scholarly journals B Cell Transcriptional Coactivator POU2AF1 (BOB-1) Is an Early Transcription Factor Modulating the Protein Synthesis and Ribosomal Biogenesis in Multiple Myeloma: With Therapeutic Implication

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2670-2670
Author(s):  
Zuzana Chyra ◽  
Mehmet K. Samur ◽  
Anil Aktas-Samur ◽  
Yao Yao ◽  
Sanika Derebail ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
B Cell ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Plasma Cells ◽  
Proteasome Inhibition ◽  
Advisory Committees ◽  
Active Enhancer ◽  
Polymerase I

Abstract Multiple Myeloma (MM) is a malignancy driven by numerous genetic and epigenetic alterations. Recurrent IgH translocations, somatic mutations and copy number abnormalities all contribute to myelomagenesis, however true drivers of the disease have not been well defined. To identify new targetable dependencies in MM, we generated high-quality active enhancer landscape using large cohort of primary patient myeloma cells (n=70), MM cell lines, and normal plasma cells. We integrated this data with an in-house curated atlas of 600+ active enhancer profile across a wide range of tumor types and normal tissues. Combining these data with gene expression and genetic dependency (CRISPR KO) enabled a multidimensional integration of how transcriptional regulation intersects with tumor specific dependencies. We identified that many of the specific and potent dependencies in MM are transcription factors, especially those establishing plasma cell identity. Among these, the POU2AF1 gene, which encodes the OCA-B/BOB-1, a B cell transcriptional coactivator protein, represented the most striking dependency in MM. Although BOB-1 is expressed throughout B-cell development, we found it to be highly expressed in CD138+ plasma cells from patients with precursor conditions (MGUS and SMM) as well as symptomatic MM compared to normal plasma cells. To functionally validate the role of BOB-1 in MM, we performed loss-of-function studies using shRNA, siRNAs and antisense GapMers specific for BOB-1 and observed a significant impact on MM cell viability and cell cycle arrest. Transcriptomic analysis upon BOB-1 depletion by RNA-sequencing revealed a small set of genes commonly modulated in all 3 MM cell lines tested including the plasma cell differentiation related transcription factor XBP1 and heme oxygenase (HMOX1). Importantly, we observed ribosome biogenesis, RNA polymerase 1A transcription and mRNA translation and elongation processes to be significantly enriched among genes modulated by BOB-1 depletion in MM cells. Bob1 KD resulted in a rapid and robust decrease in the level of transcription of rDNA by RNA polymerase I as determined by qRT-PCR quantification of pre-rRNA (47S). In addition, ChiP assay revealed decreased binding of RNA polymerase 1A to the 18S ribosomal DNA promoter region in BOB-1 depleted cells compared to control. These data indicate that BOB1 downregulation results in the suppression of RNA-polymerase I activity in MM cells. RNA Pol I-dependent transcription governs abundance of rRNA and directly regulates cellular translational and proliferative capacity. Since high protein load is a feature of MM, we evaluated the role of BOB-1 in the translational efficiency of MM cells. We observed that in MM cells compared to control cells, BOB-1 KD decreased, while its overexpression significantly enhanced de novo protein synthesis. As MM is characterized by excess production of monoclonal immunoglobulins, we evaluated impact of BOB-1 perturbation on intracellular light chains (kappa or lambda) production. We observed changes in the intracellular abundance of the light chains with BOB-1 modulation in all MM cell lines tested. As a result of decreased protein production, BOB-1 depletion was associated with induction of resistance to proteasome inhibition suggesting that high expression of BOB-1 may be one the factors driving the exquisite sensitivity of MM cells to proteasome inhibitor. Interestingly, mass spectrometry analysis revealed BOB1 in a protein complex with mTOR, Raptor and mLST8 proteins which are members of mTORC1 complex which is also involved in ribosomal function and may suggest the mechanism of action of Bob-1 at molecular level. In conclusion, here we report BOB1 as a specific proximal dependency in MM cells with potential role in modulating the protein load/capacity balance and ribosomal biogenesis essential for MM cell protein production function and therefore their sensitivity to proteasome inhibition. Disclosures Shirasaki: FIMECS: Consultancy. Mitsiades: BMS: Research Funding; Nurix: Research Funding; H3 Biomedicine: Research Funding; Novartis: Research Funding; Abbvie: Research Funding; Arch Oncology: Research Funding; Janssen/Johnson & Johnson: Research Funding; Fate Therapeutics: Consultancy, Honoraria; Karyopharm: Research Funding; Sanofi: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; FIMECS: Consultancy, Honoraria; Ionis Pharmaceuticals: Consultancy, Honoraria. Hajek: BMS: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharma MAR: Consultancy, Honoraria. Munshi: Abbvie: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Pfizer: Consultancy; Legend: Consultancy; Bristol-Myers Squibb: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Adaptive Biotechnology: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Novartis: Consultancy.

scholarly journals Exploring POU2AF1 (BOB-1) Dependency and Transcription Addiction in Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 49-49
Author(s):  
Zuzana Chyra ◽  
Mehmet Kemal Samur ◽  
Anil Aktas-Samur ◽  
Yan Xu ◽  
Eugenio Morelli ◽  
...  
Keyword(s):  
B Cell ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Target Genes ◽  
Plasma Cells ◽  
Private Company ◽  
Advisory Committees ◽  
Normal Tissues ◽  
Normal Plasma

Multiple Myeloma (MM) is a complex disease driven by numerous genetic and epigenetic alterations. Recurrent IgH translocations, copy number abnormalities and somatic mutations all contribute to myelomagenesis, yet true drivers of the disease have yet to be identified. In order to investigate the enhancer landscape and identify new dependencies and actionable therapeutic targets in MM, we have generated high-quality active enhancer landscape in MM cell lines as well as in large cohort of primary patient myeloma cells (n=70) and normal plasma cell. We integrated this data with an in-house curated atlas of 600+ active enhancer profile across a wide range of tumor types and normal tissues. Combining this data with gene expression and genetic dependency (CRISPR KO) enables a multidimensional integration of how transcriptional regulation intersects with tumor specific dependencies. We identified genes that are super-enhancer associated, expressed, and required specifically in MM versus other tumor types or normal tissues. Many of the specific and potent dependencies in MM are transcription factors, and especially those establishing plasma cell identity. Among others, the POU2AF1 gene, which encodes the OCA-B/BOB-1, a B cell transcriptional coactivator protein, represented the most striking dependency in MM. BOB-1 is a gene regulatory factor that regulates B-cell development, maturation and GC formation. Although BOB-1 is expressed throughout B-cell development, we found it to be highly expressed in CD138+ plasma cells from patients with precursor conditions (MGUS and SMM) as well and established MM compared to normal plasma cells, giving us the rationale to study the transcriptional program associated with BOB-1 in MM cells. To confirm CRISPR data described above, we have performed loss-of-function (LOF) studies using shRNA, siRNAs as well as antisense GapMers specific for BOB-1. Downregulation of BOB-1 in a panel of MM cell lines caused MM cell growth inhibition in a time-dependent manner which was associated with G2/M cell cycle arrest and induction of MM cell apoptosis. Transcriptomic analysis by RNA-sequencing revealed a set of 72 genes (adj p-value=0.2) commonly modulated in AMO-1 and NCI-929 MM cell lines upon BOB-1 depletion, as compared with scrambled cells. Among the top 5 downregulated genes in BOB-1 knockdown samples, we found AMPD1, KCNN3, BHLHA15, HID1 and XBP1. The modulation of BOB1 target genes was confirmed at the protein level by Western blot analysis in BOB-1 LOF and gain-of-function (GOF) cell systems. Analysis of patient expression datasets revealed that these genes are positively correlated with BOB-1 expression in primary MM cells and overexpressed in MM cells compared to normal plasma cells. One of these 5 genes positively controlled by BOB-1, BHLHA15, is the gene coding for the transcription factor Mist1, which functions as a coregulator of the unfolded protein response (UPR) master transcription factor XBP1. Target genes of the XBP1- BHLHA15 axis are involved in lipid synthesis, protein folding and secretion, and ER-associated protein degradation. As a result, BOB-1 knockdown increased de novo protein synthesis in MM cells compared to control cells. Interestingly, Mist1 gene expression is induced during ER stress by XBP1, but as ER stress subsides, Mist1 serves as a feedback inhibitor. Moreover, we identified that LOF studies in two MM cell lines confirmed increased expression of BOB-1 and XBP1 upon KD of Mist1. However, XBP1 and/or Mist1 are dispensable for MM cell survival, as gene depletion did not affect MM cell viability. Among the genes most significantly upregulated by BOB-1 depletion was heme oxygenase 1 HMOX1 gene, whose expression is significantly lower in-patient MM cells compared to normal plasma cells. Moreover, its lower expression significantly correlated with poor clinical outcome; and siRNA depletion of HMOX1 increased MM cell growth in MM cell lines, suggesting important role in MM. Mechanistic studies aimed at investigating the functions of HMOX1 in MM and its relation with BOB-1 are ongoing and will be presented. In conclusion, we here report BOB1 as an important transcriptional regulator in MM and its two down-stream novel target genes (BHLHA15 and HMOX1) with potential significant biological role in MM. Disclosures Hajek: BMS: Consultancy, Honoraria, Research Funding; Oncopeptides: Consultancy; PharmaMar: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Fulciniti:NIH: Research Funding. Munshi:Janssen: Consultancy; C4: Current equity holder in private company; Karyopharm: Consultancy; Amgen: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy; Legend: Consultancy; Adaptive: Consultancy; AbbVie: Consultancy; Takeda: Consultancy.

scholarly journals First Description of B Cell Maturation Antigen Expression in Light Chain Amyloidosis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5452-5452
Author(s):  
Susan Bal ◽  
Allison Sigler ◽  
Alexander Chan ◽  
David J. Chung ◽  
Ahmet Dogan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cell ◽  
Board Of Directors ◽  
Light Chain ◽  
Research Funding ◽  
Plasma Cells ◽  
Staining Intensity ◽  
Advisory Committees ◽  
Light Chain Amyloidosis ◽  
B Cell Maturation

Background B-cell maturation antigen (BCMA) is a transmembrane protein belonging to the tumor necrosis factor (TNF) superfamily involved in the regulation of B cell proliferation and survival as well as maturation/differentiation into plasma cells. In multiple myeloma cells, overexpression of BCMA has been shown to activate mitogen activated protein kinase pathways (AKT, ERK1/2, and NF-κB) and upregulates anti-apoptotic proteins (MCL1, BCL2, BCL-xL) resulting in cellular proliferation. Immunotherapeutic strategies targeting BCMA are showing great promise in heavily pre-treated refractory multiple myeloma. Light Chain Amyloidosis (AL) is a multisystem disorder of clonal plasma cells that results in the production of an abnormal light chain which misfolds and deposits in the organs leading to disruption of tissue architecture, cellular stress, dysfunction and eventually, death. The smaller burden and lower proliferative potential of the offending clonal plasma cells in amyloidosis may potentially lend itself favorably to immunotherapeutic strategies targeting BCMA. Given the efficacy of this approach in MM, the evaluation of BCMA expression on the surface of amyloidogenic plasma cells is warranted. Methods All patients diagnosed with Light chain Amyloidosis at Memorial Sloan Kettering Cancer Center, NY between January 1, 2012, and December 31, 2018, who had unstained bone marrow samples were identified. These unstained BM biopsy samples were prospectively stained for BCMA expression using Immunohistochemistry (IHC). We utilized a clinical-grade assay (clone D6; catalog sc-390147; company Santa-Cruz; monoclonal antibody; dilution 1:400) in a CLIA compliant setting. We scored the biopsies for BCMA expression, intensity, and site of staining. We also obtained their demographic details, staging, and cytogenetic information for the patients with available samples. Results During the queried period, 28 unstained samples were available for testing from the time of disease diagnosis. The median age of the population was 63 years (range 41-73). 64% of patients were male and consistent with the literature; a majority of patients (75%) had lambda-typic clonal plasma cells. Cytogenetic abnormalities using fluorescence in situ hybridization (FISH) were reviewed, t(11;14) was seen in 36% patients, and chromosome 1q and del 13q were each seen in 32% of patients. No patient had t(4;14) or del 17p. The median clonal PC burden in BM at diagnosis was 10% (range2-80%) and 36% had > 10% plasma cells. In clonal PCs, the median BCMA expression was 80% (range 20-100%). Only one patient had a staining intensity under 50% (20%). Membranous staining was noted in 82% of patients and a Golgi pattern in 11%. The median staining intensity was 2 (range 1-3). Of the patients with baseline diagnostic samples available for testing, six patients had additional unstained bone marrow samples for staining at the time of relapse. The majority of patients (83%) who relapsed had >10% plasma cells with a higher median plasma cell burden of 35% (range 10-80). The median BCMA expression was 65% (range 50-80) with no patient having <50% expression. The staining pattern was membranous in 50%, Golgi in 17%, and Golgi-membranous in 33%. At the time of relapse, the median clonal PC burden was 13% (range 5-30). BCMA expression continued to be present at the time of relapse with a median 75% (range 50-100) with predominantly membranous staining (83%). The median staining intensity in both diagnostic and relapsed tissue within the six samples studied was 1. Conclusions Our study represents the first description of BCMA expression on the surface of amyloidogenic plasma cells to our knowledge. BCMA is uniformly expressed by pathologic PCs in AL amyloidosis both at the time of diagnosis and relapse. Given the efficacy of BCMA directed therapy in multiple myeloma, further investigation of these agents in light-chain amyloidosis are warranted and may provide an effective therapeutic strategy in this devastating disease. Figure Disclosures Dogan: Corvus Pharmaceuticals: Consultancy; Celgene: Consultancy; Seattle Genetics: Consultancy; Novartis: Consultancy; Takeda: Consultancy; Roche: Consultancy, Research Funding. Giralt:Takeda: Consultancy, Research Funding; Johnson & Johnson: Consultancy, Research Funding; Kite: Consultancy; Novartis: Consultancy; Actinium: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Miltenyi: Research Funding; Spectrum Pharmaceuticals: Consultancy. Hassoun:Novartis: Consultancy; Janssen: Research Funding; Celgene: Research Funding. Landau:Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees; Caelum: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Expression and in Vitro Binding of Protease Activated Receptor1 (PAR1) with Novel Anti-PAR1 Molecules: Data on Fresh Myeloma Marrow Plasma Cells and Human Myeloma Cell Lines

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4440-4440
Author(s):  
Meral Beksac ◽  
Pinar Ataca ◽  
Berna Atesagaoglu ◽  
Klara Dalva ◽  
Andry Nur Hidayat ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Lower Percentage ◽  
Advisory Committees ◽  
In Silico Docking ◽  
Human Myeloma Cell

Abstract Introduction and Aim: Myeloma plasma cells are dependent on stromal support which is mediated through cell adhesion. Heparanase activity has been shown to be associated with aggressive behavior or Bortezomib resistance and can lead to increased levels of proteases as well as shedding of heparan sulfate proteoglycan syndecan-1(CD138) from myeloma cells. We have recently published in vivo anti-myeloma effects of low molecular weight heparin (Beksac et al Acta Haematol 2015). Protease activated Receptor (PAR1) is a thrombin receptor. PAR1 gene and antigen expression on myeloma patient samples and cell lines (HMCL) has been recently reported by University of Arkansas (UAMS) group (Tian et al ASH 2011). They were able to find HMCLs H929, U266, JJN3 to express PAR1. Also expression was found to be highest among patients with 5q amplification where the PAR1 gene is located. Patients and Methods: We analyzed PAR1 expression (WEDE15 PE, Beckman Coulter) by flow cytometry, on CD38+CD138+/-CD27+/- cells obtained from fresh patient bone marrow samples obtained either at diagnosis (n: 84)(NDMM) or relapse (n: 54)(RRMM) and were compared with marrow samples taken from patients without MM (n: 43). Our group in Ankara University had previously synthesized and published novel benzamide and phenyl acetamide derivatives. We performed an in silico docking analysis on these molecules, and eleven (TD10,TD12,TD12A,TD12B,TD13,TD14,TD14B,XT2,XT2B,XT5,XT11) were found to bind to PAR1. These molecules were screened using 72 hour MTT assay on primary and refractory cell lines (U266BR ,U266, JJN3BR, JJN3, H929R, OPM2, OPM2R, KMS28PE). Results: PAR1 expression was highest on platelets followed by myeloma plasma cells (0-81.9%) and did not correlate with ISS. PAR1 expression (Threshold: >2.5 % or >5%) could be detected in NDMM (35 % or 14%) and RRMM (31% or 19%) of patients (Table1). PAR1+CD38+138+ cells were more frequent among patients with lower percentage of plasma cells in RRMM group (2,98 ± 4,5 vs 1,93 ± 3,96, P=0.028) but not NDMM. PAR1 was similarly highly expressed on HCML. Two of the novel PAR1 binding molecules (XT5 and XT2B) were found to have the lowest IC50. The IC50 were similar for all HMCLs, primary and refractory, with XT5. With XT2B the IC50 was less (U266) or higher (JJN3) or similar (OPM2) for refractory compared to the primary HMCL. PAR1 expression and anti-myeloma IC50 values of cell lines are summarized in Table 2. Conclusion: PAR1 expression is detectable at very low or very high percentages on CD138+plasma cells. Expression is higher on cells with CD27 expression (patient samples) or lacking CD27 (HMCL). Inverse correlation between PAR1 expression and plasma cell percentage among myeloma patients is detected among RRMM but not on NDMM samples. This finding may point to expression of PAR1 on quiescent plasma progenitors as suggested by Tian et al previously. The intensity or frequency of PAR1 expression on HMCL did not influence the anti-myeloma effects of these novel molecules. PAR1 binding molecules, in particular XT5, are promising as they are effective even on Bortezomib refractory HCML. However their mechanism of action and the role of PAR1 require further investigations. This study has been supported by a research grant from Turkish Academy of Sciences. Table 1. Frequency of PAR1 expression (> 2.5 %) on total plasma cells (CD38+138+) and on quiescent plasma cells (CD38+138+27+) Control (n=43) NDMM (n=84) RRMM (n=54) P CD38+138+ (%) 0,56± 0,66 4,48 ± 7,67 5,44 ± 12,13 0,007 PAR1+ among CD38+138 (%) 6,18 ± 13,14 4,14 ± 11,00 3,42 ± 8,81 0,394 PAR1+ among CD38+138+27+(%) 5,44 ± 12,13 3,42 ± 8,81 3,58 ± 8,57 0,207 Table 1. Comparison of Flow Cytometric PAR1 expression and IC50 (in uM after 72 hours)of the two novel molecules on three Human Myeloma Cell Lines. H929 RPMI8221 U266 IC50 XT2B 33.9 >100 34.3 IC50 XT5 8.12 5.45 9.77 CD38+138+ (total%) 85 % 75 % 80 % PAR1% and (MFI) within CD38+138+ 83 %(13,6) 90 % (2,1) 85 % (2,1) Disclosures Beksac: Celgene: Consultancy, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Off Label Use: Elotuzumab is an investigational agent being studied for the treatment of multiple myeloma.. Usmani:Millennium: Honoraria, Speakers Bureau; Sanofi: Honoraria, Research Funding; Onyx: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Array BioPharma: Honoraria, Research Funding; Pharmacyclics: Research Funding; Janssen Oncology: Honoraria, Research Funding. Tian:University of Arkansas for Medical Sciecnes: Employment.

scholarly journals In Vitro and inVivo Activity of Melflufen in Amyloidosis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3100-3100 ◽  
Author(s):  
Ken Flanagan ◽  
Muntasir M Majumder ◽  
Romika Kumari ◽  
Juho Miettinen ◽  
Ana Slipicevic ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Plasma Cell ◽  
Light Chain ◽  
Research Funding ◽  
Plasma Cells ◽  
Al Amyloidosis ◽  
Advisory Committees

Background: Immunoglobulin light-chain (AL) amyloidosis is a rare disease caused by plasma cell secretion of misfolded light chains that assemble as amyloid fibrils and deposit on vital organs including the heart and kidneys, causing organ dysfunction. Plasma cell directed therapeutics, aimed at preferentially eliminating the clonal population of amyloidogenic cells in bone marrow are expected to reduce production of toxic light chain and alleviate deposition of amyloid thereby restoring healthy organ function. Melphalan flufenamide ethyl ester, melflufen, is a peptidase potentiated alkylating agent with potent toxicity in myeloma cells. Melflufen is highly lipophilic, permitting rapid cellular uptake, and is subsequently enzymatically cleaved by aminopeptidases within cells resulting in augmented intracellular concentrations of toxic molecules, providing a more targeted and localized treatment. Previous data demonstrating multiple myeloma plasma cell sensitivity for melflufen suggests that the drug might be useful to directly eliminate amyloidogenic plasma cells, thereby reducing the amyloid load in patients. Furthermore, the increased intracellular concentrations of melflufen in myeloma cells indicates a potential reduction in systemic toxicity in patients, an important factor in the fragile amyloidosis patient population. To assess potential efficacy in amyloidosis patients and to explore the mechanism of action, we examined effects of melflufen on amyloidogenic plasma cells invitro and invivo. Methods: Cellular toxicity and apoptosis were measured in response to either melflufen or melphalan in multiple malignant human plasma cell lines, including the amyloidosis patient derived light chain secreting ALMC-1 and ALMC-2 cells, as well as primary bone marrow cells from AL amyloidosis patients, using annexin V and live/dead cell staining by multicolor flow cytometry, and measurement of cleaved caspases. Lambda light chain was measured in supernatant by ELISA, and intracellular levels were detected by flow cytometry. To assess efficacy of melflufen in vivo, the light chain secreting human myeloma cell line, JJN3, was transduced with luciferase and adoptively transferred into NSG mice. Cell death in response to melflufen or melphalan was measured by in vivo bioluminescence, and serum light chain was monitored. Results: Melflufen demonstrated increased potency against multiple myeloma cell lines compared to melphalan, inducing malignant plasma cell death at lower doses on established light chain secreting plasma cell lines. While ALMC-1 cells were sensitive to both melphalan and melflufen, the IC50 for melphalan at 960 nM was approximately 3-fold higher than melflufen (334 nM). However, ALMC-2 cells were relatively insensitive to melphalan (12600 nM), but maintained a 100-fold increase in sensitivity to melflufen (121 nM). Furthermore, while 40% of primary CD138+ plasma cells from patients with diagnosed AL amyloidosis responded to melflufen treatment in vitro, only 20% responded to melphalan with consistently superior IC50 values for melflufen (Figure 1). Light chain secreting cell lines and AL amyloidosis patient samples were further analyzed by single cell sequencing. We further examined differential effects on apoptosis and the unfolded protein response in vitro in response to either melflufen or melphalan. This is of particular interest in amyloidosis, where malignant antibody producing plasma cells possess an increased requirement for mechanisms to cope with the amplified load of unfolded protein and associated ER stress. As AL amyloidosis is ultimately a disease mediated by secretion of toxic immunoglobulin, we assessed the effects of melflufen on the production of light chain invitro, measuring a decrease in production of light chain in response to melflufen treatment. Finally, we took advantage of a recently described adoptive transfer mouse model of amyloidosis to assess the efficacy of melflufen and melphalan in eliminating amyloidogenic clones and reducing the levels of toxic serum light chain in vivo. Conclusions: These findings provide evidence that melflufen mediated toxicity, previously described in myeloma cells, extends to amyloidogenic plasma cells and further affects the ability of these cells to produce and secrete toxic light chain. This data supports the rationale for the evaluation of melflufen in patients with AL amyloidosis. Figure 1 Disclosures Flanagan: Oncopeptides AB: Employment. Slipicevic:Oncopeptides AB: Employment. Holstein:Celgene: Consultancy; Takeda: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; Genentech: Membership on an entity's Board of Directors or advisory committees; Sorrento: Consultancy. Lehmann:Oncopeptides AB: Employment. Nupponen:Oncopeptides AB: Employment. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding.

scholarly journals BAR-Bodies: B-Cell Receptor Antigens As the Targeting Moiety of Antibodies in Substitution for the Variable Region of Heavy and Light Chains

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2940-2940
Author(s):  
Moritz Bewarder ◽  
Lorenz Thurner ◽  
Frank Neumann ◽  
Natalie Fadle ◽  
Evi Regitz ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphoma Cell ◽  
Lymphoma Cells ◽  
Advisory Committees

Abstract Background Chronic antigenic stimulation of the B-cell receptor (BCR) seems to play a critical role in the pathogenesis of B-cell lymphomas. We recently identified ARS2 and LRPAP1 as the autoantigenic targets of the B-cell receptors of approximately 25% of diffuse large B cell lymphomas (DLBCLs) of the ABC type and 45% of mantle cell lymphomas (MCLs), respectively. These BCR antigens can be used to target lymphoma cells in an approach we designated as BAR (B-cell receptor antigens for reverse targeting). The optimal therapeutic format BARs can be integrated in has yet to be found. Since the most established approach to deliver therapeutic payloads to specific targets are antibodies which have well-defined pharmacokinetics, we constructed and tested an antibody like construct (BAR-body) incorporating the DLBCL-BAR ARS2 in substitution for the variable domains of the heavy and light chains. Material and methods To create the ARS2 BAR-body, we exchanged the heavy and light chain variable region sequences of an IgG1 antibody with a sequence of similar length (approximately 120 amino acids) of the ARS2 protein (aa 343 - 466) containing the DLBCL reactive epitope (aa 343 - 375). The construct was assembled in a pCR2.1 vector, then transferred to a pSfi FLAG Tag vector for fusion with the FLAG tag and transfected into HEK293 cells for production. Purification of the BAR-body was performed via anti-FLAG antibody affinity chromatography. The BAR-body was detected by western blot analysis and binding capacity to the ARS2-reactive lymphoma cell lines U2932 and OCI-Ly3 and the not ARS2-reactive control DLBCL cell line TMD8 was assessed by flow cytometry. ARS2 BAR-body induced cytotoxicity of lymphoma cells with an ARS2 reactive BCR was measured by LDH release assays with human PBMCs as effector cells at an E:T ratio of 10:1. Results We cloned, expressed and characterized an ARS2 containing BAR-body incorporating 4 molecules of the lymphoma-reactive epitope of ARS2 resulting in an antibody like construct using a BAR (ARS2) as binding moiety instead of normal variable regions. The ARS2 BAR-body could successfully be cloned and expressed as confirmed by western blot analysis, which showed the construct at approximately 150 kD as was to be expected. The BAR-body bound specifically to the ARS2-reactive lymphoma cell lines U2932 and OCI-Ly3 and did not bind to the DLBCL cell line TMD8, which has a B-cell receptor of different specificity or to lymphoma cell lines of different entities. In LDH release assays with 5 x 104 PBMCs and 5 x 103 lymphoma cells (E:T ratio of 10:1) the ARS2 BAR-body induced PBMC mediated specific lysis of the ARS2 reactive lymphoma cell lines U2932 and OCI-Ly3 but not the control DLBCL cell line TMD8 starting at a concentration of 0,1µg/ml. Cytotoxic effects were dose dependent, reached a maximum of 50% specific lysis at a concentration of 1µg/ml and did not increase at concentrations of 10µg/ml. Conclusion Here, we show that BARs can substitute for the variable domains as binding moiety in antibody like constructs to target the BCR of B-cell lymphomas. Because approaches using their specific cognate antigen for targeting the malignant B cells have an exclusive specificity for the BCR of the malignant clone, they can be expected to be less toxic than the currently available antibody derived therapies targeting B-cells, because they leave normal B-lymphocytes unaffected. By incorporating BARs into the well-known format of an antibody we hope to capitalize on years of experience with this therapeutic format from conducting and interpreting in vivo experiments to the translation of the BAR approach into the clinic. Disclosures Stilgenbauer: Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals High-Dimensional Heterogeneity of Waldenström Macroglobulinemia within Its Immune Tumor Microenvironment

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3975-3975
Author(s):  
Jana Jakubikova ◽  
Danka Cholujova ◽  
Gabor Beke ◽  
Zachary R Hunter ◽  
Teru Hideshima ◽  
...  
Keyword(s):  
B Cells ◽  
Tumor Microenvironment ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Effector Memory ◽  
Advisory Committees ◽  
Effector Memory Cells ◽  
B Cell Precursors

Waldenström macroglobulinemia (WM), a malignant B-cell lymphoplasmacytic lymphoma, is a rare subtype of non-Hodgkin lymphoma representing about 1% of all cases. To better understand the WM pathogenesis, we performed large-scale data-driven proteomic profile of WM tumor cells associated with tumor-driven immune changes in the tumor microenvironment of 66 bone marrow (BM) samples from WM patients compared to 10 age-matched healthy donors (HD) by time-of-flight mass cytometry (CyTOF) technology. Our workflow has been designed based on extensive 3 CyTOF antibody panels to evaluate WM tumor within B cell lymphopoiesis concurrently with immune landscape of the tumor microenvironment in WM by state-of-art technology CyTOF. To map B cell lymphomagenesis in WM, we defined whole spectrum of maturation of B cell development, from hematopoietic stem cells and B cell precursors through immature B cells, transitional B cells, and naïve B cells together with memory un-switched and switched B cells, plasmablasts and plasma cells in BM samples of WM patients by positive and negative co-expression of 13 B cell-stage specific markers. Various immunophenotyping aberrancies within WM B lymphomagenesis were associated with WM clones characterized by significant increase of 11 B subset clusters from un-switched and switched memory B cells to plasma cells. Interestingly, WM clusters differ in intra-clonal expression of activation surface molecules (CD23, CD24, CD25, CD81, CD329, CD200, and CD319); transcriptional factors and regulators controlling B cell development (MYD88, Bcl-6, IRF-4, sXBP-1, and FGFR-3) and stemness-related markers (Oct3/4, Nanog, Sox-2, c-Myc, and Notch-1) in WM supporting the idea of sub-clonal heterogeneity insight of WM tumor. Moreover, decrease in cell frequency of B cell precursors (pro-B and pre-BI), naive B cells, and plasmablasts were observed in WM patients versus HD. To generate a comprehensive view of the tumor microenvironment, we observed significant upregulation of g/dT cells, CD4+ and CD8+ T effector cells, CD8+ T effector memory cells, monocytes, and neutrophils immune subsets and downregulation of immature T cells, CD8+ T naïve cells, plasmacytoid dendritic cells, myelo/mono progenitor clusters. Ibrutinib (IBRU) treatment has been effective in relapsed/refractory WM patients; therefore highest numbers of WM patients were receiving IBRU therapy in our cohort. IBRU treated WM patients had decreased frequency of naive B, CD4+ T naive cells and specific clusters of un-switched and switched memory B cells. Moreover, responder versus non-responders to IBRU therapy revealed increase of CD8+T effector memory cells. In sum, correspondence analysis reflecting data of each patient and immune subsets revealed stratification of WM patients with reflection on their clinical outcome, therefore providing the rational for prediction of WM patient status. This study was supported by APVV-16-0484 and VEGA 2/0076/17. Disclosures Hunter: Janssen: Consultancy. Richardson:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees. Kastritis:Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Prothena: Honoraria; Genesis: Honoraria. Treon:BMS: Research Funding; Janssen: Consultancy; Pharmacyclics: Research Funding. Anderson:Celgene: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

Analysis of Adct-602 Pre-Clinical Activity in B-Cell Lymphoma Models and Identification of Potential Biomarkers for Its Activity

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 10-11
Author(s):  
Eugenio Gaudio ◽  
Chiara Tarantelli ◽  
Luciano Cascione ◽  
Filippo Spriano ◽  
Gaetanina Golino ◽  
...  
Keyword(s):  
B Cell ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
Advisory Committees ◽  
Travel Grant

Introduction. CD22 is a cell surface marker expressed by the vast majority of normal and neoplastic B-cells. ADCT-602 is an antibody drug conjugate (ADC) composed of Emab-C220, an engineered version of the anti-CD22 humanized IgG1 antibody epratuzumab, site-specifically conjugated to SG3249, which includes the DNA minor groove crosslinking pyrrolobenzodiazepine (PBD) dimer SG3199 linked to the antibody via a protease-cleavable linker (Zammarchi et al, ASH 2016). ADCT-602 is currently being tested in a phase I/II clinical trial (NCT03698552) in recurrent or refractory B-cell acute lymphoblastic leukemia (B-ALL) patients. Here, we assessed its in vitro anti-lymphoma activity, also exploring for potential biomarkers and mechanisms of resistance. Methods. Fifty-seven human lymphoma cell lines were exposed to ADCT-602, an isotype-control ADC and the PBD dimer SG3199 as single agents for 96h, followed by MTT proliferation assay and IC50 calculation. Quantum Simply Cellular (QSC) microspheres were used for the quantitative determination of cellular CD22 antigen expression (Bangs Laboratories. Inc). Differences in IC50 values among lymphoma subtypes were calculated using the Wilcoxon rank-sum test. Statistical significance was defined by P values of 0.05 or less. Sensitivity analysis to ADCT-602 was performed by integrating different omics data, such as Illumina HT-12 microarray data (GSE94669), HTG EdgeSeq Oncology Biomarker Panel data (GSE103934) and DNA copy number variations. Results. The median IC50 for ADCT-602 was 200 pM (95%C.I, 90-400 pM) in 48 B-cell lymphoma lines (including three Hodgkin lymphoma cell lines), and 1850 pM in nine T-cell lymphoma lines (95%C.I, 700-15000 pM). ADCT-602 was more active in B- than in T-cell lymphomas, as expected based on the pattern of CD22 expression (P &lt; 0.005). Focusing on B-cell lymphomas, ADCT-602 in vitro activity was not correlated with its target expression measured both at the cell surface protein level (absolute quantitation, n=48, r=0.06 P=ns) and at the RNA level (Illumina HT-12 arrays, n=42, r=0.28, P=ns; HTG EdgeSeq Oncology Biomarker Panel, n=36, r=0.24, P=ns). In vitro activity was stronger in marginal zone lymphoma (MZL) cell lines than other B-cell lymphoma models (median IC50s 62.5 vs 312.5 pM; P = 0.03) as well as in diffuse large B-cell lymphoma (DLBCL) cell lines with BCL2 and MYC translocations (DHT DLBCL) versus DLBCL with none or a single translocation (median IC50s 25 vs 400 pM, P = 0.03). No associations were seen with TP53 status or other histology (mantle cell lymphoma, DLBCL, DLBCL cell of origin). We then exploited the gene expression profiling data using the Illumina HT-12 microarray gene expression technology. Within all the B-cell lymphoma cell lines (sensitive, n= 25; resistant, n= 23) we identified 1.207 genes down-regulated (FC-) and 1,104 genes up-regulated (FC+) in resistant cell lines. To delineate the pathways associated with the different degrees of sensitivity to ADCT-602, we performed a gene set enrichment analysis (GSEA; GSEA hallmarks and c2.common pathways) on the pre-ranked limma data. Transcripts up-regulated in resistant cell lines were enriched of genes coding for proteins involved in respiratory electron transport, oxidative phosphorylation and proteasome. Conversely, transcripts up-regulated in the sensitive cell lines were enriched of genes coding for proteins involved in inflammation, chemokine signaling, p53 response, IL2/STAT5 signaling, hypoxia, TGF-beta and interferon response. Similar gene signatures were picked up using the HTG platform, which can be applied to formalin-fixed paraffin-embedded clinical specimens, despite the smaller number of investigated genes. Conclusion. ADCT-602 showed in vitro anti-tumor activity across a large panel of B-cell lymphoma models of various histology. Expression signatures and other features (MZL or DHT DLBCL histology), but not the expression levels of its target, were associated with different sensitivity to the ADC. Our data supports the clinical evaluation of ADCT-602 in patients with B-cell lymphoma in addition to B-ALL. Disclosures Zucca: Kite: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Beigene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Other: Travel Grants; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel Grants, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Research Funding; Celltrion Healthcare: Membership on an entity's Board of Directors or advisory committees. Stathis:PharmaMar: Other: Travel Grant; Member of the steering committee of the trial of this abstract: Other; Loxo: Honoraria, Other, Research Funding; Cellestia: Research Funding; Roche: Other, Research Funding; Novartis: Other, Research Funding; Bayer: Other, Research Funding; Merck: Other, Research Funding; Pfizer: Other, Research Funding; MEI Pharma: Other, Research Funding; ADC Therapeutcis: Other, Research Funding; Abbvie: Other: Travel Grant. Van Berkel:ADC-Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zammarchi:ADC-Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bertoni:ADC-Therapeutics: Research Funding; Bayer AG: Research Funding; Helsinn: Research Funding; Menarini Ricerche: Consultancy, Research Funding; NEOMED Therapeutics 1: Research Funding; Nordic Nanovector ASA: Research Funding; Astra Zeneca: Other: travel grant; Amgen: Other: travel grant.

Role of TORC1 and TORC2 in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1815-1815
Author(s):  
Patricia Maiso ◽  
Yi Liu ◽  
Abdel Kareem Azab ◽  
Brittany Morgan ◽  
Feda Azab ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Cell Lines ◽  
Stromal Cells ◽  
Research Funding ◽  
Plasma Cells ◽  
Advisory Committees ◽  
Cycle Arrest

Abstract Abstract 1815 Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 (Raptor) and TORC2 (Rictor). TORC1 leads to the phosphorylation of p70S6 kinase and 4E- BP1, while TORC2 regulates phosphorylation of Akt and other kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin and its analogues have not shown significant activity in MM, likely due to the lack of inhibition of TORC2. In this study, we dissected the baseline activity of the PI3K/Akt/mTOR pathway TORC1/2 in MM cell lines with different genetic abnormalities. Methods: Eight different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, immunochemistry, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. In vivo homing was checked by in vivo flow. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: Raptor (TORC1) and Rictor (TORC2) knockdowns led to significant inhibition of proliferation of MM cells even in the presence of bone marrow stromal cells, this effect was also accompanied by inactivation of p-Akt, p-rS6 and p-4EBP1. We used INK128, a dual and selective TORC1/2 kinase inhibitor with similar effects to Raptor plus Rictor knockdown. We examined the protein expression levels of both mTOR complex and their downstream effectors in MM plasma cells from patients and cell lines. mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all samples. We showed that dual TORC1/2 inhibition is much more active than TORC1 inhibition alone (rapamycin) even in the presence of cytokines or stromal cells. INK128 induced cell cycle arrest, autophagy and apoptosis in cell lines and primary plasma cells even in the presence of bone marrow stromal cells (BMSCs). INK128 also showed a significant effect inhibiting cell adhesion in our in vivo homing model. Oral daily treatment with INK128 highly decreased the percentage of CD138+ tumor plasma cells in mice implanted with MM cells and reduced the levels of p-Akt and p-4EBP. These results suggest that potent and complete blockade of mTOR as part of TORC1 and TORC2 is potential therapeutic strategy to induce cell cycle arrest, apoptosis and disruption of MM cells interaction with the BM microenvironment. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Liu: Intellikine: Employment. Roccaro:Roche: Research Funding. Rommel:Intellikine: Employment. Ghobrial:Celgene: Consultancy; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Genetic Factors Predicting The Response To BET Bromodomain Inhibitors In Lymphoma Lead To New Synergistic Combinations

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3070-3070
Author(s):  
Eugenio Gaudio ◽  
Ivo Kwee ◽  
Andrea Rinaldi ◽  
Michela Boi ◽  
Elena Bernasconi ◽  
...  
Keyword(s):  
Dna Repair ◽  
Glucose Metabolism ◽  
B Cell ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Target Genes ◽  
Cell Lymphoma ◽  
Targeted Agents ◽  
Advisory Committees

Abstract Epigenome deregulation in cancer cells affects transcription of oncogenes and tumor suppressor genes. BET Bromodomain proteins recognize chromatin modifications and act as epigenetic readers contributing to gene transcription. BET Bromodomain inhibitors showed promising pre-clinical activity in hematological and solid tumors and are currently in phase I studies. The mechanism of action and relevant affected genes are not fully characterized and there are no established response predictors. We have shown activity of BET Bromodomain OTX015 in lymphoma cell lines (ASH 2012; ICML 2013). This study aimed at elucidating pathways and genes affecting response/resistance to BET Bromodomain inhibitors in lymphomas. Methods Baseline gene expression profiles (GEP) were obtained in 38 cell lines [22 diffuse large B-cell lymphoma (DLBCL), 8 anaplastic large T-cell lymphoma, 4 mantle cell lymphoma, 3 splenic marginal zone lymphoma, 1 chronic lymphocytic leukemia] with Illumina HumanHT-12 v4 Expression BeadChip. Genetic and biologic information were collected from literature. GEP/IC50 correlation (ASH 2012; ICML 2013) was assessed by Pearson correlation. Associations in two-way tables were tested for statistical significance using either chi-square or Fisher exact test, as appropriate. Differential expression analysis was performed using LIMMA, followed by multiple test correction using the BH method. Enrichment of functionally-related genes was evaluated by GSEA. For combination studies, 3 germinal center B-cell (GCB) and 2 activated B-cell (ABC) DLBCL were exposed to increasing doses of OTX015 alone or in combination with increasing doses of targeted agents for 72 hours, followed by MTT assay. Synergy was assessed by Chou-Talalay combination index (CI) with Synergy R package. Results Transcripts associated with resistance to OTX015 were significantly enriched of genes involved in cell cycle regulation, DNA repair, chromatin structure, early B-cell development, E2F/E2F2 target genes, IL6-dependent genes, and mRNA processing. Conversely, transcripts associated with OTX015 sensitivity were enriched of hypoxia-regulated genes, interferon target genes, STAT3 targets, and involved in glucose metabolism. Genes associated with OTX015 sensitivity included LDHA, PGK1 (glucose metabolism) and VEGFA (hypoxia), while BCL2L1/BCLXL, BIRC5/survivin (anti-apoptosis), ERCC1 (DNA repair), TAF1A and BRD7 (transcription regulation) were correlated with reduced sensitivity. GEP identified 50 transcripts differentially expressed, including IL6, HCK, SGK1, MARCH1 and TRAFD1, between cells undergoing or not apoptosis after OTX015 exposure. GSEA showed significant enrichment of genes involved in IL-10 signaling pathway. While there was no association between response to OTX015<500nM and presence of translocated MYC, analysis of genetic and biologic features identified the ABC phenotype (P=.008) and presence of concomitant somatic mutations in MYD88 and CD79B or CARD11 genes and wild type TP53 (P=.027) as associated with apoptosis. Based on these observations and since mutated MYD88 interacts with BTK and MYD88/CD79B mutations have been associated with clinical responses with the BTK inhibitor ibrutinib, we evaluated OTX015 combination with this compound. Synergy was observed in particular in ABC-DLBCL with a median CI of .04 (range .02-.1). The demonstrated down-regulation of the MYD88/JAK/STAT pathway after OTX015 treatment, as shown by additional GEP, highlighted the importance of this pathway for OTX015 activity. Other targeted agents (everolimus, lenalidomide, rituximab, decitabine, vorinostat) appeared to synergize with OTX015 (Fig 1). The mTOR inhibitor everolimus presented a very strong synergism with a median CI of .11 (.1-.2), in accordance with the association between OTX015 sensitivity to high glucose metabolism and high levels of SGK1 in cells undergoing apoptosis. Conclusions Our study identified genetic mechanisms contributing to the response to BET Bromodomain inhibitors and promising combination schemes, such as OTX015/everolimus, to be further investigated. Disclosures: Stathis: Oncoethix: NCT01713582 PI Other. Herait:Oncoethix: Membership on an entity’s Board of Directors or advisory committees. Noel:Oncoethix: Membership on an entity’s Board of Directors or advisory committees. Inghirami:Oncoethix: Research Funding. Bertoni:Oncoethix: Research Funding.

BI 836826, a Novel Fc-Engineered Antibody in Combination with Phosphoinositide-3-Kinase Inhibitor for Treatment of High Risk Chronic Lymphocytic Leukemia and Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2767-2767
Author(s):  
Deborah M Stephens ◽  
Kyle A. Beckwith ◽  
Priscilla Do ◽  
Carolyn Cheney ◽  
Xiaokui Mo ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Viability ◽  
B Cell ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Single Agent ◽  
Annexin V ◽  
Lymphocytic Leukemia ◽  
Advisory Committees

Abstract Background Targeting new antigens in chronic lymphocytic leukemia (CLL) and lymphoma may increase flexibility in the clinic and help circumvent resistance. The tetraspanin CD37 domain mediates transduction of survival and apoptotic signals (Lapalombella et al.,Cancer Cell, 2014), and has been clinically validated by recent trials of otlertuzumab (TRU-016) in CLL and Non-Hodgkin Lymphoma . Ligation of CD37 by this reagent simultaneously induced pro-apoptotic signaling and inhibited pro-survival signaling of phosphoinositide 3-kinase δ (PI3Kδ), which introduces a unique opportunity to use combination strategies employing activation of CD37 and inhibition of PI3Kδ. A new agent BI 836826 is an Fc-engineered anti-CD37 IgG1 that displays improved effector activities as well as crosslinker-independent direct cytotoxicity. We have evaluated the efficacy of BI 836826 combined with the PI3Kδ-selective inhibitor idelalisib in diffuse large B-cell lymphoma (DLBCL) cell lines and primary human CLL B-cells in the University and then by industry to validate the synergistic finding initially reported. Methods Cell viability assays usedCellTiterGlo to measure inhibition of antibody, isotype control, idelalisib or a combination of antibody and compound over 72h in culture. The cell viability of vehicle is measured at the time of dosing (T0) and after seventy-two hours (T72). A GI reading of 0% represents no growth inhibition, GI 100% represents complete growth inhibition, and a GI 200% represents complete death of all cells in the culture well. Annexin V-FITC and propidium iodide measure by flow cytometry was used to assess enhanced killing of primary CLL cells, with incubation of BI 836826 (0.1 µg/mL) and/or idelalisib (1 µM) at 37°C for 24 hours. Trastuzumab included as a non-specific IgG1 control. Data was reported as percentage of viable cells (Annexin V negative, PI negative) normalized to untreated control. Results DLBCL cell lines were variably sensitive to single agent BI 836826. In most of the cell lines tested, the cell viability was inhibited by 40%-50% with BI 836826 in the concentration range of 1-1000 ng/mL (Figure 1A). A synergistic effect was noted in several DLBCL cell lines when BI 836826 was combined with idelalisib. When the maximal effect of BI 836826 was greater than isotype control (GI% > 12, dotted line) and the effect of idelalisib showed a GI50 < 1uM, 3/5 cell lines showed synergy in combination (red dot, Figure 1B). A shift in the EC50of idelalisib can be seen with the addition of increasing amounts of BI 836826 (Figure 1C). In primary CLL B-cell cultures, 1 µM idelalisib displayed weak single agent activity following 24-hour incubation. The cytotoxicity of BI 836826 at 0.1 µg/mL was more variable, although treatment of samples from most CLL patients resulted in 20-50% B-cell death. The combination of these 2 agents resulted in enhanced cytotoxic activity (Figure 2A), and this effect was not attenuated by the presence of del(17)(p13.1), as there was no significant difference in cytotoxicity against these cells compared to those with lower risk cytogenetics (Figure 2B,C). Additionally, the combination was beneficial in CLL B-cells isolated from patients who were refractory to ibrutinib (Figure 2D). Conclusions This collaborative industry and academic endeavor with cross validation of initial mechanistic studies of synergy between CD37 and idelalisib demonstrates that addition of idelalisib to BI 836826 augments cytotoxicity against DLBCL cell lines and primary human CLL B-cells in an additive-to-synergistic manner. In addition, it maintains efficacy against CLL B-cells with del(17)(p13.1) and those from ibrutinib-refractory patients. Further exploration of this therapeutic strategy in clinical trials is strongly warranted. Disclosures Jones: AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Awan:Innate Pharma: Research Funding; Pharmacyclics: Consultancy; Novartis Oncology: Consultancy. Grosmaire:Gilead: Employment. Jones:Gilead: Employment. DiPaolo:Gilead: Employment. Tannheimer:Gilead Sciences: Employment. Heider:4Boehringer Ingelheim RCV: Employment.

Sign in / Sign up

Export Citation Format

Share Document