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

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.

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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 ◽

10.1182/blood-2021-152121 ◽

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.

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

Blood ◽

10.1182/blood.v122.21.3070.3070 ◽

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.

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High Levels of APOBEC3B Gene Expression Contribute to Poor Prognosis in Multiple Myeloma Patients

Blood ◽

10.1182/blood-2018-99-117067 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3897-3897

Author(s):

Valeriy V Lyzogubov ◽

Pingping Qu ◽

Cody Ashby ◽

Adam Rosenthal ◽

Antje Hoering ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Board Of Directors ◽

Research Funding ◽

Poor Prognosis ◽

Target Genes ◽

Plasma Cells ◽

Nuclear Fraction ◽

Advisory Committees ◽

Significant Difference

Abstract Introduction: Poor prognosis and drug resistance in multiple myeloma (MM) is associated with increased mutational load. APOBEC3B is a major contributor to mutagenesis, especially in myeloma patients with t(14;16) MAF subgroup. It was shown recently that presence of the APOBEC signature at diagnosis is an independent prognostic factor for progression free survival (PFS) and overall survival (OS). We hypothesized that high levels of APOBEC3B gene expression at diagnosis may also have a prognostic impact in myeloma. To consider APOBEC3B as a potential target for therapy more studies are necessary to understand how APOBEC3B expression is regulated and how APOBEC3B generates mutations. Methods: Gene expression profiling (GEP, U133 Plus 2.0) of MM patients was performed. APOBEC3B gene expression levels were investigated in plasma cells of healthy donors (HD; n=34), MGUS (n=154), smoldering myeloma (SMM; n=219), MM low risk (LR; n=739), MM high risk (HR; n=129), relapsed MM (RMM; n=74), and primary plasma cell leukemia (pPCL; n=19) samples. The samples from relapse were taken on or after the progression/relapse date but within 30 days after progression/relapse from Total Therapy trials 3, 4, 5 & 6. GEP70 score was used to separate samples into LR and HR groups. We also investigated APOBEC3B expression in different MM molecular subgroups and used logrank statistics with covariate frequency distribution to determine an optimal cut off APOBEC3B expression value. Gene expression was compared in cases with low expression of APOBEC3B (log2<7.5) and high expression of APOBEC3B (log2>10), and an optimal cut-point in APOBEC3B expression was identified with respect to PFS. To explore the role of MAF and the non-canonical NF-ĸB pathway we performed functional studies using a cellular model of MAF downregulation. TRIPZ lentiviral shRNA MAF knockdown in the RPMI8226 cell lines was used to explore MAF-dependent genes. NF-ĸB proteins, p52 and RelB, were investigated in the nuclear fraction by immunoblot analysis. Results: Expression of APOBEC3B in HD control samples (log2=10.9) was surprisingly higher than in MGUS (log2=9.51), SMM (log2=9.09), and LR (log2=9.40) and was comparable to HR (log2=10.4) and RMM (log2=10.6) groups. Expression levels of APOBEC3B were gradually increased as disease progressed from SMM to pPCL. The high expression of APOBEC3B in HD places plasma cells at risk of APOBEC induced mutagenesis where the regulation of APOBEC3B function is compromised. The correlation between APOBEC3B expression and GEP70 score in MM was 0.37, and there was a significant difference in APOBEC3B expression between GEP70 high and low risk groups (p=0.0003). An optimal cut-point in APOBEC3B expression of log2=10.2 resulted in a significant difference in PFS (median 5.7 yr vs.7.4 yr; p=0.0086) and OS (median 9.1 yr vs. not reached; p<0.0001), between high and low expression. The highest APOBEC3B expression was detected in cases with a t(14;16). We analyzed t(14;16) cases with the APOBEC mutational signature and compared them to t(14;16) cases without the APOBEC signature and found elevated MAF (2-fold) and APOBEC3B (2.7-fold) gene expression in samples with the APOBEC signature. No APOBEC signature was detected in SMM cases, including those with a t(14;16). High APOBEC3B levels in myeloma patients was associated with overexpression of genes related to response to DNA damage and cell cycle control. Significant (p<0.05) increases of NF-κB target genes was seen in high APOBEC3B cases: TNFAIP3 (4.4-fold), NFKB2 (1.7-fold), NFKBIE (1.9-fold), RELB (1.4-fold), NFKBIA (2.0-fold), PLEK (2.5-fold), MALT1 (2.5-fold), WNT10A (2.4-fold). However, in t(14;16) cases there was no significant increase of NF-κB target genes except BIRC3 (2.5-fold) and MALT1 (2.0-fold). MAF downregulation in RPMI8226 cells did not lead to changes in NF-κB target gene expression but MAF-dependent genes were identified, including ETS1, SPP1, RUNX2, HGF, IGFBP2 and IGFBP3. Analysis of nuclear fraction of NF-ĸB proteins did not show significant changes in expression of p52 and RelB in RPMI8226 cells after MAF downregulation. Conclusions: Increased expression of APOBEC3B is a negative prognostic factor in multiple myeloma. MAF is a major factor regulating expression of APOBEC3B in the t(14;16) subgroup. NF-ĸB pathway activation is most likely involved in upregulation of APOBEC3B in non-t(14;16) subgroups. Disclosures Davies: TRM Oncology: Honoraria; MMRF: Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; ASH: Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy. Morgan:Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding; Takeda: Consultancy, Honoraria.

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

Blood ◽

10.1182/blood-2019-127332 ◽

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.

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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 ◽

10.1182/blood.v126.23.4440.4440 ◽

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.

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FLT3 and LSD1 Inhibitor Combinations Synergistically Repress Growth of FLT3-Mutant Acute Myeloid Leukemia Via Blockage of MYC Function

Blood ◽

10.1182/blood-2020-143029 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 31-32

Author(s):

Daniel J. Coleman ◽

Brittany M. Smith ◽

Cody Coblentz ◽

Rowan L. Callahan ◽

Jake VanCampen ◽

...

Keyword(s):

Transcription Factor ◽

Board Of Directors ◽

Transcriptional Activation ◽

Myeloid Leukemia ◽

Research Funding ◽

Target Genes ◽

Publicly Traded ◽

Private Company ◽

Advisory Committees ◽

Flt3 Inhibitor

Internal Tandem Duplication mutations of Fms Related Receptor Tyrosine Kinase 3 (FLT3), known as FLT3-ITD mutations, are associated with poor prognosis in Acute Myeloid Leukemia (AML). The clinical efficacy of inhibiting FLT3 in AML is limited by the rapid development of drug resistance and relapse, underscoring a need for more potent and durable treatment strategies. The early persistence of leukemic blasts during FLT3 inhibition is a key driver of resistance. We find that in combination, inhibitors of Lysine Specific Demethylase 1 (LSD1) potentiate the activity of FLT3 inhibitors, driving synergistic cell death. This novel therapeutic approach has the potential to drive deeper therapeutic responses in FLT3-Mutant AML, delaying or preventing the development of resistance. LSD1 is a dynamic DNA-associated protein that functions as a chromatin modifier and transcription factor. LSD1 removes methylation on both lysine 4 of histone H3 (H3K4), associated with transcriptional activation, and lysine 9 (H3K9), associated with transcriptional repression. Additionally, LSD1 has been reported to function as a transcription factor independent of its catalytic demethylase function. LSD1 inhibition reduces cell proliferation in several cancer types. In AML specifically, inhibition of LSD1 has been reported to activate enhancers associated with genes that promote differentiation. We hypothesized that combining LSD1 inhibition with FLT3 inhibition in FLT3-ITD AML would result in synergistic effects on cell viability through reactivating differentiation pathways and more strongly blocking proliferation. In this study, we aimed to examine the efficacy, transcriptional effects, and changes in chromatin dynamics when combining LSD1 inhibition with FLT3 inhibition in a FLT3-ITD mutant cell line and patient samples. We used matrix combination screening to determine that combining the FLT3 inhibitor Quizartinib with LSD1 inhibitors (GSK-2879552 or ORY-1001) synergistically represses cell viability in the FLT3-ITD mutant MOLM-13 cell line and in multiple primary AML samples. RNA-seq followed by Gene Set Enrichment Analysis revealed that combining LSD1 and FLT3 inhibition synergistically represses target genes of the oncogenic transcription factor MYC. This finding was corroborated through high-throughput genome-wide profiling of histone marks, using the recently developed technique Cleavage Under Targets and Tagmentation (CUT&Tag). Specifically, we discovered several promoter regions in which acetylation of lysine 27 of Histone H3 (H3K27Ac), associated with transcriptional activation, was repressed by combining LSD1 and FLT3 inhibition. The genes associated with these regions were strongly enriched for known MYC target genes. Through additional genomic profiling methods including ChIP-seq and ATAC-seq, we have established potential roles for several DNA-binding transcription factors including CEBPA, RUNX1, STAT5, and LSD1 itself, that may mediate repression of MYC function resulting from combining LSD1 and FLT3 inhibition. Together, our work establishes LSD1 and FLT3 inhibitor combinations as a promising treatment strategy in FLT3-ITD AML. Importantly, this study identifies combined FLT3 and LSD1 inhibition as an effective strategy to indirectly target MYC function, as MYC is often referred to as an "undruggable" target. Furthermore, it has the potential to drive deeper molecular responses in FLT3-mutant AML, decreasing the likelihood of treatment resistance. Disclosures Druker: Bristol-Myers Squibb: Research Funding; Blueprint Medicines: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; VB Therapeutics: Membership on an entity's Board of Directors or advisory committees; Millipore (formerly Upstate Biotechnology): Patents & Royalties; Pfizer: Research Funding; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Patient True Talks: Consultancy; Oregon Health & Science University: Patents & Royalties; Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; MolecularMD (acquired by ICON): Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Iterion Therapeutics (formerly Beta Cat Pharmaceuticals): Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics Inc. (formerly Lorus): Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Merck & Co: Patents & Royalties; GRAIL: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; Leukemia & Lymphoma Society: Research Funding; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Dana-Farber Cancer Institute: Patents & Royalties; EnLiven: Consultancy, Research Funding. Maxson:Gilead Sciences: Research Funding; Ionis Pharmaceuticals: Other: Joint oversight committee for a collaboration between OHSU and Ionis Pharmaceuticals.

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Descriptive Analysis of Isatuximab Use Following Prior Daratumumab in Patients with Relapsed/Refractory Multiple Myeloma

Blood ◽

10.1182/blood-2020-140526 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 20-21

Author(s):

Melody R Becnel ◽

Sandra B. Horowitz ◽

Sheeba K. Thomas ◽

Swami P. Iyer ◽

Krina K. Patel ◽

...

Keyword(s):

Multiple Myeloma ◽

High Risk ◽

North America ◽

Board Of Directors ◽

Research Funding ◽

Plasma Cells ◽

Laboratory Research ◽

Private Company ◽

Advisory Committees ◽

Advisory Boards

Background: Anti-CD38 monoclonal antibodies (mAb) like daratumumab (dara) have become integral in managing relapsed/refractory (RR) and newly diagnosed (ND) multiple myeloma (MM). Isatuximab (isa), a newer CD38 mAb, induces direct rather than indirect apoptosis of MM cells. However, little is known about whether the use of one prior CD38 mAb will alter the efficacy of another in subsequent lines of therapy. Methods: All patients (pts) with MM treated at MD Anderson with isa after receiving dara in prior lines of therapy were identified. We conducted a retrospective analysis with data points including patient and disease characteristics, responses to dara, response to isa, the presence of high risk features, and the presence of t(11,14). Results: 9 pts were identified, ages 56-72. 5 pts (55%) were male. 5 pts (55%) were alive at the time of data cutoff. 5 pts were Hispanic, 3 White, and 1 Black. 8 pts (89%) had high risk features as represented by the presence of del17p, t(4,14), t(14,16), t(14,20), p53 mutations, gain 1q, extramedullary disease (EMD), CNS disease, early relapse (within 1 year) after autologous transplant, or an increased (>5%) peripheral blood plasma cells (PBPC). 2 (22%) had t(11,14). 4 (44%) had IgG MM. 2 (22%) with light chain disease, 2 (22%) with IgA MM, and 1 (11%) with IgD MM. Dara was initially used in lines 2-7. Dara combinations with pomalidomide (pom), bortezomib (bor), thalidomide (thal), lenalidomide (len), or carfilzomib (car); and pom combinations that also included elotuzumab (elo) or Cytoxan (cytox) are noted in table 1. Dara was discontinued (dc'd) in 8 pts due to progressive disease (PD) and in 1 pt due to toxicity. 8 pts (89%) experienced a best overall response (ORR) of partial response (PR) to dara; 1 pt had stable disease (SD). All pts received prior len and 8 pts received prior pom at some time during the treatment of MM. All pts received isa in combination with pom/dexamethasone (dex). Best ORR to isa/pom/dex: 5 pt (55%) had PR, 2 pt with minimal response (MR), 1 SD, 1 PD. Median treatment duration of isa/pom/dex was 5 weeks (2-14 weeks) at data cutoff. 3 pts dc'd isa/pom/dex due to infections, and 2 due to later progression. 2 pts remain on therapy. 1 pt chose to dc all MM therapy for quality of life purposes despite PR with isa/pom/dex. 1 pt died from cardiac disease unrelated to MM or treatment. Conclusions: Our current study of heavily pretreated pts with RRMM demonstrates that despite prior anti-CD38 therapy with dara, most patients (77%) experienced a response of MR or better with treatment with another anti-CD38 therapy isa. To our knowledge, this is the first report of outcomes to isa in patients with prior dara therapy. Further long term follow up will be needed to determine the length of response. Additional studies are planned to further evaluate this patient population. Table 1 Disclosures Thomas: Pharmacyclics: Other: Advisory Boards; BMS: Research Funding; Ascentage: Membership on an entity's Board of Directors or advisory committees, Research Funding; X4 Pharma: Research Funding; Xencor: Research Funding; Genentech: Research Funding. Iyer:Rhizen: Research Funding; CRISPR: Research Funding; Spectrum: Research Funding; Merck: Research Funding; Curio Biosciences: Honoraria; Target Oncology: Honoraria; Afffimed: Research Funding; Daiichi Sankyo: Consultancy; Legend Biotech: Consultancy; Trillium: Research Funding; Seattle Genetics, Inc.: Research Funding. Patel:Celgene: Consultancy, Research Funding; Cellectis: Research Funding; Nektar: Consultancy, Research Funding; Oncopeptides: Consultancy; Poseida: Research Funding; Precision Biosciences: Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding. Manasanch:Adaptive Biotechnologies: Honoraria; GSK: Honoraria; Sanofi: Honoraria; BMS: Honoraria; Takeda: Honoraria; Quest Diagnostics: Research Funding; Merck: Research Funding; JW Pharma: Research Funding; Novartis: Research Funding; Sanofi: Research Funding. Kaufman:Janssen: Research Funding; Bristol Myers Squibb: Research Funding; Karyopharm: Honoraria. Lee:Genentech: Consultancy; GlaxoSmithKline: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Sanofi: Consultancy; Daiichi Sankyo: Research Funding; Regeneron: Research Funding; Genentech: Consultancy. Orlowski:Sanofi-Aventis, Servier, Takeda Pharmaceuticals North America, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen, Inc., AstraZeneca, BMS, Celgene, EcoR1 Capital LLC, Forma Therapeutics, Genzyme, GSK Biologicals, Ionis Pharmaceuticals, Inc., Janssen Biotech, Juno Therapeutics, Kite Pharma, Legend Biotech USA, Molecular Partners, Regeneron Pharmaceuticals, Inc.,: Honoraria, Membership on an entity's Board of Directors or advisory committees; STATinMED Research: Consultancy; Founder of Asylia Therapeutics, Inc., with associated patents and an equity interest, though this technology does not bear on the current submission.: Current equity holder in private company, Patents & Royalties; Laboratory research funding from BioTheryX, and clinical research funding from CARsgen Therapeutics, Celgene, Exelixis, Janssen Biotech, Sanofi-Aventis, Takeda Pharmaceuticals North America, Inc.: Research Funding.

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In Vitro and inVivo Activity of Melflufen in Amyloidosis

Blood ◽

10.1182/blood-2019-125952 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3100-3100 ◽

Cited By ~ 2

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.

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Activity of Carfilzomib (CFZ) in Acute Myeloid Leukemia (AML) As a Single Agent and in Novel Combinations

Blood ◽

10.1182/blood-2020-141459 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 6-7

Author(s):

Mao Yu Peng ◽

Yasmin Abaza ◽

Martina Mcdermott ◽

Monica Mead ◽

Dennis J. Slamon ◽

...

Keyword(s):

Cell Cycle ◽

Synergistic Effect ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Fatty Acid Synthase ◽

Single Agent ◽

Therapeutic Strategies ◽

Private Company ◽

Advisory Committees

Background:Recent advances in targeted therapy have expanded the available therapeutic optionsfor patients with AML. However, many patients still have suboptimal outcomes, particularly in the relapsed/refractory setting, underscoring the need for novel therapeutic strategies. Proteasome inhibitors (PIs), such as bortezomib, exhibit antitumor activity in AML through inhibition of the nuclear factor κB pathway and induction of apoptosis. CFZ, a second-generation PI, has preferential preclinical activity in AML compared to bortezomib making it an agent of interest in AML therapy. Here we assessed the activity of CFZ as a single agent and in novel combinations with Ara-C and/or other agents targeting potential vulnerabilities in AML cell lines. Methods:20 AML cell lines were treated with a single dose of CFZ for 7 days, proliferation inhibition was measured using an IC50 cutoff for CFZ of 10 nM. 2 sensitive (ML2 and MV411) and 2 resistant (AML193 and NOMO1) cell lines were selected for further analysis. Apoptosis, cell cycle, and cell senescence analysis were performed after 72 hours of CFZ exposure at 10 nM. Combination assays using CFZ 10 nM and Ara-C 200 nM were performed to evaluate for potential interaction in the form of antagonism or potentiation. Proteomic analysis was performed at baseline using reverse phase protein assay (RPPA). Cell lines were aligned according to CFZ IC50. Several proteins involved in various physiological pathways exhibited a potential correlation with CFZ sensitivity. Combination treatments with CFZ and agents targeting these pathways were carried out in selected cell lines. Results:Single-agent CFZ induced apoptosis with apoptotic rates >85% in sensitive cell lines and only 10% in resistant cell lines. Similarly, CFZ resulted in G0/G1 cell cycle arrest in sensitive, but not resistant AML cell lines. Lack of difference in cellular senescence confirmed apoptosis as the major mechanism of CFZ-induced growth inhibition in AML cell lines. No antagonism was noted when CFZ was combined with Ara-C. RPPA revealed that AML cell lines with higher expression of autophagy-related proteins (Atgs) were more resistant to CFZ treatment. Combining autophagy inhibitor hydroxychloroquine (HCQ) or ROC-325 with CFZ produced a synergistic effect to induce apoptosis in several CFZresistant cell lines. RPPA also revealed that lower basal levels of fatty acid synthase (FASN), a key enzyme involved in lipogenesis, correlated with CFZ sensitivity and CFZ resistant lines tendedto have higher basal FASN levels. The combination of CFZ with a FASN inhibitor resulted in a significant synergistic apoptosis-inducing effect that was observed in the AML lines tested. Conclusion:CFZ demonstrated single agent activity in the nanomolar range in human AML cell lines. The addition of standard-of -care chemotherapy to CFZ did not show antagonism. Combining CFZ with agents targeting autophagy or lipid-metabolism showed synergistic effect in apoptosis. These results suggest a role for CFZ in combination therapeutic strategies for AML patients. Disclosures Mcdermott: TORL Biotherapeutics:Current equity holder in private company;1200 Pharma:Current equity holder in private company.Slamon:TORL Biotherapeutics:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees;1200 Pharma:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees;Novartis:Consultancy, Research Funding;Eli Lilly:Consultancy;Bayer:Consultancy, Research Funding;Pfizer:Consultancy, Other: stock, Research Funding;Syndax:Research Funding;Aileron:Research Funding;Genetech:Research Funding;Biomarin:Membership on an entity's Board of Directors or advisory committees;Seattle Genetics:Other: Stock;Amgen:Other: Stock.Larson:BMS, Bioline, Celgene, Juno, Janssen:Research Funding;TORL Biotherapeutics:Current equity holder in private company.

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

Blood ◽

10.1182/blood-2018-99-115348 ◽

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.

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