scholarly journals The Spliceosome As a New Therapeutic Target in Cytarabine-Resistant Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3334-3334
Author(s):  
María Luz Morales ◽  
Roberto Garcia-Vicente ◽  
Alba Rodríguez García ◽  
Noemí Álvarez Sánchez-Redondo ◽  
Alejandra Ortiz-Ruiz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Board Of Directors ◽  
Rna Splicing ◽  
Ex Vivo ◽  
Sr Proteins ◽  
Splicing Factors ◽  
Advisory Committees ◽  
Paired Samples

Abstract Introduction. Despite the recent approval of several drugs for the treatment of AML, the 3 + 7 regimens remain as the standard of care for many patients. Its lack of efficacy represents the main cause of death, since only 10% of patients who show refractoriness/relapse overcome the disease. Therefore, there is still an urgent need for seeking more effective treatments. Aberrant RNA splicing has been described in AML, but its relevance as mechanism of resistance is unclear. In this study, we deepen the mechanism of resistance to cytarabine and the role of splicing factors SR proteins, involved in the spliceosome functionality, to seek more effective therapies for AML. Methods. First, the expression levels of genes encoding SR proteins were analyzed with the GEPIA2 platform, comparing the data from the TCGA-LAML (AML patients) and GTEx (healthy) projects. Then, the gene expression of one of the most overexpressed genes, SRRM2, was validated by qPCR in samples of AML patients compared to controls and other myeloid disorders, as MDS and MPN (n=54). The resistance-associated phospho-proteomic profile was analyzed by LC-MS / MS after IMAC enrichment in paired samples from 3 AML patients. The expression of SR proteins and their phosphorylated forms was studied by immunohistochemistry (IHC) before and after resistance in paired bone marrow samples from 3 AML patients. We also analyzed by IHC the prognostic value of phospho-SR proteins at the moment of diagnosis in 64 patients with different responses to cytarabine (non-responders and responders). In order to validate an altered function of SR proteins, the analysis of the differential use of exons of paired samples from 25 AML patients was performed using RNAseq. Then, we evaluated in vitro the efficacy of some splicing modulators, and its combination with other approved drugs, in cytarabine-sensitive and resistant cells. The combination of H3B-8800, a spliceosome inhibitor, with venetoclax was tested in ex vivo samples from AML patients and healthy donors. Results. We found that the gene expression levels of SRSF9, SRSF12 and SRRM2 were altered in AML (Fig 1A-B). Immunohistochemical studies revealed that, although at the protein level no differences were found in SR proteins expression between the diagnosis and relapse moment, an increase in the levels of phosphorylated SR proteins was associated at the time of relapse (Fig 1C). Indeed, the phosphorylation levels of SRRM2, among other SR proteins, were found to be increased during cytarabine resistance by phospho-proteomics (Fig 1D). Moreover, the phosphorylation levels of SR proteins predicted the response to cytarabine treatment, as AML patients that were non-responders presented significantly higher levels compared to responders ones (Fig 1E). The observed alterations in the phosphorylation of these proteins were correlated with a differential use of exons in some of their known targets, when comparing the diagnostic condition and drug resistance moment. Based on this evidence, the efficacy of combining different therapeutic options was evaluated in vitro using sensitive or cytarabine-resistant cell models (Fig 1F). The combination of H3B-8800 together with venetoclax was the most effective in vitro and also presented synergic effects ex vivo in AML patients samples (Fig 1G). Furthermore, this combination did not show toxicity over healthy hematopoietic progenitors, since the same doses that were effective in AML did not show toxicity in a healthy context (Fig 1H). Conclusions. The results of this work shed light on the role of the RNA splicing process in cytarabine resistance in AML. Interestingly, the high levels of phosphorylated splicing factors SR proteins at diagnosis in refractory patients, would allow us to use them as a predictive biomarker of response to cytarabine treatment. Otherwise, due to the need to search effective and safe treatments in this disease, we have found that the combination of splicing inhibitors with venetoclax should be a good strategy for the treatment of AML. Acknowledgment. This work has been possible thanks to the granting of the project PI19/01518 from the Carlos III Health Institute and the CRIS Against Cancer Foundation. ML.M. enjoys a research grant from the Spanish Society of Hematology and Hemotherapy and R.GV. a FPU grant from the Ministry of Science, Innovation and Universities. Figure 1 Figure 1. Disclosures Sanchez: Altum sequencing: Current Employment. Ayala: Incyte Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Celgene: Honoraria.

Deregulation of TNFRSF18 (GITR) Through Promoter CpG Island Methylation Induces Tumor Proliferation in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2424-2424
Author(s):  
Yang Liu ◽  
Yong Zhang ◽  
Phong Quang ◽  
Hai T Ngo ◽  
Feda Azab ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Cpg Island ◽  
Advisory Committees ◽  
Brdu Assay

Abstract Abstract 2424 Introduction Tumor necrosis factor receptor super families (TNFRSFs) play an important role in activation of lymphocyte and cell apoptosis. However the function of TNFRSFs in multiple myeloma (MM) remains unknown. Loss of function mutation of Fas antigen (TNFRSF6) was identified in MM cells, thus suggesting the possible role of TNFRSFs in regulating MM pathogenesis. We therefore investigated the epigenetic mechanisms that may mediate inactivation of TNFRSFs and its functional role in MM. Methods Dchip software was utilized for analyzing gene expression dataset. DNA was extracted from both primary CD138+ MM plasma cells and MM cell lines using blood & tissue DNA isolation kit (Qiagen, Inc.). Expression of GITR in primary CD138+ plasma cells was detected by Imunohistochemistry (IHC) DNA methylation was analyzed by methylated DNA immunoprecipitation (Medip) assay and bisulfate sequencing. 5'azacytidine was used to demethylate genomic DNA. Gene expression was detected by qRT-PCR and confirmed at the protein level by flow cytometry and western-blot. Over-expression of GITR was obtained in MM1.S cells by using GITR recombinant plasmid and electroporation. Apoptosis was determined using Annexin/PI staining and flow cytometry analysis. Activation of apoptotic signaling was studied by western blot. Cell survival and proliferation were analyzed by MTT and BrdU assay, respectively. Recombinant GITR-lentivirus was obtained from the supernatant of culture medium after 72 hours transfection in 293 cells. GFP positive MM cells were sorted and analyzed by flow cytometry. In vivo effect of GITR on MM tumor growth was determined by injection of GITR over-expressing MM cells in null mice. Mice skull, femur and vertebrae were isolated after 4 weeks injection. Anti-human CD138+ mAb microbead was used to detect MM cells extracted from mice tissue by flow cytometry. Results Gene-expression profiling showed down-regulation of TNFRSFs, including TNFRSF11A, TNFRSF11B, TNFRSF8, TNFRSF10C, TNFRSF9, TNFRSF21, TNFRSF1B, TNFRSF1A and TNFRSF18, compared to normal plasma cells. Moreover, Our IHC results also showed that GITR expression was positive in primary CD138+ plasma cells from 9 normal bone marrow, but negative in 9 MM samples. Importantly, we found that low GITR expression significantly correlated with MM progression. Indeed, GITR gene levels were lower in smoldering and active MM patients compared to MGUS patients and normal donors. Promoter CpG island (CGI) methylation of GITR was indentified in 5 out of 7 MM primary bone marrow (BM)-derived CD138+ cells but not in normal BM-derived plasma cells. Bisulfate sequencing and Medip assay showed that methylation of GITR was significantly associated with GITR expression in 5 MM cell lines, including MM1.S, OPM1, U266, RPMI and INA6. Promoter CGI of GITR was highly methylated leading to complete silencing of GITR in MM1.S cell line. GITR expression was significantly up-regulated in MM cells upon treatment with the 5'azacytidine. MTT and BrdU assay revealed that the proliferation and survival of MM1.S cells was disrupted in the GITR over-expressing MM1.S cells, notably with inhibition of cell proliferation compared to control vector infected cells. Moreover induction of cytotoxicity in GITR over-expressing cells was confirmed by using GFP competition assay. GITR-induced apoptosis was supported by induction of caspase 8 and 3 cleavage. The inhibition of human CD138+ plasma cell growth in the bone marrow of SCID mice using a disseminated MM xenograft model was observed in the experimental group injected with GITR expressing cells compared to the control group after 4 weeks injection. Conclusion Our findings uncovered a novel epigenetic mechanism contributing to MM pathogenesis, showing the role of GITR methylation as a key regulator of MM cell survival. Disclosures: Roccaro: Roche:. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; 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.

Evaluation of IGHV Ultra-Deep Sequences for Activation-Induced Deaminase Characteristics in CLL Cells after T Cell Stimulation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2583-2583
Author(s):  
Charles C. Chu ◽  
Piers E.M. Patten ◽  
Thomas MacCarthy ◽  
Xiao-Jie Yan ◽  
Jacqueline C. Barrientos ◽  
...  
Keyword(s):  
T Cell ◽  
Board Of Directors ◽  
Deep Sequencing ◽  
Ex Vivo ◽  
Advisory Committees ◽  
Polymerase Eta ◽  
Activation Induced Deaminase ◽  
T Cell Help

Abstract Ultra-deep sequencing has revolutionized our ability to acquire large amounts of genetic data. We have applied this technology towards understanding the mutational process in B-cell chronic lymphocytic leukemia (CLL), which may be a key to understanding CLL pathogenesis. Acquisition of new cytogenetic aberrations and gene mutations in the CLL clone is associated with worse patient outcome. CLL is not unique in this aspect, as new somatic mutations and DNA rearrangements are also found during the evolution of other solid and liquid tumors. In many of these, activation-induced deaminase (AID), an enzyme normally expressed in germinal center B lymphocytes to induce IGHV-D-J mutations and isotype class switch recombination, is abnormally expressed. Its mutational activity, acting outside of the Ig loci, is implicated in the evolution to more aggressive disease. In CLL, the detection of leukemic cells expressing AID ex vivo correlates with significantly shorter patient survival. To test if AID mutational activity is functional in CLL cells and therefore could contribute to CLL evolution, we analyzed mutations in IGHV-D-J, the preferred substrate for AID. Because the rate of AID-induced mutation is low and only a small percentage of CLL cells express AID ex vivo, we used ultra-deep sequencing to analyze CLL cells that were activated under conditions that simulate the CLL microenvironment. Specifically, CLL cells were activated (1) in vitro by simulating the provision of T-cell help or (2) in vivo after adoptive transfer into alymphoid recipient mice, which requires the presence of T-cells for CLL cell growth. Each of these conditions induce AID in a large fraction of CLL cells. To analyze IGHV-D-J mutations, the specific CLL clone IGHV was amplified from cDNA obtained on day 0 or from the activated CLL samples using IGHV family-specific and IGHM primers to enable subsequent comparison of IGHV-D-J with IGHM mutation frequencies. Three unmutated IGHV CLL (U-CLL) and 3 mutated IGHV (>2% compared to germline) CLL (M-CLL) samples were sequenced with the Roche 454 FLX system, resulting in a total of 1,367,522 sequence reads. After using the Roche 454 algorithm to trim sequence reads, they were prepared using custom R scripts that separated 5’ IGHV and 5’ IGHM primer sequences, aligned sequences to the CLL clone IGHV-D-J rearrangement, and removed poor quality (<20) sequences, insertions, and deletions. Beginning at the 5’ end, the script also extracted blocks of sequences of the same length for day 0 and activated samples, which are required for subsequent analyses. After these preparations, the resulting 724,855 sequence blocks were subjected to clonal analyses with custom R scripts. The dominant CLL clone accounted for 94.5% (684,691) of the sequences. Subclone sequences occurring more than once were extracted. After comparison to day 0, new subclones could be identified in all samples after activation (3.22 – 28.70 new subclones / read bp *106). To evaluate AID mutational characteristics in new subclones, SHMTool (http://scb.aecom.yu.edu/shmtool) was employed to calculate mutation frequencies in IGHV-D-J relative to the IGHM constant region, at AID mutation hotspot sites (GYW or WRC), at AID mutation coldspot sites (SYC or GRS), at C/G base pairs, and at error-prone DNA polymerase eta repair hotspot sites (WA or TW). To calculate statistical significance, we utilized a custom R script that used a bootstrap method to account for the large sample sizes provided by ultra-deep sequencing as well as to correct for differences in sequencing sample size. All samples showed an increase in IGHV-D-J versus IGHM mutations after T cell activation. Five of 6 cases showed an increase in AID hostpot mutation frequency. AID coldspot mutation frequency decreased in 3/6 CLL cases. Percent transition mutation at C/G sites was higher than random in 2/6 CLL cases, which correlated with low frequencies of DNA polymerase eta hotspot mutation. In the other 4/6 CLL cases, the lower percent transitions at C/G sites may reflect the contribution of error-prone DNA repair. In summary, we developed a method to analyze ultra-deep IGHV-D-J sequences that revealed AID mutational characteristics in both U-CLL and M-CLL cells after activation with T-cell help in vitro or in vivo. These data are consistent with the hypothesis that AID, perhaps along with error-prone DNA repair, creates new mutations leading to the evolution of aggressive CLL. Disclosures: Rai: Sanofi: Membership on an entity’s Board of Directors or advisory committees; GSK: Membership on an entity’s Board of Directors or advisory committees; Teva: Membership on an entity’s Board of Directors or advisory committees; Genentech: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees.

scholarly journals Multiple Myeloma Pathogenesis: The Role of Junb in Bone Marrow Angiogenesis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4341-4341
Author(s):  
Fengjuan Fan ◽  
Stefano Malvestiti ◽  
Yujia Shen ◽  
Eugenio Morelli ◽  
Yuji Mishima ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Research Funding ◽  
Angiogenic Factors ◽  
University Research ◽  
Advisory Committees ◽  
Cell Clones ◽  
In Vitro Angiogenesis

A significant increase in bone marrow (BM) angiogenesis represents a key event in early, microenvironment-dependent, multiple myeloma (MM). Angiogenic growth factor- and cytokine- production and secretion is a complex process regulated by a plethora of transcription factors (TFs). Over the past years, members of the AP-1 family of TFs have emerged as potential new therapeutic targets. Our recent work demonstrated for the first time a pivotal role for the AP-1 family member JunB in MM pathogenesis (Fan et al., 2017). Whether JunB also contributes to MM BM angiogenesis is currently unknown. In silico and immunohistochemical analyses revealed a correlative increase of JunB and angiogenic growth factors in samples isolated from healthy donors to MGUS and MM patients; and a decrease in samples isolated from patients with plasma cell leukemia. These data were supported by the utilization of an innovative in vivo MM model of clonal evolution. Specifically, JunB as well as selected angiogenic factors were significantly increased in tumor cell clones at primary sites (bone chips) versus tumor cell clones at metastatic (distant BM) sites, as evidenced by whole exome and RNA sequencing. Functionally, doxycyclin- induced inhibition of stroma cell: MM cell co-culture- as well as of IL-6- mediated JunB upregulation in TetR-shJunB/ MM.1S cells significantly reduced production and secretion of angiogenic factors; and consequently inhibited in vitro angiogenesis. Conversely, 4-hydroxytamoxifen (4-OHT)-mediated upregulation of JUNB activity in JUNB-ER/MM cells strongly increased the expression and secretion of angiogenic factors and in vitro angiogenesis. The interaction of JunB with angiogenic factor- encoding DNA in MM cells was further confirmed utilizing chromatin immunoprecipitation (ChIP)- sequencing. Finally, treatment with doxycycline effectively inhibited JunB levels and consistently reduced microvessel density in immunodeficient NSG mice inoculated with TetR-shJUNB/ MM.1S, but not TetR-SCR/ MM.1S. In conclusion, our findings demonstrate a pivotal role of JUNB in MM BM angiogenesis; they thereby provide further evidence that JUNB is a promising therapeutic target particularly in early MM. Disclosures Vallet: Pfizer: Honoraria; Roche Pharmaceuticals: Consultancy; MSD: Honoraria. Roccaro:Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; AstraZeneca: Research Funding; Transcan2-ERANET: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Transcan2-ERANET: Research Funding; AstraZeneca: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; European Hematology Association: Research Funding; European Hematology Association: Research Funding. Goldschmidt:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; John-Hopkins University: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Research Funding; Dietmar-Hopp-Stiftung: Research Funding; John-Hopkins University: Research Funding; Chugai: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Molecular Partners: Research Funding. Podar:Takeda: Consultancy; Celgene: Consultancy, Honoraria; Amgen Inc.: Honoraria; Janssen Pharmaceuticals: Consultancy, Honoraria; Roche Pharmaceuticals: Research Funding.

scholarly journals Comparative Analysis of Independent Ex Vivo functional Drug Screens Identifies Predictive Biomarkers of BCL-2 Inhibitor Response in AML

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2763-2763 ◽  
Author(s):  
Brian S. White ◽  
Suleiman A. Khan ◽  
Muhammad Ammad-ud-din ◽  
Swapnil Potdar ◽  
Mike J Mason ◽  
...  
Keyword(s):  
Gene Expression ◽  
Board Of Directors ◽  
Research Funding ◽  
Drug Response ◽  
Ex Vivo ◽  
Predictive Performance ◽  
Data Sets ◽  
Advisory Committees ◽  
Data Set ◽  
Equity Ownership

Abstract Introduction: Therapeutic options for patients with AML were recently expanded with FDA approval of four drugs in 2017. As their efficacy is limited in some patient subpopulations and relapse ultimately ensues, there remains an urgent need for additional treatment options tailored to well-defined patient subpopulations to achieve durable responses. Two comprehensive profiling efforts were launched to address this need-the multi-center Beat AML initiative, led by the Oregon Health & Science University (OHSU) and the AML Individualized Systems Medicine program at the Institute for Molecular Medicine Finland (FIMM). Methods: We performed a comparative analysis of the two large-scale data sets in which patient samples were subjected to whole-exome sequencing, RNA-seq, and ex vivo functional drug sensitivity screens: OHSU (121 patients and 160 drugs) and FIMM (39 patients and 480 drugs). We predicted ex vivo drug response [quantified as area under the dose-response curve (AUC)] using gene expression signatures selected with standard regression and a novel Bayesian model designed to analyze multiple data sets simultaneously. We restricted analysis to the 95 drugs in common between the two data sets. Results: The ex vivo responses (AUCs) of most drugs were positively correlated (OHSU: median Pearson correlation r across all pairwise drug comparisons=0.27; FIMM: median r=0.33). Consistently, a samples's ex vivo response to an individual drug was often correlated with the patient's Average ex vivo Drug Sensitivity (ADS), i.e., the average response across the 95 drugs (OHSU: median r across 95 drugs=0.41; FIMM: median r=0.58). Patients with a complete response to standard induction therapy had a higher ADS than those that were refractory (p=0.01). Further, patients whose ADS was in the top quartile had improved overall survival relative to those having an ADS in the bottom quartile (p<0.05). Standard regression models (LASSO and Ridge) trained on ADS and gene expression in the OHSU data set had improved ex vivo response prediction performance as assessed in the independent FIMM validation data set relative to those trained on gene expression alone (LASSO: p=2.9x10-4; Ridge: p=4.4x10-3). Overall, ex vivo drug response was relatively well predicted (LASSO: mean r across 95 drugs=0.62; Ridge: mean r=0.62). The BCL-2 inhibitor venetoclax was the only drug whose response was negatively correlated with ADS in both data sets. We hypothesized that, whereas the predictive performance of many other drugs was likely dependent on ADS, the predictive performance of venetoclax (LASSO: r=0.53, p=0.01; Ridge: r=0.63, p=1.3x10-3) reflected specific gene expression biomarkers. To identify biomarkers associated with venetoclax sensitivity, we developed an integrative Bayesian machine learning method that jointly modeled both data sets, revealing several candidate biomarkers positively (BCL2 and FLT3) or negatively (CD14, MAFB, and LRP1) correlated with venetoclax response. We assessed these biomarkers in an independent data set that profiled ex vivo response to the BCL-2/BCL-XL inhibitor navitoclax in 29 AML patients (Lee et al.). All five biomarkers were validated in the Lee data set (Fig 1). Conclusions: The two independent ex vivo functional screens were highly concordant, demonstrating the reproducibility of the assays and the opportunity for their use in the clinic. Joint analysis of the two data sets robustly identified biomarkers of drug response for BCL-2 inhibitors. Two of these biomarkers, BCL2 and the previously-reported CD14, serve as positive controls credentialing our approach. CD14, MAFB, and LRP1 are involved in monocyte differentiation. The inverse correlation of their expression with venetoclax and navitoclax response is consistent with prior reports showing that monocytic cells are resistant to BCL-2 inhibition (Kuusanmäki et al.). These biomarker panels may enable better selection of patient populations likely to respond to BCL-2 inhibition than would any one biomarker in isolation. References: Kuusanmäki et al. (2017) Single-Cell Drug Profiling Reveals Maturation Stage-Dependent Drug Responses in AML, Blood 130:3821 Lee et al. (2018) A machine learning approach to integrate big data for precision medicine in acute myeloid leukemia, Nat Commun 9:42 Disclosures Druker: Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Fred Hutchinson Cancer Research Center: Research Funding; Celgene: Consultancy; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; Patient True Talk: Consultancy; Millipore: Patents & Royalties; Monojul: Consultancy; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Bristol-Meyers Squibb: Research Funding; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; ARIAD: Research Funding; Novartis Pharmaceuticals: Research Funding. Heckman:Orion Pharma: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Porkka:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Tyner:AstraZeneca: Research Funding; Incyte: Research Funding; Janssen: Research Funding; Leap Oncology: Equity Ownership; Seattle Genetics: Research Funding; Syros: Research Funding; Takeda: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Aptose: Research Funding; Agios: Research Funding. Aittokallio:Novartis: Research Funding. Wennerberg:Novartis: Research Funding.

scholarly journals Dysregulation of Splicing in Multiple Myeloma: The Splicing Factor SRSF1 Supports MM Cell Proliferation Via Splicing Control

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4500-4500
Author(s):  
Mariateresa Fulciniti ◽  
Michael A Lopez ◽  
Anil Aktas Samur ◽  
Eugenio Morelli ◽  
Hervé Avet-Loiseau ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Alternative Splicing ◽  
Board Of Directors ◽  
Research Funding ◽  
Splicing Factor ◽  
Splicing Factors ◽  
Rna Seq ◽  
Advisory Committees ◽  
Disease Biology

Abstract Gene expression profile has provided interesting insights into the disease biology, helped develop new risk stratification, and identify novel druggable targets in multiple myeloma (MM). However, there is significant impact of alternative pre-mRNA splicing (AS) as one of the key transcriptome modifier. These spliced variants increases the transcriptomic complexity and its misregulation affect disease behavior impacting therapeutic consideration in various disease processes including cancer. Our large well annotated deep RNA sequencing data from purified MM cells data from 420 newly-diagnosed patients treated homogeneously have identified 1534 genes with one or more splicing events observed in at least 10% or more patients. Median alternative splicing event per patient was 595 (range 223 - 2735). These observed global alternative splicing events in MM involves aberrant splicing of critical growth and survival genes affects the disease biology as well as overall survival. Moreover, the decrease of cell viability observed in a large panel of MM cell lines after inhibition of splicing at the pre-mRNA complex and stalling at the A complex confirmed that MM cells are exquisitely sensitive to pharmacological inhibition of splicing. Based on these data, we further focused on understanding the molecular mechanisms driving aberrant alternative splicing in MM. An increasing body of evidence indicates that altered expression of regulatory splicing factors (SF) can have oncogenic properties by impacting AS of cancer-associated genes. We used our large RNA-seq dataset to create a genome wide global alterations map of SF and identified several splicing factors significantly dysregulated in MM compared to normal plasma cells with impact on clinical outcome. The splicing factor Serine and Arginine Rich Splicing Factor 1 (SRSF1), regulating initiation of spliceosome assembly, was selected for further evaluation, as its impact on clinical outcome was confirmed in two additional independent myeloma datasets. In gain-of (GOF) studies enforced expression of SRSF1 in MM cells significantly increased proliferation, especially in the presence of bone marrow stromal cells; and conversely, in loss-of function (LOF) studies, downregulation of SRSF1, using stable or doxy-inducible shRNA systems significantly inhibited MM cell proliferation and survival over time. We utilized SRSF1 mutants to dissect the mechanisms involved in the SRSF1-mediated MM growth induction, and observed that the growth promoting effect of SRSF1 in MM cells was mainly due to its splicing activity. We next investigated the impact of SRSF1 on allelic isoforms of specific gene targets by RNA-seq in LOF and confirmed in GOF studies. Splicing profiles showed widespread changes in AS induced by SRSF1 knock down. The most recurrent splicing events were skipped exon (SE) and alternative first (AF) exon splicing as compared to control cells. SE splice events were primarily upregulated and AF splice events were evenly upregulated and downregulated. Genes in which splicing events in these categories occurred mostly did not show significant difference in overall gene expression level when compared to control, following SRSF1 depletion. When analyzing cellular functions of SRSF1-regulated splicing events, we found that SRSF1 knock down affects genes in the RNA processing pathway as well as genes involved in cancer-related functions such as mTOR and MYC-related pathways. Splicing analysis was corroborated with immunoprecipitation (IP) followed by mass spectrometry (MS) analysis of T7-tagged SRSF1 MM cells. We have observed increased levels of SRSF phosphorylation, which regulates it's subcellular localization and activity, in MM cell lines and primary patient MM cells compared to normal donor PBMCs. Moreover, we evaluated the chemical compound TG003, an inhibitor of Cdc2-like kinase (CLK) 1 and 4 that regulate splicing by fine-tuning the phosphorylation of SR proteins. Treatment with TG003 decreased SRSF1 phosphorylation preventing the spliceosome assembly and inducing a dose dependent inhibition of MM cell viability. In conclusions, here we provide mechanistic insights into myeloma-related splicing dysregulation and establish SRSF1 as a tumor promoting gene with therapeutic potential. Disclosures Avet-Loiseau: Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, 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. Munshi:OncoPep: Other: Board of director.

scholarly journals Erythron to the Liver: Send Iron

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. SCI-37-SCI-37
Author(s):  
Elizabeta Nemeth ◽  
Tomas Ganz ◽  
Léon Kautz
Keyword(s):  
Bone Marrow ◽  
Iron Overload ◽  
Board Of Directors ◽  
Ex Vivo ◽  
Iron Loading ◽  
Erythropoietic Activity ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Deficient Mice

For successful expansion of erythropoiesis, the activity of the hormone erythropoietin (EPO) must be coordinated with the supply of iron to erythroid precursors. Increased iron supply for erythropoiesis is ensured by the suppression of hepcidin, the iron-regulatory hormone produced by the liver. Low hepcidin levels allow greater absorption of dietary iron and greater mobilization of iron from the stores in the spleen and the liver. The mechanisms coordinating erythropoietic activity with iron delivery are not well understood. We recently identified erythroferrone as a new mediator of hepcidin suppression during stress erythropoiesis1. Erythroferrone (ERFE) is a member of the C1q/TNF-related protein (CTRP) family of metabolic mediators. ERFE is produced in response to EPO by erythroblasts of the bone marrow and spleen of mice. The induction of ERFE by EPO was dependent on Jak2/Stat5 signaling. Ex vivo treatment of human erythroblasts with EPO also resulted in a dramatic induction of ERFE expression. The essential role of ERFE in acute hepcidin suppression by erythropoiesis was demonstrated in ERFE-deficient mice. In contrast to wild-type mice which suppressed hepcidin ~10-fold within hours after hemorrhage or erythropoietin injection, no hepcidin suppression was observed in ERFE knockout mice within 24 h. As a consequence, ERFE-deficient mice exhibited delayed recovery of hemoglobin after hemorrhage or severe inflammation. Treatment of mice or hepatocytes with recombinant ERFE protein confirmed the hepcidin-suppressive activity of the protein. It remains to be seen whether administration of ERFE protein would be useful for the treatment of anemia of inflammation mediated by elevated hepcidin. In iron-loading anemias including β-thalassemia, hepcidin is chronically suppressed by the exuberant but ineffective erythropoietic activity. This is the cause of iron overload in untransfused thalassemia patients and may contribute to iron loading even in transfused patients. We found that ERFE expression is greatly increased in the bone marrow and spleen of mice with β-thalassemia intermedia (th3 model). Transgenic ablation of ERFE in th3 mice normalized hepcidin and partially corrected their iron overload. Although human studies of the role of ERFE in health and disease are clearly needed, ERFE is a promising candidate for the pathological suppressor of hepcidin in anemias with ineffective erythropoiesis. References: 1. Kautz L, Jung G, Valore EV, et al. Identification of erythroferrone as an erythroid regulator of iron metabolism. Nat Genet. 2014; 46: 678-684. Disclosures Nemeth: Intrinsic LifeSciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Merganser Biotech: Equity Ownership. Ganz:Intrinsic LifeSciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Keryx Pharma: Consultancy; Merganser Biotech: Consultancy, Equity Ownership.

scholarly journals Dasatinib Inhibits FLT3/ITD and PTPN11mutated Acute Myeloid Leukemia Cells Overexpressing SRC Tyrosine Kinases

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1451-1451
Author(s):  
Sigal Tavor ◽  
Tali Shalit ◽  
Noa Chapal Ilani ◽  
Yoni Moskovitz ◽  
Nir Livnat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Drug Sensitivity ◽  
Kinase Inhibitors ◽  
Advisory Committees ◽  
Acute Myeloid

Background: Recent advances in acute myeloid leukemia(AML) targeted therapy improve overall survival. While these targeted therapies can achieve prolonged remissions, most patients will eventually relapseunder therapy. Our recent studies suggest that relapse most often originates from several sub-clones of leukemic stem cells (LSCs), present before therapy initiation, and selected due to several resistance mechanisms. Eradication of these LSCs during treatment induction /remission could thus potentially prevent relapse. The overall goal of the current study was to identify drugs which can be safely administrated to patients at diagnosis and that will target LSCs. Since simultaneously testing multiple drugs in vivo is not feasible, we used an in vitrohigh throughput drug sensitivity assay to identify new targets in primary AML samples. Methods: Drug sensitivity and resistance testing (DSRT) was assessed in vitro (N=46 compounds) on primary AML samples from patients in complete remission (N=29). We performed whole exome sequencing and RNAseq on samples to identify correlations between molecular attributes and in vitro DSRT. Results:Unsupervised hierarchical clustering analysis of in vitro DSRT, measured by IC50, identified a subgroup of primary AML samples sensitive to various tyrosine kinase inhibitors (TKIs). In this subgroup, 52% (9/17) of AML samples displayed sensitivity to dasatinib (defined as a 10-fold decrease in IC50 compared to resistant samples). Dasatinib has broad TKI activity, and is safely administered in the treatment of leukemia. We therefore focused our analysis on predicting AML response to dasatinib, validating our results on the Beat AML cohort. Enrichment analysis of mutational variants in dasatinib-sensitive and resistant primary AML samples identified enrichment of FLT3/ITD (p=0.05) and PTPN11(p=0.05) mutations among dasatinib responders. Samples resistant to dasatinib were enriched with TP53 mutations (p=0.01). No global gene expression changes were observed between dasatinib-sensitive and resistant samples in our cohort, nor in the Beat AML cohort. Following this, we tested the differential expression of specific dasatinib-targeted genes between dasatinib-responding and resistant samples. No significant differences were identified. However, unsupervised hierarchical clustering of dasatinib targeted genes expression in our study and in the Beat AML cohort identified a subgroup of AML samples (enriched in dasatinib responders) that demonstrated overexpression of three SRC family tyrosine kinases:FGR, HCK and LYN as well as PTK6, CSK, GAK and EPHB2. Analysis of the PTPN11 mutant samples revealed that the IC50 for dasatinib in 23 carriers of the mutant PTPN11 was significantly lower compared to the IC50 of PTPN11 wild type samples (p=0.005). LYN was also upregulated (p&lt;0.001) in the mutant samples. We therefore hypothesized that gene expression of dasatinib-targeted genes could be used as a predictive biomarker of dasatinib response among FLT3/ITD carriers. We found that among FLT3/ITD AML carriers in the Beat AML cohort LYN, HCK, CSK and EPHB2 were significantly over-expressed in the dasatinib responding samples (N=27) as compared to the dasatinib resistant samples (N=35). To predict response to dasatinib among FLT3/ITD carriers we used a decision tree classifier based on the expression levels of these four genes. Our prediction model yielded a sensitivity of 74% and specificity of 83% for differentiating dasatinib responders from non-responders with an AUC of 0.84. Based on our findings, we selected FLT3/ITD AML samples and injected them to NSG-SGM3 mice. We found that in a subset of these samples, dasatinib significantly inhibited LSCs engraftment. This subset of FLT3/ITD AML samples expressed higher levels of LYN, HCK,FGR and SRC as compared to the FLT3/ITD samples that were not sensitive to dasatinib therapy in vivo. In summary, we identified a subgroup of AML patients sensitive to dasatinib, based on mutational and expression profiles. Dasatinib has anti-leukemic effects on both blasts and LSCs. Further clinical studies are needed to demonstrate whether selection of tyrosine kinase inhibitors, based on specific biomarkers, could indeed prevent relapse. Disclosures Tavor: Novartis: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS companies: Membership on an entity's Board of Directors or advisory committees.

Functional Analysis of the CML Blast Crisis Transcriptome and Epigenome Using Crispr-CAS9 and Pharmacologic Approaches

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2764-2764
Author(s):  
Tun Kiat Ko ◽  
Xin Xuan Sheila Soh ◽  
Willie Yu ◽  
Peter S. Winter ◽  
Thushangi Pathiraja ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Blast Crisis ◽  
Genetic Alterations ◽  
Mapk Signaling ◽  
Rational Approach ◽  
Structural Variations ◽  
Advisory Committees ◽  
Paired Samples

Abstract Current models of CML blast crisis (BC) propose that expression of BCR-ABL results in genomic instability and the acquisition of genetic alterations that affect cell proliferation and survival, self-renewal and differentiation. To characterize the molecular events that underlie progression, we performed whole genome sequencing of paired samples of the same patient at CP and at BC (n = 12), as well as expression and methylation arrays of these samples and a larger validation cohort of unpaired CD34-selected samples (n = 38). Contrary to expectations, we found that the CML BC genome is relatively quiescent with regards to SNVs, indels and structural variations. In contrast, we observed widespread hyper-methylation in BC that was associated with distinct changes in expression and was independent of lineage/differentiation state. These findings suggest that in addition to genetic alterations, epigenomic events are likely to contribute substantively to BC progression. To understand the functional effects of the dysregulated transcriptome and epigenome in BC CML, we employed both pharmacologic and genetic methods to target candidate genes of interest identified in our earlier studies. To induce de-methylation of the BC genome, we treated primary samples with low doses of decitabine, a DNMT inhibitor. We found that decitabine impaired colony formation ability of BC CD34+ progenitors and concomitantly activated regulators of myeloid differentiation that were both hyper-methylated and down-regulated in BC CD34+ progenitors, such as MPO and KLF1. These results suggest that hyper-methylation does contribute to BC CD34+ progenitor function, and support the use of epigenetic therapies as a rational approach to targeting BC. The genetic approach we chose was a CRISPR-based in vitro pooled screen. We created a custom library targeting 200 genes, with an average of 5 sgRNAs per gene, and 50 non-targeting controls. We transduced K562 with the library and harvested samples at different time-points post-transduction/selection - Day 0, 7 and 21 - for deep sequencing. As expected, sgRNAs targeting essential genes such as MYC and MCM 2-7 were recurrently depleted in the population over time. More importantly, enriched sgRNAs targeted genes including TET2, which has been previously reported to be inactivated in myeloid malignancies, as well as novel candidates including RREB1, a transcription factor that binds to RAS-responsive elements (RREs) and may be involved in MAPK signaling. We will validate these targets by knocking them out individually and assessing their effect on the ability of CP cells to serially replate and/or engraft immune-deficient mice. Disclosures Chuah: Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Chiltern International: Honoraria. Takahashi:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Masis: Consultancy; Otsuka: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau; Sysmex: Research Funding, Speakers Bureau; Astellas: Speakers Bureau. Valent:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria.

scholarly journals Evaluation of in Vitro Macrophage Characterization in Gaucher Type 1 (GD1) Patients

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3592-3592
Author(s):  
Paola Delbini ◽  
Viola Ghiandai ◽  
Maria Domenica Cappellini ◽  
Lorena Duca ◽  
Isabella Nava ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Iron Metabolism ◽  
Macrophage Activation ◽  
Gene Expression Analysis ◽  
Advisory Committees ◽  
Macrophage Population

Gaucher disease type 1 is the most common inherited lysosomal storage disorder caused by the deficiency of lysosomal β-glucocerebrosidase (GBA, acid-β-glucosidase), required for the degradation of glycosphingolipids. The deficiency of the enzyme results in the widespread accumulation of glucosylceramide in macrophages, leading to anemia, thrombocytopenia and coagulopathy, visceral (hepatosplenomegaly, lungs) and skeletal manifestations (deformities, fractures, avascular osteonecrosis). However, GD manifestations are caused not only by the burden of glucosylceramide storage, but also by macrophage activation. It seems that GD reflects the downstream consequences of inappropriate macrophage activation, with the release of pro-inflammatory cytokines and other responses to storage material. The aim of this study is to assess in vitro phenotypic characterization, functional properties and gene expression anaysis of GD1 macrophages in order to understand their possible role in inflammation and in impairment of iron metabolism occurring in GD patients. Monocytes were isolated from buffy coats of GD patients (n=3) and controls (n=3) by applying an in vitro model protocol. Monocytes were expanded in ImmunoCult™ medium and 1% ZellShield® antibiotic cocktail. Differentiation was induced in ImmunoCult™ medium, 50 ng/mL M-CSF and 50 ng/mL GM-CSF. To mimic the in vivo condition, macrophage population was loaded with erythrocytes ghosts isolated from GD patients. Cell morphology was analyzed on cytocentrifuged preparations stained with May-Grünwald Giemsa (MGG). Surface marker expression (CD11, CD33, CD68, CD64) were examined by flow cytometry to evaluate macrophages differentiation and phenotype. Gene expression analysis of iron metabolism-related genes was evaluated through Real-Time Quantitative PCR. Biochemical indices (NTBI, GDF15, sTfR and chitotriosidase) were analyzed in supernatant through ELISA assay. Flow cytometry analysis (Fig.1) revealed that without erythrocytes ghosts (preM∅), the proportion of CD11+/CD68+ macrophage population was similar between GD patients and control. However, in GD macrophages, when loaded with Gaucher erythrocytes ghosts (M∅+ghosts), the proportion of CD11++/CD68++ cells increased (36.4%), as a reflection of a more pro-inflammatory phenotype. Treated controls showed no differences. To further characterize these different macrophages subpopulations in GD patients, we used an additional parameter, CD64, because CD64/CD68 markers are specific for M1/M2 polarization. GD M∅+ghosts showed an higher percentage of CD64++/CD68++ population (78%) in comparison of GD pre-M∅ (45%) and controls (36%), confirming a M1 proinflammatory phenotype of GD macrophages loaded with erythrocytes ghosts. Under microscope evaluation, GD M∅+ghosts presented high number of spindle-shaped fibroblastoid, typically M1 phenotype cells, rather than large flat-round cells (M2 cells). Moreover, morphological staining of these cells confirmed the typical features of GD cells with basophilic cytoplasm with characteristic crinkles. Controls showed no differences in macrophage features when loaded with erythrocytes ghosts. To confirm the pro-inflammatory potential of GD M∅+ghosts, high levels of pro-inflammatory mediators TNF-α, IL-1β and (s)TfR) were found in supernatant of GD M∅+ghosts (72,9±8,5, 24,7±2,7 and 3,1±4,4, respectively) compared to pre-M∅ and controls. High GDF15 expression in GD M∅+ghosts (14,77±5,87) respect to preM∅ (1,80±1,1) and controls (1,67±0,8) was observed. By gene expression analysis we observed in GD M∅+ghosts an higher HAMP expression (28,13±5,63) compared to preM∅ (2,49±1,5) and controls (0,71±0,02), and a lower SLC40A1 expression (0,01±0,00) compared to GD preM∅ (0,16±0,05) and controls (0,79±0,25). No significant differences in TFRC and GDF11 expression between GD preM∅ and M∅+ghosts in GD patients were observed. These preliminary data suggest that GD macrophages, when stimulated, display a proinflammatory potential. These activated M1 macrophages could contribute to an inflammatory-producing environment, triggering hepcidin and ferroportin expression by an autocrine/paracrine mechanism and leading to dysregulation of iron distribution. Disclosures Cappellini: CRISPR Therapeutics: Membership on an entity's Board of Directors or advisory committees; Vifor Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Honoraria; Genzyme/Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Motta:Sanofi-Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals 3D-Tissue Engineered Bone Marrow (3DTEBM) Culture Retrospectively Predicts Treatment Clinical Outcomes of Multiple Myeloma Patients

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1987-1987
Author(s):  
Amanda Jeske ◽  
Feda Azab ◽  
Pilar De La Puente ◽  
Barbara Muz ◽  
Justin King ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Ex Vivo ◽  
In Vitro Models ◽  
Advisory Committees ◽  
Treatment Plans ◽  
Equity Ownership

Abstract Background: Multiple Myeloma (MM) is the second most common hematological malignancy, and continues to be a fatal disease even with the development of novel therapies. Despite promising preclinical data in standard tissue culture models, most drugs fail in clinical trials and show lower efficacy in patients. This highlights the discrepancy between the current in vitro models, the pathophysiology of the disease in the patients, and the urgent need for better in vitro models for drug development and improved prediction of efficacy in patients. We have previously developed a patient-derived 3D-Tissue Engineered Bone Marrow (3DTEBM) culture model, which showed superior properties for proliferation of primary MM cells ex vivo, and better recapitulated drug resistance. The long-term goal of this study is to use the 3DTEBM model as a tool to perform drug screens on BM aspirates of MM patients and prospectively predict the efficacy of different therapies in individual patients, and help treatment providers develop personalized treatment plans for each individual patient. In the current study, we used the 3DTEBM model to, retrospectively, predict clinical responses of MM patients to therapy, as a proof of concept. Methods: We used whole-BM, viably frozen tissue banked samples from 20 MM patients with clear clinical response patterns of complete remission, and either very good partial response (sensitive) or progressive disease (non-sensitive). The BM aspirates were used to develop a 3DTEBM that represents each individual patient. The patient-derived 3DTEBM cultures were treated ex vivo with the same therapeutic regimen that the patient received in the clinic for 3 days. The treatment ex vivo was based on combinations at different concentrations which mimic the steady state concentrations (Css) of each drug. The efficacy of the treatment ex vivo was evaluated by digestion of the 3DTEBM matrix, extraction of the cells, and analysis for prevalence of MM cells in the treatment groups compared to the non-treated controls. Patients were defined "sensitive" if the effect reached 50% killing in the range of 10xCss. The ex vivo sensitivity data was then correlated with the clinical response outcomes. Results: We found that the 3DTEBM was predictive in approximately 80% of the cases (in about 85% of the combination therapy cases, and in about 70% of the single therapy cases). Broken down by individual drug, it was predictive in 80% of the cases treated with Bortezomib, 78% Lenalidomide, 84% Dexamethasone, 100% Daratumumab, 50% Carfilzomib, 50% Pomalidomide, and 100% Doxorubicin. Conclusions: The 3DTEBM is a more pathophysiologically relevant model which predicts clinical efficacy of drugs in multiple myeloma patients, retrospectively. This data provides the bases for future studies which will examine the ability of the 3DTEBM model to predict treatment efficacy, prospectively, for development of personalized treatment plans in individual multiple myeloma patients. Disclosures Jeske: Cellatrix LLC: Employment. Azab:Cellatrix LLC: Employment. De La Puente:Cellatrix LLC: Other: Co-founder. Vij:Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansson: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Azab:Ach Oncology: Research Funding; Cellatrix LLC: Equity Ownership, Other: Founder and owner; Glycomimetics: Research Funding; Targeted Therapeutics LLC: Equity Ownership, Other: Founder and owner.

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