zone = 1, Waldenstrom's = 1. Dose limiting organs receiving up to 27Gy included lung (30), kidney (4), and liver (2) with a median administered I-131 activity of 471 mCi (range 260–1620). Fludarabine was escalated from 10 mg/m2/day × 5 days to 30 mg/m2 × 7 days without observation of a DLT (Table). The median CD34 dose was 5.42 ×106/kg with neutrophil (ANC>500μl) and platelet (>20 K/μl) engraftment occurring a median on 10 and 12 days after ASCT, respectively. Only 2 patients developed grade 4 NCI-CTC grade 4 non-hematologic toxicities (hypokalemia/hypophosphatemia, depression), 25 (69%) remained outpatients after discharge from radiation isolation, and there were no non-relapse deaths in the first 100 days after transplant. Responses to therapy were as follows: CR/CRu = 26 (79%), PR = 2 (6%), SD = 4 (11%), and PD = 4 (11%). Currently, 23 (64%) patients are alive with 22 (61%) progression free. The estimated 3 yr overall survival, progression-free survival, relapse, and non-relapse mortality are 54%, 53%, 41%, and 7%, respectively (median follow up of 2.5yrs after ASCT, Figure). Improved survival was associated with <2 prior regimens (HR=.08, p=.02) and chemosensitive disease (HR=.35, p=.07). Conclusions: High-dose (27 Gy) I-131 tositumomab can safely be administered concurrently with up to 210 mg/m2 fludarabine in an older, high-risk patient population. This strategy warrants further investigation as a method to safely augment the antitumor activity of myeloablative RIT. Disclosures: Gopal: GSK: Research Funding; Spectrum: Research Funding; Seattle Genetics: Consultancy, Research Funding; Merck: Research Funding; Piramal: Research Funding; Cephalon: Research Funding; Millenium: Speakers Bureau; Abbott: Research Funding; Pfizer: Research Funding; SBio: Research Funding; Bio Marin: Research Funding; Biogen-Idec: Research Funding. Off Label Use: High-dose use of I-131-tositumomab. Maloney:GSK: Consultancy, Honoraria.

The Brd-Inhibitor OTX015 Is Active in Pre-Clinical Models of Mature B-Cell Lymphoid Tumors

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1657-1657 ◽  
Author(s):  
Paola Bonetti ◽  
Michela Boi ◽  
Maurilio Ponzoni ◽  
Maria Grazia Tibiletti ◽  
Anastasios Stahis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Lines ◽  
Proliferative Activity ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Rt Pcr ◽  
Down Regulation ◽  
Myc Gene ◽  
Dose Dependent

Abstract Abstract 1657 Background: Bromodomain-containing proteins play an important role in gene expression regulation, via chromatin structure remodelling. Antitumor activity has been reported in acute and chronic hematological malignancies using inhibitors of BRD2/3/4, members of the Bromodomain and Extraterminal (BET) family. Here, we report anti-proliferative activity of OTX015, a novel selective orally bioavailable BRD2/3/4 inhibitor, in a large panel of cell lines derived from mature B-cell lymphoid tumors. Material and Methods: Established human cell lines derived from 13 diffuse large B-cell lymphoma (DLBCL), 4 mantle cell lymphoma (MCL), three splenic marginal zone lymphoma (SMZL) and from three multiple myeloma (MM) were treated with increasing doses of OTX015 (OncoEthix SA) and MTT assays were performed after 72 hours exposure. For cell cycle analysis, cells were treated and stained with Click-iT Edu Flow Cytometry Assay Kits (Invitrogen) and 7-AAD and analyzed for DNA content using a FACScan flow cytometer. Results were analyzed with FlowJo 7.6.3 software. RNA extracted using the Qiagen RNAEasy kit and reverse-transcribed using the Superscript First-Strand Synthesis System for RT-PCR kit according to the manufacturer's instructions. RT-PCR was performed using Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. For senescence detection, cells were stained using a b-Galactosidase Staining Kit (Calbiochem). Results: OTX015 demonstrated anti-proliferative activity in DLBCL cell lines (median IC50 0.192μM; range 0.069–12.68μM). Similar results were obtained on SMZL (median IC50 0.165μM, range 0.105–0.24μM), and on MM cell lines (median IC50 0.449μM; range 0.06–0.7μM). Conversely, MCL cell lines appeared less sensitive to OTX015 (median IC50 2.01μM; range 1.22- >15μM). Among DLBCL cell lines, there was no significant difference based upon the cell of origin of the cell lines. OTX105 caused a cell cycle arrest in G1 in a dose-dependent manner in 5/5 DLBCL and 3/3 MM cell lines, without an increase in cell death. An increase in the percentage of senescent cells after treatment with the BRD-inhibitor was observed in 1/1 sensitive DLBCL cell line. In order to understand the mechanism of action of OTX015, we assessed MYC mRNA levels before and after 24h treatment with increasing doses. We observed a dose-dependent suppression of MYC mRNA by OTX015 in 4/5 DLBCL and in 2/2 MM cell lines. In DLBCL, down-regulation of MYC mRNA was observed within 1h after treatment with OTX015, suggesting a direct effect of the compound on the MYC gene. To determine whether the suppression of MYC gene by OTX015 was reversible, DLBCL cell lines were treated for 2h with OTX015 and then the inhibitor was removed from the media. MYC mRNA suppression appeared reversible, as shown in DLBCL cell lines, which, after 2h exposure to OTX015, showed a time-dependent restoration of MYC mRNA expression to untreated levels after 2–3h. In one of the most sensitive DLBCL cell lines no MYC mRNA down-regulation was observed after treatment, suggesting that alternative pathways can be affected by BRD-inhibition. Conclusion: OTX015 is a new potent BRD-inhibitor with evident anti-proliferative activity in several cell lines representative of mature B-cell tumors. An apparently reversible down-regulation of MYC mRNA was commonly observed, appearing as a possible mechanism of action of the compound. The compound appears worth of further investigation as a new promising therapeutic agent in mature B-cell origin malignancies. A phase I trial is scheduled to start in 2012. Disclosures: Bonetti: OncoEthix SA: Research Funding. Inghirami:OncoEthix SA: Research Funding. Noel:OncoEthix SA: Membership on an entity's Board of Directors or advisory committees. Bertoni:OncoEthix SA: Research Funding.

SYK and STAT3 Are Active in Diffuse Large B-Cell Lymphoma: Activity of Cerdulatinib, a Dual SYK/JAK Inhibitor

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 926-926
Author(s):  
Y. Lynn Wang ◽  
Jiao Ma ◽  
Wei Xing ◽  
Pin Lu ◽  
Karen Dresser ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Staining Pattern ◽  
B Cell Lymphoma ◽  
Down Regulation ◽  
Dual Inhibitor ◽  
Large B Cell Lymphoma ◽  
Bcr Signaling ◽  
Large B Cell

Abstract Non-Hodgkin Lymphoma (NHL) represents about 5 percent of all cancers diagnosed in the United States. While incidence of NHL has increased slightly over the past decade, death rates have been declining steadily. These declines in mortality can be attributed to improvements in treatment that are based on an increased understanding of the biology of the disease. Diffuse large B-cell lymphoma (DLBCL) accounts for ~30% of NHLs and greater than 80% of aggressive NHLs. Recent studies including large-scale genetic analyses have demonstrated the critical roles of the B-cell receptor’s (BCR) and JAK/STAT pathways in DLBCL. Herein, we investigated the anti-lymphoma activity of cerdulatinib (aka PRT062070), a novel compound that dually targets both SYK and JAK/STAT signaling pathways. To determine whether targeting both SYK and JAK/STAT is relevant in DLBCL, we examined the expression of p-SYK (pY525/526) and p-STAT3 (pY705) on a tissue microarray of 62 DLBCL primary tumors, including 41 GCB and 21 non-GCB cases. p-SYK expression was detected in 29 (47%) cases with a characteristic peri-membrane staining pattern. Of those 29 p-SYK positive cases, 17 were GCB type (17/41, 41%) and 12 were non-GCB type (12/21, 57%). p-STAT3 exhibits a characteristic nuclear staining pattern in DLBCL cases. A total of 26 (42%) stained positive for p-STAT3; 16 were GCB type (16/41, 39%) and 10 were non-GCB type (10/21, 48%). Interestingly, there are 19 cases (31%) with reactivity for both p-SYK and p-STAT3, among which, 11 were GCB type (27%) and 8 were non-GCB type (38%). SYK and STAT3 are also phosphorylated in a panel of nine DLBCL cell lines. Immunoblotting analyses showed that ABC and GCB subtypes of DLBCL cells appear to exhibit different JAK/STAT and BCR signaling profiles. For instance, p-AKT was highly expressed in GCB cells, whereas p-STAT3 was more strongly expressed in ABC cells. Overall, the DLBCL cells are more sensitive to the dual inhibitor than to the SYK-specific inhibitor alone. In both GCB and ABC cell lines, cerdulatinib induced apoptosis via down-regulation of MCL1 protein and PARP cleavage. The compound also blocked G1/S transition and caused cell cycle arrest through inhibition of RB phosphorylation and down-regulation of cyclin E. Further analyses of the cell signaling activities showed that STAT3 phosphorylation was sensitive to inhibition by cerdulatinib in ABC cell lines while phosphorylation of SYK, PLCg2, AKT and ERK was sensitive to inhibition by cerdulatinib in GCB cell lines. Importantly, JAK/STAT and BCR signaling can be blocked by cerdulatinib in GCB and non-GCB primary human DLBCL cells, which led to cell death of these cells. Our work provided mechanistic insights into the actions of SYK/JAK dual inhibitor cerdulatinib, suggesting that the drug may be a potent treatment of DLBCL with a broader anti-tumor activity in both ABC and GCB subtypes of the lymphoma. Disclosures Pandey: Portola Pharmaceuticals: Employment. Conley:Portola Pharmaceuticals: Employment. Coffey:Portola Pharmaceuticals: Employment.

Recurrent Mutations of the Exportin 1 Gene (XPO1) in Primary Mediastinal B-Cell Lymphoma: A Lysa Study

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 129-129 ◽  
Author(s):  
Fabrice Jardin ◽  
Anais Pujals ◽  
Laura Pelletier ◽  
Elodie Bohers ◽  
Vincent Camus ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Lines ◽  
Nuclear Export ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Wild Type ◽  
U2os Cells ◽  
Aggressive B Cell Lymphoma

Abstract Background and aim of the study Primary mediastinal B-cell lymphoma (PMBL) is an entity of aggressive B-cell lymphoma that is clinically and biologically distinct from the other molecular subtypes of diffuse large B-cell lymphoma (DLBCL). We recently detected by Whole exome sequencing a recurrent point mutation in the XPO1 (exportin 1) gene (also referred to as chromosome region maintenance 1; CRM1), which resulted in the Glu571Lys (p.E571K) missense substitution in 2 refractory/relapsed PMBL (Dubois et al., ICML 2015; Mareschal et al. AACR 2015). XPO1 is a member of the Karyopherin-b superfamily of nuclear transport proteins. XPO1 mediates the nuclear export of numerous RNAs and cellular regulatory proteins, including tumor suppressor proteins. This mutation is in the hydrophobic groove of XPO1 that binds to the leucine-rich nuclear export signal (NES) of cargo proteins. In this study, we investigated the prevalence, specificity, and biological / clinical relevance of XPO1 mutations in PMBL. Patients and methods High-throughput targeted or Sanger sequencing of 117 PMBL patients and 3 PMBL cell lines were performed. PMBL cases were defined either molecularly by gene expression profile (mPMBL cohort) or by standard histological method (hPMBL cohort) and enrolled in various LYSA (LYmphoma Study Association) clinical trials. To assess the frequency and specificity of XPO1 mutations, cases of classical Hodgkin lymphoma (cHL) and primary mediastinal grey zone lymphoma (MGZL) were analysed. Cell experiments were performed to assess the impact of the E571 mutation on the activity of selective inhibitor of nuclear export (SINE) molecules. Results XPO1 mutations were present in 28/117 (24%) PMBL cases but were rare in cHL cases (1/19, 5%) and absent from MGZL cases (0/20). A higher prevalence (50%) of the recurrent codon 571 variant (p.E571K) was observed in PMBL cases defined by gene expression profiling (n = 32), as compared to hPMBL cases (n = 85, 13%). No difference in age, International Prognostic Index (IPI) or bulky mass was observed between the PMBL patients harboring mutant and wild-type XPO1 in the overall cohort whereas a female predominance was noticed in the mPMBL cohort. Based on a median follow-up duration of 42 months, XPO1 mutant patients exhibited significantly decreased PFS (3y PFS = 74% [CI95% 55-100]) compared to wild-type patients (3y PFS = 94% [CI95% 83-100], p=0.049) in the mPMBL cohort. In 4/4 tested cases, the E571K variant was also detected in cell-free circulating plasmatic DNA, suggesting that the mutation can be used as a biomarker at the time of diagnosis and during follow-up. Importantly, the E571K variant was detected as a heterozygous mutation in MedB-1, a PMBL-derived cell line, whereas the two other PMBL cell lines tested, Karpas1106 and U-2940, did not display any variants in XPO1 exon 15. KPT-185, the SINE compound that blocks XPO1-dependent nuclear export, induced a dose-dependent decrease in cell proliferation and increased cell death in the PMBL cell lines harbouring wild type or mutated alleles. To test directly if XPO1 mutation from E571 to E571K alters XPO1 inhibition by SINE compounds, the mutated protein was tested in vitro. The E571XPO1 mutated allele was transiently transfected into osteosarcoma U2OS cells which stably express the fluorescently labelled XPO1 cargo REV. Cells were treated with the clinical SINE compound selinexor, which is currently in phase I/II clinical trials and nuclear localization of REV-GFP was analysed in red transfected cells. The results showed that the nuclear export of the mutated XPO1 protein was inhibited by selinexor similarly to the wild-type XPO1 protein (Figure 1). Conclusion Although the oncogenic properties of XPO1 mutations remain to be determined, their recurrent selection in PMBL strongly supports their involvement in the pathogenesis of this curable aggressive B-cell lymphoma. XPO1 mutations were primarily observed in young female patients who displayed a typical PMBL molecular signature. The E571K XPO1 mutation represents a novel hallmark of PMBL but does not seem to interfere with SINE activity. Rev-GFP (green fluorescent) expressing U2OS cells were transfected with wild type XPO1-RFP (red fluorescent protein), XPO1-C528S-RFP, XPO1-E571K-mCherry, and XPO1-E571G-mCherry. The cells were then treated with 1µM KPT-330 for 8 hours. Figure 1. Rev-GFP expressing U2OS cells transfected with XPO1 variants. Figure 1. Rev-GFP expressing U2OS cells transfected with XPO1 variants. Disclosures Landesman: Karyopharm Therapeutics: Employment. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Argueta:Karyopharm Therapeutics: Employment. Milpied:Celgene: Honoraria, Research Funding.

scholarly journals EZH1/2 Dual Inhibitor Valemetostat Tosylate (DS-3201b) Acts Differently from EZH2 Selective Inhibitor on Epigenetic Landscape to Exert Greater Anti-Tumor Effect Against Diffuse Large B-Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2948-2948
Author(s):  
Toshihiro Banjo ◽  
Yasuhiro Hama ◽  
Emi Nosaka ◽  
Yoshimi Takata ◽  
Daisuke Honma ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tumor Suppressor ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Epigenetic Landscape ◽  
Complex Activity ◽  
Dual Inhibitor ◽  
Chip Sequencing

Abstract Enhancer of zeste homologous (EZH) 2 and its close homolog EZH1 are catalytic subunits of polycomb repressive complex (PRC) 2 protein complex, and play redundant and crucial role for the maintenance of transcriptional repression by tri-methylating histone H3 lysine 27 (H3K27). Hyper trimethylation of H3K27 has been associated with malignant lymphoma and myeloma progression, thus several small molecules suppressing PRC2 complex activity has been developed for hematological malignancy therapy. We have developed valemetostat tosylate (DS-3201b, also known as valematostat), a potent dual inhibitor of EZH1/2, and demonstrated its superior anti-proliferative effect against DLBCL cells to tazemetostat (EPZ-6438, E7438) a selective EZH2 inhibitor currently in clinic. In addition, valemetostat synergized with wide variety of 1st and 2nd line drugs used in DLBCL therapy both in vitro and in vivo proposing its potential combination opportunities. However, it is still elusive how valemetostat modulates epigenetic landscape and represses malignant B-cell proliferation more potently than selective EZH2 inhibitors. Therefore, impact on epigenetic landscape between valemetostat and tazemetostat was analyzed by RNA/ChIP-sequencing. Though these two inhibitors significantly reduced cellular global H3K27me3 level, we observed ectopic EZH1/2 accumulation in several tumor suppressor gene loci after tazemetostat treatment resulting in partial reduction in H3K27me3 and de-repression of silenced gene expression. Meanwhile valemetostat treatment evidently triggered gene expression by depleting H3K27me3 and enhancing H3K27Ac mark without inducing ectopic enrichment of EZH1/2, suggesting that valemetostat has a distinct effect on genome wide distribution of EZH1/2 from tazemetostat. In conclusion, these results suggest that valemetostat has a capacity of averting ectopic relocation of EZH1/2 on tumor suppressor genes mainly induced by EZH2 specific inhibition and thereby exerts greater anti-B cell tumor effect than EZH2 preferential inhibitor. A phase 2 clinical study of valemetostat is now ongoing for patients with Relapse/Refractory B-cell Lymphoma [ClinicalTrials.gov Identifier: NCT04842877] Disclosures Banjo: Daiichi Sankyo Co., Ltd.: Current Employment. Hama: Daiichi Sankyo Co., Ltd.: Current Employment. Nosaka: Daiichi Sankyo Co., Ltd.: Current Employment. Takata: Daiichi Sankyo Co., Ltd.: Current Employment. Honma: Daiichi Sankyo Co., Ltd.: Current Employment. Kitagawa: Daiichi Sankyo Co., Ltd.: Current Employment. Yamamoto: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Wada: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Yoshida: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Lim: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Okamoto: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Sato: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Katayama: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Sato: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Goto: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Abe: Daiichi Sankyo Co., Ltd.: Current Employment.

scholarly journals Targeting NF-κB with First in Class N-Quinoline Benzenesulfonamides (NQBS) Reveals Potent Activity in Models of Diffuse Large B-Cell Lymphoma (DLBCL)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4435-4435
Author(s):  
Matko Kalac ◽  
Michael Mangone ◽  
Alison Rinderspacher ◽  
Shi-Xian Deng ◽  
Luigi Scotto ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
B Cell ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Research Funding ◽  
Nuclear Translocation ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract The first two authors contributed equally to this work Identifying pharmacologic strategies to inhibit the activation of NF-κB and its target genes has been a major research pursuit. To date, no direct inhibitors of the NF-κB subunits have been explored in the clinic. Based on the constitutive activation of NF-κB in diffuse large B-cell lymphoma (DLBCL), we used this disease model to develop drugs targeting NF-κB. Using a fluorescence-based high throughput screening (HTC) approach, a unique N-quinoline-benzenesulfonamide (NQBS) scaffold was identified as potential small molecule inhibitor of the NF-κB pathway. A confocal microscopy based HTC assay performed in human umbilical vein endothelial cells (HUVEC) identified hit compounds that contained a unique NQBS core structure. The assay screened for compounds that inhibited nuclear translocation of NF-κB subunits in TNFα-induced HUVEC cells. To date over 100 NQBS analogs have been synthesized with varying potency and cytotoxicity in inhibiting growth of DLBCL lines (OCI-Ly10, RIVA, HBL-1 and OCI-Ly3). Cytotoxicity assays demonstrated that the most potent compounds exhibit IC50s in the 0.5 to 1.5 µM range. These most potent NQBS analogs identified as CU-O42 CU-O47 and CU-O75 were also able to induce apoptosis and caspase activation. Apoptosis was preceded by exclusion of the NF-κB proteins from the nucleus. To analyze the localization of NF-κB proteins within the cell compartments before and after the treatment with CU-O42, CU-O47 and CU-O75, we used confocal microscopy, electromobility shift (EMSA) and ELISA assays. Control cells tested positive for p50/p65 both within the cytoplasm and the nucleus. Following treatment with CU-O42 NF-κB was sequestered within the cytoplasm of the cell which occurred as early as 3h after exposure. In addition, all three analogs reduced the nuclear levels of NF-κB in a concentration-dependent manner when measured by EMSA and ELISA. Furthermore, CU-O47 and CU-O75 were able to inhibit TNFα induced luciferase expression in a HEK293T cell model where luciferase is controlled by an NF-κB promoter. A KINOMEscan platform (examining the activity of over 450 different kinases) showed that no NQBS analog screened (CU-O42 and CU-O75) inhibited any of the kinases in the assay. In addition, a proteasome inhibition assay tested negative for trypsin-like and chromotrypsin-like protease activity (CU-O42, CU-O47 and CU-O75). Stabilization of the inactive trimer of p50, p65 and IκBα was hypothesized as a potential mechanism of action of CU-O42 and CU-O75 through Internal Coordinate Mechanics (ICM) software. This binding hypothesis was further corroborated by cellular thermal shift assays (CETSA) with an increase of the IκBα melting temperatures (2.5-3°C) in whole cell lysates following rapid (30min) exposure to CU-O42 and CU-O75. Using a genome-wide regulatory network perturbation analysis (DeMAND) based on the RNA-Seq data collected from OCI-Ly10 cells treated with CU-O75, we identified IκBα as one of the potential targets of the compounds. Gene set enrichment analysis demonstrated NF-κB target gene downregulation using IC20 of CU-O75 at 24h (p=0.045). In vivo experiments were conducted in two models: (1) xenografts with human DLBCL cell lines of both ABC and GC subtype; and (2) myc cherry luciferase mouse model where mice spontaneously develop aggressive lymphomas. In both models, CU-O42 was able to inhibit tumor growth. Interestingly, in the xenograft model, malignant cell growth was inhibited in both ABC (HBL-1) and GC (OCI-Ly1) cells when compared to controls (p=0.01 and p=0.02). However, overall survival of mice with ABC xenografts treated with CU-042 significantly exceeded the survival of mice with GC xenografts (p<0.01) suggesting a more sustainable response in this subtype of disease, consistent with its dependency on NF-κB. Identification of a unique NQBS scaffold has led to the chemical synthesis of over 100 structural analogs with a potent inhibition on NF-κB nuclear translocation. They display potent activity across a panel of lymphoma cell lines, producing a survival benefit in mice implanted with an ABC-subtype of lymphoma. ICM, CETSA and DeMAND suggest that this is a direct effect mediated on the proteins within the p65/p50/IκBα complex. These findings point to a novel mechanism of action and warrant further research into potential clinical translation of this class of small molecules. Disclosures Califano: Thermo Fischer Scientific: Consultancy; Ipsen pharmaceuticals: Consultancy; Cancer Genetics Inc: Consultancy; Therasis Inc: Employment. O'Connor:Spectrum Pharmaceuticals: Consultancy, Honoraria, Research Funding; Takeda Millennium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Research Funding; Bristol-Myers Squibb Company: Consultancy; Novartis: Consultancy, Honoraria; Seattle Genetics: Consultancy; Bayer: Consultancy, Honoraria; Mundipharma: Consultancy, Honoraria, Research Funding; Acetylon Pharmaceuticals, INC: Consultancy.

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