Multiple Myeloma Plasma Cells Research Articles

DNA Polymerases Pol θ/Pol η Involved in Error-Prone DNA Repair Are Highly Expressed in Multiple Myeloma and Upregulated By DNA Damage

Background: DNA polymerases (DNA pols) are essential enzymes for DNA replication. In mammalian cells, DNA pols are divided into four families: A (Pol θ, Pol γ, and Pol ν), B (Pol α, Pol δ, Pol ε, and Pol ζ), X (Pol β, Pol λ, Pol μ, and TDT), and Y (Pol η, Pol ι, Pol κ, and REV1). These DNA pols are required for both genome duplication and protecting cells from DNA damage induced by endogenous and exogenous agents, such as ROS, UV, and chemotherapeutic drugs. For example, Pol β, Pol λ, and Pol ι participate in base excision repair. Contrastingly, Pol ζ, REV1, Pol η, Pol ι, and Pol κ can replicate over various DNA lesions to prevent DNA replication stalling, known as translesion synthesis. Although some DNA pols are highly expressed in cancer cells, indicating chemotherapeutic resistance and poor outcome, their exact roles and expression mechanisms have not been fully elucidated. Multiple myeloma (MM) is a hematological malignancy of terminally differentiated plasma cells, with multistep progression from pre-cancer stage namely. In this study we attempted to elucidate the involvement of DNA pols in multistep oncogenesis of MM. Methods: A total of 63 MM and 29 MGUS patients, 15 controls, and 9 MM cell lines were included in the study. RNA was extracted from purified CD138+ plasma cells. DNA pol expressions were determined by RQ-PCR. Their expression levels were normalized against ACTB levels and calculated with 2-ΔΔCt value. Doxycycline-inducible p53 system (Tet-on p53) and nutlin-3 were used for analyzing the role of p53 in DNA pol expressions in MM cell lines. Melphalan, doxorubicin, and bortezomib were used to examine DNA pol expressions in damaged cells in vitro. JQ1 and CPI203 were used to evaluate the role of bromodomain in DNA pol expressions. Results: Pol α and Pol ε expressions were significantly higher in MM than in control (p=0.007 and p=0.004, respectively), but Pol ε and Pol ζ levels were not significantly different (p=0.631, p=0.0826, respectively). Pol η, REV1, Pol ι, and Pol κ expressions were significantly higher in MM than control (p<0.001, p=0.002, p<0.001, and p<0.001, respectively). Pol θ and Pol γ were expressed at a higher level in MM than in control (p<0.001 and p<0.001, respectively). Pol β and Pol λ expressions were higher in MM than in control (p=0.0088 and p=0.013, respectively). Although the expressions of many DNA pols were higher in MM plasma cells, we focused on Pol η and Pol θ, because Pol λ, Pol μ, Pol ν, and Pol ι were expressed at very low levels, and Pol ε, Pol ζ, Pol γ, Pol κ, and REV1 were expressed in PBMNCs of healthy volunteers at high level. Pol η and Pol θ expressions did not differ due to known risk factors, such as cytogenetic abnormalities and ISS. Pol η expressions were positively correlated with p53 and myc expressions (r=0.718, p<0.001, r=0.528, p<0.001 respectively). p53 overexpression by Tet-on vector or nutlin-3 treatment enhanced Pol η expression, indicating that Pol η expression is regulated by p53. Melphalan or doxorubicin increased Pol η expression, but bortezomib or lenalidomide did not, suggesting that Pol η is upregulated by DNA damage via p53 pathway. Overall survival of the patients with high Pol η expression tended to be worse than with low Pol η expression (24 months survival: 69.6% vs. 57.9%, p=0.29). Pol θ expression was weakly correlated with p53. Melphalan induced Pol θ expression but doxorubicin did not. JQ1 significantly reduced Pol θ expression suggesting that Pol θ was regulated by bromodomain. Conclusion: We found that Pol θ and Pol η are highly expressed in MM, and upregulated by DNA damage. These DNA pols are involved in drug resistance and genomic instability leading to poor prognosis. Thus, DNA pols can be used as novel therapeutic targets and prognostic markers. Disclosures Handa: Ono: Research Funding.

Ixazomib Modulates Bone Remodeling and Actives Sonic Hedgehog Pathways

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by an accumulation of clonal plasma cells (PC) in the bone marrow (BM) leading to bone destruction and BM failure. Osteolytic bone disease is a common manifestation of MM that leads to progressive skeleton destruction and is the most severe cause of morbidity in MM patients. Pathogenetic mechanisms of MM bone destruction are closely linked to MM PC and osteoclasts (OCs) hyperactivity coupled with defective osteoblast (OB) function unable to counteract bone resorption. We recently demonstrated that the proteasome inhibitor (PI) Bortezomib, commonly used to treat MM, was capable to inhibit osteoclastic differentiation modulating chitinase family genes. In this work we investigated the effect of Ixazomib (IXA), a third generation PI, on osteoclastogenesis and osteogenic differentiation. Human monocytes were differentiated in OCs in presence of OC medium (supplemented with RANKL and M-CSF), and/or 10nM IXA. We observed that IXA was able to inhibit the expression of different OCs markers such as RANK, CTSK, TRAP, and MMP9 when added in OC medium in respect to OC medium alone (p<0.001). In addition, IXA treatment reduced CHIT1 enzymatic activity and downregulated CHIT1 and YKL40 (both mRNA and proteins). Immunofluorescence evaluation confirmed that IXA inhibited the mature OCs formation with five or more nuclei. Moreover, IXA was able to stimulate osteogenic differentiation of human mesenchymal stromal cells (MSCs). After 21 days of treatment, IXA alone or added to osteogenic medium increased the osteogenic markers genes (BMP2, RUNX2, Osteocalcin and Osteonectin). Immunofluorescence assay confirmed the increase of BMP2 after IXA treatment alone or in combination with osteogenic medium. Sonic Hedgehog (Shh) is one of the intricate signal transduction mechanisms that govern the precisely regulated developmental processes of multicellular organisms. Its signaling cascade plays an important role in bone homeostasis, and reducing Shh pathway protects against age-related bone loss. In this work we observed that IXA, but not Bortezomib, was able to bind the Smoothened (Smo) receptor which is the transducing molecule of the extracellular signal of Shh. Interestingly, using cellular thermal shift assay (CETSA), we demonstrated that IXA was able to bind directly Smo receptor activating the Shh cascade that leads to the nuclear translocation of Gli1 an effector of this pathway. Inhibiting Shh signaling by using an inhibitor of Smo activation (Cyclopamine), IXA-activated ostegenic differentiation-related genes were down-regulated. In conclusion, our data demonstrated that IXA regulates bone remodeling decreasing osteoclastogenesis and promoting osteogenic differentiation and therefore represents a good therapeutic option to improve the complex pathological condition of patients with MM. Disclosures Conticello: Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Palumbo:Amgen: Honoraria; Celgene: Honoraria; Hospira: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Teva: Honoraria. Di Raimondo:Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy; Celgene: Consultancy, Honoraria, Research Funding.

Oncogenic MAF in Co-Operation with IRF4 Confers Extensive Chromatin Re-Arrangement in Plasma Cells and Generates ‘Neo-Enhancers’ That Regulate Genes Critical for Myeloma Biology

Overexpression of the transcription factor MAF, as a result of its juxtaposition to the IgH enhancer [MAF-translocated t(14;16)], is a myeloma-initiating event in 3-5% of patients with multiple myeloma (MM) and confers a poor prognosis. MAF is also overexpressed in another 40% of cases, often in co-operation with the oncogene MMSET. The mechanisms by which MAF overexpression impacts on the regulatory genome to generate the MAF-driven oncogenic transcriptome and its direct targets are not known. To address this, we employed a multi-layer -omics approach using primary myeloma plasma cells (PC) as well as myeloma cell lines (MMCL). First, we determined the chromatin accessibility and transcriptome profiles of MAF-translocated myeloma by performing ATAC-seq and RNA-seq, respectively, in purified bone marrow CD138+ PC from two patients with t(14;16) and three healthy donors. We identified 6,640 differentially accessible regions, 87% of which displayed enhanced chromatin accessibility in MAF samples compared to normal PC. Secondary analysis comparing this with ATAC-seq data from a set of 28 other MM samples, including hyperdiploid, MMSET and CCND1-translocated MM, revealed 33% of those regions to be MAF subgroup specific (1,949 regions), with the rest shared between MAF and other cytogenetic groups. Gene annotation and pathway enrichment analysis using GREAT confirmed overrepresentation of the MF myeloma patient signature, as previously identified in microarray datasets. RNA-seq detected significant upregulation of approximately 900 genes in MAF samples compared to normal counterparts, including MAF itself (top 4th hit) as well as its presumed targets (CCND2, ITGB7 and NUAK1). Next, we obtained the MAF cistrome using ChIP-seq in the MAF-translocated MMCL MM1.S and integrated it with the primary PC ATAC-seq data. This revealed that 31% (618/1,949) of the differentially accessible regions in MAF-translocated MM PC are also MAF-bound. Additional overlay with ENCODE ChromHMM epigenome map showed that 47% of MAF binding sites are on active enhancers and 42% on active promoters signifying potential direct regulation of the corresponding genes. Next, we superimposed the accessible and MAF-bound loci on the epigenomic landscapes of normal PC and other B-cell types using their corresponding ChromHMM maps (Blueprint consortium data). Interestingly, 56% (345/618) of the MAF-specific regions were not active in any stage of B cell development. This suggests that aberrant MAF overexpression and chromatin binding in PC is associated with de novo activation of these chromatin regions, over half of which (200/345; 58%) are enhancers; we termed these 'neo-enhancers'. Upon de novo motif analysis of MAF ChIP-seq in MAF-translocated JJN3 and MM1.S MMCL, we confirmed MAF as the first and, interestingly, IRF4 as the second top hit, suggesting a possible MAF-IRF4 functional interaction in myelomagenesis. Indeed, overlay of the accessible MAF-bound loci with IRF4 ChIP-seq data in MM1.S revealed 63% co-occupancy (including 62% of "neo-enhancers"), proposing a novel and extensive co-operative chromatin-based network between the two transcription factors. Final integration of the accessible MAF-bound regions with the paired transcriptomes of primary myeloma PC revealed a set 206 candidate enhancer-gene pairs. Strikingly, we identified two IRF4-cobound "neo-enhancers" linked to overexpression of TLR4 and CCR1, two genes known for their roles in myeloma cell proliferation and migration. We confirmed significant downregulation of both genes upon shRNA-mediated knockdown of MAF in the two MAF-translocated MMCL, MM1.S and JJN3, as well as the lethality of MAF depletion. Further, MAF overexpression in MAF-negative myeloma backgrounds led to transcriptional upregulation of these genes, further validating them as MAF targets. While CRISPR/Cas9i experiments targeting TLR4 are ongoing, preliminary results validated the functional role of the "neo-enhancer" in CCR1 gene expression. In conclusion, we demonstrate for the first time an extensive re-organisation of the PC chromatin conferred by oncogenic MAF in MM; we reveal its extensive co-operation with IRF4 in this process; we validate the directly MAF-regulated genes and functionally characterise neo-enhancers of key MAF-dependent genes that in addition to MAF itself are also critical for myeloma biology. Disclosures Hatjiharissi: Janssen: Honoraria. Caputo:GSK: Research Funding. Karadimitris:GSK: Research Funding.

Aberrant RHAMM Splicing in Multiple Myeloma (MM) and Its Implications for Immunotherapy

Background: Receptor for hyaluronan-mediated motility (RHAMM) or CD168 has been a promising target for MM immunotherapy because it is overexpressed in MM cells. RHAMM has been tested as a target for an anti-RHAMM peptide vaccination approach in MM and other hematological malignancies. Although RHAMM peptide-induced immune response in patients, clinical outcomes were mixed, that can be a result of equal expression levels of RHAMM in different subpopulation of bone marrow (BM) cells in MM patients and healthy donors (HD). To enhance current RHAMM-peptide and future immunotherapeutic approaches, we investigated the cause of altered RHAMM mRNA splicing in MM patients. mRNA splicing has the potential to produce numerous mis-spliced genes, creating novel disease markers; some resulting proteins are likely to contain neoantigens selectively expressed on MM tumor cells. Methods/results: Splicing alterations can be caused by single nucleotide variations (SNVs) that affect splicing regulatory elements (SREs), or by deregulated splicing factors (SFs). We evaluated the incidence of SNVs located in the vicinity of the RHAMM. We identified a total of 57 SNVs: 72% SNVs are in the intronic region, and 28% are in the RHAMM coding region. We used the "HEXplorer" tool and predicted that four SNVs have the potential to contribute to aberrant RHAMM splicing in MM either by altering SF binding to SREs or by impacting splice site selection. Predicted SNVs were evaluated using an in vivo splicing assay to identify SNV-clusters causing aberrant RHAMM splicing. We have observed progressive overexpression of core SF PTBP1/2 (polypyrimidine track-binding protein) in MM patients and associated with disease progression. Since SNVs on the RHAMM modulate canonical SF binding sites, we tested the effects of PTBP1/2 deregulation on RHAMM splicing. We expressed PTBP1/2 in H929 cells, and then evaluated the RHAMM splicing pattern in transfected cells at a single cell (SC) level. SC analyses showed that overexpression of PTBP1/2 increased (2.5-fold) the RHAMM-V3:FL ratio in MM cells. SC analyses also identified overexpression of the RHAMM-V3 splice variant in 18% of H929 SCs expressing PTBP1, and in 37% of cells expressing PTBP2, confirmed at the single cell (SC) level. In BM-infiltrating myeloid cells, analyses showed 50% of myeloid cells express the RHAMM-V3 variant alone, and 79% of plasma cells (PCs) express this variant in combination with RHAMM-FL. Moreover, the RHAMM-V3/FL ratio in PCs is elevated (2.6-fold), further confirming a correlation between the RHAMM variant ratio and the clinical outcome. Next, we determined RHAMM-V3/FL ratios in BM stromal cells from 16 MM patients: MM-BMSC samples exclusively express the RHAMM-V3 in combination with RHAMM-FL and the RHAMM-V3:FL is 1.8 fold. In BMSC samples derived from healthy donors (HD), we detected relatively low-level expression of RHAMM-FL as compared to expression levels of RHAMM-FL in MM patients, while RHAMM-V3 transcripts were undetectable. SC analysis of RHAMM FL and splice variant transcripts in MM BMSC and HD-BMSC agreed with the analyses done on the MM HD-BMSC bulk population. We did not detect any MM BMSC cells expressing RHAMM-V3 alone and the RHAMM V3/FL ratio was 1.6-fold, which is lower than that in MM-PCs. MM-BMSC screening also identified a new splice variant of RHAMM, that was absent in MM PCs or in MM myeloid cells. Conclusions: Our study suggests that aberrant RHAMM splicing in MM can result from SNPs/SNVs affecting SRE due to the upregulation of PTBP1/2. Our study is the first to show that the RHAMM-V3 variant is associated with PTBP2 overexpression. The identification of cell type-specific RHAMM splicing events identifies novel targets for improved immunotherapy in MM. Disclosures Chu: Celgene: Honoraria; AstraZeneca: Honoraria; Gilead: Honoraria; Teva: Consultancy; Amgen Inc.: Honoraria. Anderson:C4 Therapeutics: Other: Scientific founder ; OncoPep: Other: Scientific founder ; Gilead Sciences: Other: Advisory Board; Janssen: Other: Advisory Board; Sanofi-Aventis: Other: Advisory Board.

Real-World Treatment Patterns from the HUMANS Study in Multiple Myeloma in Denmark

Introduction Current treatment for multiple myeloma (MM), an incurable but treatment sensitive plasma cell cancer, aims to extend time to disease progression, prolong survival and improve quality of life. Nevertheless, epidemiological knowledge regarding MM treatment is mostly derived from randomized controlled trials, which are limited by strict inclusion criteria, study designs that assess drug efficacy in optimal clinical settings, and short follow-up. Current treatment options for MM are associated with complex and varying treatment-related side effect profiles. However, real-world evidence is available only for a limited selection of treatment regimens. Thus, there is a need for studies to further investigate treatment patterns in clinical settings that reflect real-world practice. The Health Outcome and Understanding of Myeloma - a multi-national real-world evidence (HUMANS) study - aimed to characterize patient characteristics, treatment patterns, and outcomes for newly diagnosed patients with MM who received first-line treatment. Here we report first results from the Danish study. Methods This population-based, retrospective, longitudinal, observational study used secondary data from the Danish Cancer Register (DCR) and National Patient Register (NPR) for patients diagnosed with MM. Patients were stratified by autologous stem cell transplantation (ASCT) and pharmacological treatment (bortezomib-based, lenalidomide-based, or other first-line therapy) and characterized using descriptive statistics. To analyse recent treatment patterns and also include patients with long duration before treatment start, eligible patients had first MM diagnosis from 2005-2016 in the NPR and DCR (diagnosis date identified from the DCR), first MM-specific treatment from 2010-2018 in the NPR, no other hematologic cancer records in the NPR and DCR, and no MM treatment before diagnosis. Treatment duration (time between start and end of treatment period, with set grace period of 60 days and assumed treatment supply of 7 and 28 days per treatment event for bortezomib and lenalidomide, respectively) and overall survival (OS) were estimated by Kaplan-Meier method. Results The study population comprised 2,451 patients with MM, of which 887 patients (36%) underwent ASCT. In the non-ASCT population (n=1564), the majority (n=838, 54%) received bortezomib as first-line treatment, 102 patients (7%) received lenalidomide, and for 631 patients (40%), first-line treatment could not be identified (referred to as the other non-ASCT cohort). Mean (standard deviation) age overall at first MM diagnosis was 68 (±11) years, and was 72 (±8), 75 (±9), 77 (±7), and 59 (±8) years in the bortezomib, lenalidomide, other non-ASCT and ASCT cohorts, respectively. A higher number of men (57%) than women were diagnosed with MM. From 2015 onwards, the proportion of patients who received lenalidomide increased, whereas for patients who received other MM specific drugs, the proportion decreased (see Table 1). The median OS (95% confidence interval [CI]) from administration of first-line treatment for the bortezomib and lenalidomide cohorts was 52.9 (46.2-58.2) and 69.3 (54.7-108.4) months, respectively. For the ASCT cohort the median OS was 117.2 (104.2-133.8) months from MM diagnosis. Patients followed a once or twice weekly regimen of bortezomib treatment, i.e. 3/4 or 7 days between treatments (Figure 1). Patients in the bortezomib cohort remained treated with a median bortezomib treatment duration of 4 months (CI 4.04-4.60) and an estimated 10% remained on treatment at 10 months. In the lenalidomide cohort, patients remained treated with a median duration of 7 months (CI 4.67-10.12) and an estimated 10% remained on treatment at 23 months (Figure 2). Conclusion In this study, we present population-based treatment patterns and outcomes for MM in Danish clinical practice. The 4 month median treatment duration of bortezomib was lower than the target treatment suggested by prior clinical trials. The differences in overall survival and treatment duration should be interpreted with caution, as patients in the different cohorts have varying baseline characteristics. Linked data from the DCR and NPR may provide real-world evidence of treatment patterns in clinical practice. Research regarding time to progression in a multiple myeloma real-world setting is warranted. Disclosures Abildgaard: Amgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding; Celgene: Research Funding. Szilcz:Parexel International: Employment. Ma:Parexel International: Employment. Ørstavik:Takeda Pharmaceuticals International AG: Employment. Bent-Ennakhil:Takeda Pharmaceuticals International AG: Employment. Freilich:Parexel International: Employment. Gavini:Takeda Pharmaceuticals International AG: Employment.

PARP1-Mediated Alt-NHEJ Repair Is a Therapeutic Target in Multiple Myeloma

Background- Multiple Myeloma (MM) is a hematologic malignancy strongly characterized by genomic instability, which promotes disease progression and drug resistance. We previously demonstrated that LIG3-dependent Alt-NHEJ repair is involved in genomic instability, drug resistance and survival of MM cells. On these premises, we investigated PARP1 as driver component of Alt-NHEJ pathway and new therapeutic target in MM. Materials and methods- Cell proliferation and apoptosis were evaluated with CellTiter-Glo assay and Annexin V staining. Alt-NHEJ repair was evaluated using EJ2-GFP. PARP1, Caspase 3, MYC and DNA Damage Response protein levels were analyzed by Western blot of whole protein extracts. In vivo anti-MM activity was evaluated in NOD-SCID mice bearing subcutaneous H929 and AMO-1 Bortezomib resistant (ABZB) xenografts, daily treated with Olaparib (Selleckchem) via oral gavage. Results - By interrogating public available datasets, we found significant correlation between higher mRNA expression of PARP1 and shorter survival of MM patients. On these findings, we investigated the effect of available PARP inhibitors (PARPi) on MM cell survival. We found that Olaparib, a clinically available PARPi induced a significant reduction of proliferation and clonogenic growth of MM cell lines at low micromolar concentrations. Importantly, Olaparib impaired viability of MM cell lines or primary malignant plasmacells co-cultured with stromal cells, thus overcoming the bone marrow microenvironment supportive effect for MM survival. As result of PARP-mediated Alt-NHEJ repair inhibition, anti-proliferative effects were associated to increase of DNA double-strand breaks (DSBs), activation of DNA damage response, cell cycle arrest and finally apoptosis. To identify predictive biomarkers for PARPi in MM, a published sensitivity gene expression signature was applied to our MM gene expression profiling (GEP) data. Interestingly, this signature was particularly enriched in TC2 MM and secondary plasma cell leukemia (PCL). Therefore, in order to evaluate concordantly modulated sets of genes that were possibly associated to PARPi signature in MM, PARPi-positive and PARPi-negative MM-TC2 cases were compared by GSEA analysis. Interestingly, groups of genes regulated by MYC or involved in DNA repair resulted among the most significantly up-regulated in PARPi-positive versus PARPi-negative MM-TC2 cases. Accordingly, MYC transcript reached the highest median expression levels in sPCL and HMCLs across PC dyscrasia groups, and in MM-TC2 class. Consistently U266 cells, which was quiet insensitive to PARP knockdown or PARP inhibitor Olaparib, were null for c-MYC as compared to multiple myeloma cell lines evaluated in this study. Conversely, as formal proof of our hypothesis, over-expression of c-MYC in U266 cells (MYC-OE) induced cell death upon PARP silencing or PARP inhibitor treatment. Notably, we found that c-MYC-PARP1 loop was also hyper-activated in Bortezomib resistant cells, thus confirming pivotal role of Alt-NHEJ repair in drug resistance development. Remarkably, to demonstrate the in vivo relevance of our findings, we showed that clinically available Parp-inhibitor Olaparib exerted a significant anti-MM activity on both Bortezomib sensitive (H929) and resistant (ABZB) MM cells injected in immunocompromised mice. Conclusion Taken together, our findings indicate that MM cells are dependent on PARP-mediated Alt-NHEJ repair pathway, which therefore represents a novel druggable target pathway in MYC-driven MM cells. Disclosures No relevant conflicts of interest to declare.

Bortezomib Overcomes Poor Prognosis Conferred By High Sictp in Untreated Plasma Cell Disorders

Introduction To date, various biochemical markers of bone remodeling have been investigated in multiple myeloma (MM) patients. Serum C-terminal telopeptide of type I collagen (sICTP) is a well-known biochemical marker of bone remodeling. Although several reports showed the relationship between a high level of sICTP and poor prognosis in MM patients, little is known about the efficacy of bortezomib in high sICTP patients. In this single center retrospective study, we assessed the association between sICTP and the prognosis of plasma cell disorder patients, particularly in patients firstly treated with bortezomib-containing regimen. Methods We retrospectively reviewed untreated MM and other plasma cell disorder patients who were diagnosed at The University of Tokyo Hospital between January 2001 and December 2016. The clinical data of patients whose sICTP was measured before the start of first-line therapy were collected. Serum ICTP was measured at diagnosis in 59 patients: 56 patients with MM, 2 patients with plasmacytoma, and 1 patient with light chain amyloidosis. These patients were divided into high sICTP and low sICTP groups according to the median of sICTP level. Additionally, patients firstly treated with bortezomib-containing regimen were divided into two groups according to the median of sICTP level. Overall survival (OS) and bone fracture-free survival (FFS) were calculated from initiation of the first-line therapy using the Kaplan-Meier method and log-rank tests. Results In 59 patients, the median age was 65 years (range, 45-85 years) and median follow-up was 387 days (range, 22-2019 days). Thirty-three patients (55.9%) were male and 26 (44.1%) were female. IgG was the most frequent subtype of M-protein (58%), followed by IgA (19%), Bence Jones proteins (19%), and IgE (3%). International scoring system classified 16 (27.1%), 24 (40.7%), and 19 (32.2%) as stage I, II, and III, respectively. Levels of sICTP were high in 31 (52.5%) patients and low in 28 (47.4%) patients. The level of sICTP differed significantly (P<0.01) between the groups ISS stage I, II, and III, and increased parallel with the progression of the disease. Twenty-eight (47.4%) patients were administered bortezomib-containing regimen as first-line therapy, which was consisted of BD (bortezomib + dexamethasone), VCD (bortezomib + cyclophosphamide + dexamethasone), and VMP (bortezomib + melphalan + prednisolone). The remaining 31 (52.5%) patients were administered without proteasome inhibitors as first-line therapy. Nineteen patients (32.2%) achieved at least very good partial response after first-line therapy and 9 (15%) patients fractured after start of first-line therapy. Median OS for the entire cohort was 1874 days. In the Kaplan-Meier survival analysis, high sICTP (above upper limit: 6.0 mg/l) significantly associated with worse 2-year OS (63% versus 96%, P=0.02) and worse 2-year FFS (51% versus 86%, P=0.04). Serum ICTP may be a useful marker to predict survival and future bone fracture in our cohort, and these results are consistent with previous reports. Interestingly, in the patients firstly treated with bortezomib-containing regimen, the level of sICTP had no effect on 2-year FFS (90% with high sICTP versus 100% with low sICTP, P=0.53) and 2-year OS (95% with high sICTP versus 100% with low sICTP, P=1). Conclusion In general, high sICTP is considered to be a poor prognostic factor in MM patients, and similar results were also demonstrated in our cohort. Importantly, our data suggest that bortezomib may overcome poor prognosis conferred by high sICTP in plasma cell disorder patients. Further studies based on more cases are warranted to elucidate the efficacy of bortezomib in high sICTP patients. In addition, further studies on other proteasome inhibitors, such as carfilzomib or ixazomib are also needed. Disclosures Honda: Hitachi, Ltd.: Speakers Bureau. Toyama:Bristol-Myers Squibb: Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau; Celgene K.K.: Speakers Bureau; Daiichi Sankyo Conpany: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Speakers Bureau; Takeda Pharmaceutical Company Limited.: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Speakers Bureau; Chugai Pharmaceutical Company: Speakers Bureau. Kurokawa:Shire Japan K.K.: Speakers Bureau; Novartis Pharma K.K.: Research Funding; Daiichi Sankyo Conpany: Speakers Bureau; Pfizer Japan Inc.: Research Funding; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Speakers Bureau; Celgene K.K.: Consultancy, Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co.,Ltd.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Yakult Honsha Company: Speakers Bureau; Bioverativ Japan ltd.: Consultancy; Chugai Pharmaceutical Company: Consultancy, Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; MSD K.K.: Consultancy, Research Funding, Speakers Bureau; Shionogi & Co., Ltd: Consultancy, Honoraria; Teijin Limited: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Janssen Pharmaceutical K.K.: Speakers Bureau.

High-Throughput Functional Drug Screening and Genomic Analysis to Guide Individualized Therapy for Relapsed/Refractory Multiple Myeloma

Background: The response to treatment for patients with relapsed/refractory multiple myeloma (MM) is highly variable. This may in part be related to the broad genomic heterogeneity that has been reported across individual patients. We hypothesize that in vitro functional drug screening and correlated genomic analyses of patient tumor cells is feasible and may improve clinical treatment response. To address this, we developed a custom high-throughput drug sensitivity (HTS) assay enabling simultaneous testing of 170 chemotherapy drugs and targeted inhibitors, as well as correlative gene expression and mutational analysis by next-generation sequencing. We report on the feasibility of this approach in a clinical trial enrolling patients with relapsed/refractory MM (NCT03389347). Patients and Methods: Twenty patients with relapsed/refractory MM or secondary plasma cell leukemia were enrolled in the study and 15 (9 female, 6 male) had a sufficient number of plasma cells for HTS. The average number of prior treatment regimens among the tested patients was 5.8 (range 3-11) and all had previously received a proteasome inhibitor and immunomodulatory drug and 8 had prior autologous stem cell transplants. Nine patients had stage III disease by the Revised International Staging System (R-ISS) and 4 had stage II disease. Eight patients exhibited high-risk cytogenetics, including 4 patients with del(17p), 5 patients with t(4;14), and 2 patients with t(14;16). The sources of plasma cells utilized for the HTS assay included bone marrow aspirate or core biopsies, soft tissue biopsies of plasmacytomas, and blood samples from patients with circulating plasma cells. Additional blood and bone marrow samples were obtained for RNA sequencing, whole-exome sequencing, and targeted sequencing of circulating tumor DNA. The CLIA approved HTS assay was performed at the Quellos HTS Core at the University of Washington using CD138+plasma cells isolated by magnetic bead separation. Tumor cells were tested against a panel of 170 drugs that includes conventional agents and targeted inhibitors, some FDA approved and others investigational. Cell viability was assessed at 72 hours by a luminescent cell viability assay (Cell Titer Glo, Promega) and drug response was determined using the concentration of experimental compound required to achieve 50% in vitro response inhibition (IC50) and area under the dose-response curve (AUC). Results: We report that the HTS assay can be completed in a median of 5 days from sampling (range 4-6 days), establishing feasibility. The mean percentage of bone marrow plasma cells among patients who could be tested was 56% compared to 5% for patients who had insufficient cells. We observed heterogeneity in patient genomic profiles and in vitro drug responses. The drugs with the highest in vitro response include those commonly used in MM such as bortezomib, ixazomib, carfilzomib, and panobinostat, as well as those under investigation including venetoclax and selinexor. Sensitivity was observed for drugs that have not been investigated for the treatment of MM including alvocidib and omacetaxine. Progression on prior therapy was not consistently predictive of in vitro drug responses and could be a consequence of some myeloma cells retaining sensitivity. Correlative genomic analyses were associated with in vitro drug responses for a subset of compounds. Among the 8 patients who received individualized therapy guided by the assay, 6 had an evaluable response and 5 (83%) achieved effective disease control (stable disease or better). Updated clinical treatment outcomes will be reported at the ASH conference. Conclusions: In this study, we successfully demonstrate the feasibility of a CLIA approved HTS assay capable of delivering actionable results in time to inform individualized treatment decisions for patients with MM. Correlative gene expression and mutational analysis are associated with in vitro drug response. Finally, we demonstrate that some of the patients who received treatment guided by the results of the HTS assay achieved a clinical response. These data suggest that using functional drug screening to predict sensitivity or resistance to both approved and experimental agents warrants further testing. Validation of the HTS assay in a larger population of MM patients holds the potential to improve treatment outcomes. Disclosures Cowan: Cellectar: Consultancy; Janssen: Consultancy, Research Funding; Abbvie: Research Funding; Celgene: Consultancy, Research Funding; Juno: Research Funding; Sanofi: Consultancy. Green:Celgene: Consultancy; Cellectar Biosciences: Research Funding; GSK: Consultancy; Juno Therapeutics: Consultancy, Patents & Royalties, Research Funding; Seattle Genetics: Research Funding. Libby:Alnylam: Consultancy; Abbvie: Consultancy; Pharmacyclics and Janssen: Consultancy; Akcea: Consultancy. Silbermann:Janssen, Sanofi: Other: Consultant/Advisor. Becker:The France Foundation: Honoraria; Accordant Health Services/Caremark: Consultancy; AbbVie, Amgen, Bristol-Myers Squibb, Glycomimetics, Invivoscribe, JW Pharmaceuticals, Novartis, Trovagene: Research Funding. OffLabel Disclosure: We will discuss in-vitro sensitivity testing of drugs that have not been approved for the treatment of multiple myeloma.

TRIP13 Modulates Protein Deubiquitination to Promote Myeloma Progression

Thyroid Hormone Receptor 13 (TRIP13) is an exciting emerging epitope in numerous cancers and is related to genomic integrity. In multiple myeloma (MM), TRIP13 is the third most upregulated gene in the 70-gene high-risk signature and is one of the top 10 genes correlated with drug resistance and disease relapse. Expression of TRIP13 is elevated in MM cells, when compared to normal, smoldering MM, and MGUS plasma cells. MM patients with elevated TRIP13 expression had inferior event-free and overall survival. Here we introduce a novel role for TRIP13 in the modulation of ubiquitin homeostasis and in its contribution to cancer progression. Because TRIP13 has been shown to affect MAD2 stability in a proteasome-dependent fashion, we hypothesized that TRIP13 may modulate protein ubiquitination. The overexpression of TRIP13 in MM cell lines resulted in decreased cellular ubiquitination and similar decreases in ubiquitin were found in vivo upon the creation of a TRIP13 transgenic mouse (TRIP13TG). Conversely, targeted knockdown of TRIP13 through the expression of shRNA resulted in elevated cellular ubiquitin. While these results indicate that TRIP13 promotes deubiquitination, TRIP13 is known to have ATPase activity but not protease activity. Using in vitro deubiquitination assays we found that TRIP13 lacked endogenous deubiquitination capacity. However, TRIP13 did increase the efficiency of ubiquitin cleavage by USP7. To investigate the phenotypic outcome of TRIP13 overexpression, we crossed TRIP13TG mice with the Em-MYC mice, which spontaneously develop diffuse large B cell lymphoma (DLBCL) and/or Burkitts' lymphoma. Kaplan-Meier analyses demonstrated that expression of TRIP13 TG/ Em-MYC TG decreased overall survival by greater than 25% compared to TRIP13 WT/ Em-MYC TG mice. Treatment of TRIP13 TG/ Em-MYC TG mice with a USP7 inhibitor, P5091, rescued this phenotype, restored overall survival to nearly that of TRIP13 WT/ Em-MYC TG mice. These data indicate that USP7 plays a key role in TRIP13-induced tumor progression. We further investigated whether TRIP13 regulates USP7 important substrates, such as p53 and PTEN. RNA-sequencing analysis was performed on splenic premalignant B cells derived from both TRIP13 TG/ Em-MYC TG and TRIP13 WT/ Em-MYC TG mice. GSEA identified that p53 and PTEN signaling pathways were significant inhibited in TRIP13 TG/ Em-MYC TG mice compared to TRIP13 WT/ Em-MYC TG mice. Western blots confirmed that p53 protein was decreased in TRIP13 TG/ Em-MYC TG mouse tissues. Using cellular fractionation and immunoblotting, we found that PTEN was excluded from the nucleus in TRIP13 TG/ Em-MYC TG mice and TRIP13-overexpressing MM cell lines. Treatment with the USP7 inhibitor, P5091, restored p53 stabilization and nuclear PTEN localization in TRIP13 overexpressing MM cells, demonstrating that USP7 signaling pathways are involved in TRIP13-induced MM progression. These data describe an exciting new role for TRIP13 in the promotion of deubiquitination through USP7 and highlight TRIP13 as a target in MM. The elevated expression of TRIP13 has been associated with inferior outcomes in many forms of cancers suggesting that these results may have relevance beyond MM. Disclosures Zhan: BIPHARM LLC: Consultancy, Other: % Allocation of Profit.

Systematic Characterization of Genes Representing Preferential Molecular Vulnerabilities for Myeloma Cells Compared to Other Neoplasias - Implications for the Biology and Therapeutic Targeting of Myeloma

In the last 2 decades, the improved clinical outcomes for multiple myeloma (MM) patients have been driven predominantly by therapeutics which exhibit limited activity outside plasma cell (PC) dyscrasias; do not target specific oncogenic mutations in MM cells, but rather pathways which are critical for PCs and dispensable for normal or malignant cells of most other lineages. We reasoned that identification of genes that are more potently / recurrently essential for MM cells, but less so for other neoplasias, would allow us to "re-identify" targets of currently used "PC-selective" anti-MM therapies. We also reasoned that systematic identification of MM-preferential dependencies could also uncover additional, previously underappreciated, genes which can serve as targets for potential future therapies and hopefully contribute to additional improvements in the therapeutic outcome for MM. To this end, we performed genome-scale CRISPR gene-editing studies to systematically characterize the molecular vulnerabilities of 20 MM cell lines and define which of these genes are more pronounced and/or recurrent dependencies for MM vs. cell lines (n=679) from other blood cancers and solid tumors. We identified 90+ genes whose function was significantly more essential for MM lines than other neoplasias. These MM-preferential dependencies included a large collection of transcription factors (e.g. IRF4, PRDM1, MAF, NFKB1, RELB, IKZF3, IKZF1, TCF3, CCND2, CBFB, MEF2C); transcriptional cofactors (e.g. POU2AF1); epigenetic regulators (e.g. EP300, DOT1L, HDAC1,ARID1A, CARM1); kinases such as IKBKB and CHUK/IKKa (both upstream of NF-κB), PIM2, IGF1R, SIK3,STK11; genes related to endoplasmic reticulum (ER) or Golgi function (e.g. HERPUD1, SYVN1,UBE2J1, SEC23B); as well as BCL2 and SMAD7. Results for several of these genes were further supported by in vitro studies with individual sgRNAs for CRISPR-based gene editing or activation of the respective genes; "addback" experiments with CRISPR-resistant cDNAs; shRNA studies in MM lines; use of small molecule inhibitors (e.g. against PIM kinases, CBFB, CARM1); and a focused in vivo CRISPR screen with MM.1S cells implanted in mice with BM-like scaffolds harboring a "humanized" stromal compartment: this latter in vivo study examined 46 MM-preferential dependencies which are also essential for MM.1S cells in vitro and observed that 41 of these genes were also essential for MM.1Scells in the "humanized" BM-like in vivo system. Some MM-preferential dependencies are essential for subsets of leukemia or lymphoma lines, but most have more pronounced/recurrent essentiality in MM vs. other blood cancers. In terms of overexpression (in high- vs. standard-risk MM; MM vs. normal PCs; or MM vs other cancers); frequency of mutations, DNA copy number gain or proximity to superenhancers, most of the MM-preferential dependencies do not exhibit such alterations or are not ranked in the top-100 genes in terms of the magnitude or frequency of these alterations. Notably, among the MM-preferential dependencies identified in our study, the majority are universally expressed in MM patient samples, while >80% and >75% of these genes have detectable transcript levels (RPKM>1) in CD138+ cells from at least 50% or 80%, respectively, of newly-diagnosed MM patients (MMRF CoMMpass study), suggesting broad expression of these dependencies across MM patients, including individuals with high-risk disease. It was reassuring to observe that MM-preferential dependencies identified in our study include prominent known targets for therapeutics with relatively MM-selective clinical activity (e.g. thalidomide derivatives [IKZF1/IKZF3], proteasome inhibitors [NF-kappaB genes and ER function] or panobinostat [HDAC1]). The identification of these known genes as preferential MM dependencies provides a mechanistic explanation for the relatively selective clinical activity of the respective therapies in MM/PC dyscrasias and also underscores the promising therapeutic implications of the large number of additional and previously underappreciated / understudied MM-preferential dependencies identified in our CRISPR-based functional studies. Disclosures Boise: Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Gray:Gatekeeper, Syros, Petra, C4, B2S and Soltego.: Equity Ownership; Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield and Sanofi.: Equity Ownership, Research Funding. Tsherniak:Tango Therapeutics: Consultancy. Mitsiades:Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding.

In Vitro and in Vivo Activity of Melflufen in Amyloidosis

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.

Mechanical segregation and capturing of clonal circulating plasma cells in multiple myeloma using micropillar-integrated microfluidic device.

Multiple myeloma (MM), the disorder of plasma cells, is the second most common type of hematological cancer and is responsible for approximately 20% of deaths from hematological malignancies. The current gold standard for MM diagnosis includes invasive bone marrow aspiration. However, it lacks the sensitivity to detect minimal residual disease, and the nonuniform distribution of clonal plasma cells (CPCs) within bone marrow also often results in inaccurate reporting. Serum and urine assessment of monoclonal proteins, such as Kappa light chains, is another commonly used approach for MM diagnosis. Although it is noninvasive, the level of paraprotein elevation is still too low for detecting minimal residual disease and nonsecretive MM. Circulating CPCs (cCPCs) have been reported to be present in the peripheral blood of MM patients, and high levels of cCPCs were shown to correlate with poor survival. This suggests a potential noninvasive approach for MM disease progress monitoring and prognosis. In this study, we developed a mechanical property-based microfluidic platform to capture cCPCs. Using human myeloma cancer cell lines spiked in healthy donor blood, the microfluidic platform demonstrates high enrichment ratio (>500) and sufficient capture efficiency (40%-55%). Patient samples were also assessed to investigate the diagnostic potential of cCPCs for MM by correlating with the levels of Kappa light chains in patients.

MiR-16 regulates crosstalk in NF-κB tolerogenic inflammatory signaling between myeloma cells and bone marrow macrophages.

High levels of circulating miR-16 in the serum of multiple myeloma (MM) patients are independently associated with longer survival. Although the tumor suppressor function of intracellular miR-16 in MM plasma cells (PCs) has been elucidated, its extracellular role in maintaining a nonsupportive cancer microenvironment has not been fully explored. Here, we show that miR-16 is abundantly released by MM cells through extracellular vesicles (EVs) and that differences in its intracellular expression as associated with chromosome 13 deletion (Del13) are correlated to extracellular miR-16 levels. We also demonstrate that EVs isolated from MM patients and from the conditioned media of MM-PCs carrying Del13 more strongly differentiate circulating monocytes to M2-tumor supportive macrophages (TAMs), compared with MM-PCs without this chromosomal aberration. Mechanistically, our data show that miR-16 directly targets the IKKα/β complex of the NF-κB canonical pathway, which is critical not only in supporting MM cell growth, but also in polarizing macrophages toward an M2 phenotype. By using a miR-15a-16-1-KO mouse model, we found that loss of the miR-16 cluster supports polarization to M2 macrophages. Finally, we demonstrate the therapeutic benefit of miR-16 overexpression in potentiating the anti-MM activity by a proteasome inhibitor in the presence of MM-resident bone marrow TAM.

An Anti-BCMA RNA Aptamer for miRNA Intracellular Delivery

B cell maturation antigen is highly expressed on malignant plasma cells in human multiple myeloma and has recently emerged as a very promising target for therapeutic interventions. Nucleic-acid-based aptamers are small oligonucleotides with high selective targeting properties and functional advantages over monoclonal antibodies, as both diagnostic and therapeutic tools. Here, we describe the generation of the first-ever-described nuclease resistant RNA aptamer selectively binding to B cell maturation antigen. We adopted a modified cell-based systematic evolution of ligands by exponential enrichment approach allowing the enrichment for internalizing aptamers. The selected 2′Fluoro-Pyrimidine modified aptamer, named apt69.T, effectively and selectively bound B cell maturation antigen-expressing myeloma cells with rapid and efficient internalization. Interestingly, apt69.T inhibited APRIL-dependent nuclear factor κB (NF-κB) pathway in vitro. Moreover, the aptamer was conjugated to microRNA-137 (miR-137) and anti-miR-222, demonstrating high potential against tumor cells. In conclusion, apt69.T is a novel tool suitable for direct targeting and delivery of therapeutics to B cell maturation antigen-expressing myeloma cells.

Outpatient Autologous Stem Cell Transplants for Multiple Myeloma: Analysis of Safety and Outcomes in a Tertiary Care Center

BackgroundAutologous stem cell transplant (ASCT) is the preferred consolidation strategy to treat eligible patients with multiple myeloma (MM) and related plasma cell dyscrasias. Given the increasing volume of patients and longer wait time, outpatient ASCT for MM is the standard of care at the Vancouver General Hospital. Patients and MethodsPatients with MM, POEMS syndrome, and amyloidosis undergoing ASCT were included in this analysis. We analyzed patient characteristics, the number of patients requiring admission, duration of admission, 30-day and 100-day mortality, and overall survival. ResultsBetween January 2007 and June 2016, 724 patients underwent 752 ASCTs. Of these, 702 were first ASCTs, 44 were second, and 6 were third. The median age was 60 years (interquartile range [IQR], 54-65 years). Reasons for ASCTs were MM (96.9%) amyloidosis (2.4%), and POEMS syndrome (0.7%). There were 431 (59.5%) males in this group. The median time from diagnosis to transplant was 5 months. Conditioning was melphalan 200 mg/m2 for 89.6% of the patients. Admission to the inpatient ward was required by 245 (32.6%) patients within the first 30 days. The median time to admission was 9 days post-transplant (IQR, 5-13 days). The median duration of admission was 6 days (IQR, 3-9 days). The day 100 all-cause mortality rate was 0.9%, and transplant-related mortality was 0.4%. ConclusionOutpatient ASCT is a safe and feasible treatment strategy with low transplant-related mortality. Overall resource utilization is significantly lower than inpatient ASCT: however, this requires a multidisciplinary approach with close follow-up.

Effect of eIF4E on Autophagy of CD138+ Cells in Multiple Myeloma

To investigate the effect of eukaryotic translation initiation factor 4E(eIF4E) on the autophagy of CD138+ plasma cells in multiple myeloma(MM). Multiple myeloma CD138+ plasma cells were treated with eIF4E inhibitor 4EGI, the changes of autophagy-related factors LC3-II and Beclin1 were detected by fluorescent quantitative PCR and Western blot, the changes of cell proliferation inhibition were detected by MTT assay, and cell apoptosis was detected by flow cytometry. Quantitative fluorescence PCR showed that after treatment of myeloma cells with 4EGI, the expression levels of LC3-II and Beclin1 mRNA gradually increased with the enhancomer of 4EGI concentration and the prolongation of action time, and the differences were statistically significant (48 h: LC3-Ⅱ,r=0.942, Beclin1,r=0.952; 80 μg/ml: LC3-Ⅱ,r=0.966, Beclin1,r=0.998）; Western blot showed that with the enhancement of 4EGI concentration, the expression of LC3-II and Beclin1 protein gradually increased（LC3-Ⅱ,r=0.923, Beclin1,r=0.977）; CCK-8 showed that the inhibition rate of cells gradually increased （r=0.996）; the apoptotic rate of 4EGI-treated groups (23.23±4.47, 7.59±1.67, 2.03±0.19) was significantly different from that of control group (0.03±0.04) (P<0.05). The inhibition of eIF4E can activate the autophagy of CD138+ plasma cells in multiple myeloma and induce the death of myeloma cells.

DNA methyltransferase inhibitors upregulate CD38 expression and enhance daratumumab efficacy in multiple myeloma

The anti-CD38 monoclonal antibody Daratumumab is highly effective against multiple myeloma and is well tolerated. Daratumumab resistance in patients is correlated with CD38 loss (Nijhof et al, 2016). As a result, there is significant interest in elucidating the regulation of CD38 in MM. Recently All Trans Retinoic Acid (ATRA) and the FDA-approved histone deacetylase inhibitor panobinostat were both demonstrated to upregulate CD38 in MM plasma cells. We hypothesized that demethylation of the CpG island in the promoter and first exon of CD38 may lead to increased CD38 protein at the cell surface in MM plasma cells.

Inhibition of the JAK-STAT3-Mcl-1 Axis by Combining Ruxolitinib with S63845 Completely Prevents Plasmacytoma Growth in the Gp130-Regulated INA-6 Xenograft Mouse Model

Growth of malignant plasma cells in multiple myeloma is regulated by the JAK/STAT3 pathway via cytokines such as interleukin(IL)-6 which is produced in the bone marrow microenvironment. IL-6 stimulation leads to upregulation of anti-apoptotic proteins of the Bcl-2 family including Mcl-1 and/or Bcl-xL. Growth of INA-6 plasmacytomas in SCID mice depends on activation of the signal transducer gp130, the common receptor for IL-6 and other cytokines of the IL-6/gp130 family (Burger et al., Haematologica 102, 2017).

Targeting of versican, a proteoglycan in bone marrow microenvironment by microRNAs inhibit multiple myeloma progression

Abstract Background The abnormal growth of malignant plasma cells in Multiple myeloma (MM) require bone marrow (BM) niche consisting of proteoglycans, cytokines, etc. for their growth. One of the chondroitin sulfate proteoglycan, Versican (VCAN) has gained consideration in milieu of solid tumors but there is dearth of literature in MM. Hence, involvement of VCAN in MM and its regulation by microRNAs has been studied. Materials and Methods 30 MM patients & 20 controls were recruited and BM Stromal Cells (BMSCs) were harvested by primary culture. Molecular expression of VCAN and microRNAs (miR-144, miR-199 & miR-203) were examined in study subjects and MM cell lines (RPMI8226 & U266). The involvement of VCAN in myeloma pathogenesis was studied using BMSCs conditioned medium (BMSCs-CM) and VCAN-neutralizing antibody or microRNA mimics (miR-144 and miR-199). Further, signaling pathways altered by knockdown of VCAN by antibody or microRNA mimics were identified. Results Elevated expression of VCAN was found in MM patients especially in the stroma of BM niche while microRNA expression was lower in patients and showed negative correlation with VCAN levels. VCAN being produced in stroma found at lower levels in MM cell lines. Moreover, BMSCs CM showed presence of VCAN which upon supplementing to MM cells alter parameters in favour of myeloma progression, however, this effect was neutralized by VCAN antibody or miR mimics of miR-144 and miR-199. The downstream signaling of VCAN was found to activate FAK and STAT3 which subsides by using VCAN antibody or miR mimics. Conclusion The neutralization of oncogenic effect of BMSCs CM by VCAN blockage using antibody or miR mimics affirms its plausible role in progression of MM. These findings open up new avenues for exploring VCAN as a novel therapeutic target and microRNAs as a mean to target VCAN which could be exploited in clinical settings in future.

Stabilization of amyloidogenic antibody light chains as a potential therapeutic strategy in AL amyloidosis

Clonal plasma cells in multiple myeloma and related conditions secrete immunoglobulins, including free light chains. In systemic AL amyloidosis, these light chains (LCs) or their fragments can misfold and aggregate to form insoluble, non-native amyloid fibrils that cause organ damage and eventually death. Of the 3-4,000 Americans diagnosed each year with AL amyloidosis, 1,000 will die within a year of diagnosis, mainly due to heart failure. No drugs are approved specifically for AL amyloidosis, but off-label use of cytotoxic regimens approved for multiple myeloma can be effective.