Multiple Myeloma Drug Research Articles

Improved survival of multiple myeloma patients treated with autologous transplantation in the modern era of new medicine.

New drugs for multiple myeloma (MM) have dramatically improved patients’ overall survival (OS). Autologous stem cell transplantation (ASCT) remains the mainstay for transplant‐eligible MM patients. To investigate whether the post‐ASCT prognosis of MM patients has been improved by new drugs, we undertook a retrospective observational analysis using the Transplant Registry Unified Management Program database in Japan. We analyzed 7323 patients (4135 men and 3188 women; median age, 59 years; range 16‐77 years) who underwent upfront ASCT between January 2007 and December 2018. We categorized them by when they underwent ASCT according to the drugs’ introduction in Japan: group 1 (2007‐2010), group 2 (2011‐2016), and group 3 (2017‐2018). We compared the groups’ post‐ASCT OS. The 2‐year OS rates (95% confidence interval [CI]) of groups 1, 2, and 3 were 85.8% (84.1%‐87.4%), 89.1% (88.0%‐90.1%), and 92.3% (90.0%‐94.2%) (P < .0001) and the 5‐year OS (95% CI) rates were 64.9% (62.4%‐67.3%), 71.6% (69.7%‐73.3%), and not applicable, respectively (P < .0001). A multivariate analysis showed that the post‐ASCT OS was superior with these factors: age less than 65 years, performance status 0/1, low International Staging System (ISS) stage, receiving SCT for 180 days or less post‐diagnosis, better treatment response pre‐ASCT, later year of ASCT, and receiving SCT twice. A subgroup analysis showed poor prognoses for the patients with unfavorable karyotype and poor treatment response post‐ASCT. The post‐ASCT OS has thus improved over time (group 1 < 2 < 3) with the introduction of new drugs for MM. As the prognosis of high‐risk‐karyotype patients with ISS stage III remains poor, their treatment requires improvement.

USP15 antagonizes CRL4CRBN-mediated ubiquitylation of glutamine synthetase and neosubstrates.

Targeted protein degradation by the ubiquitin-proteasome system represents a new strategy to destroy pathogenic proteins in human diseases, including cancer and neurodegenerative diseases. The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide have revolutionized the treatment of patients with multiple myeloma (MM) and other hematologic malignancies, but almost all patients eventually develop resistance to IMiDs. CRBN, a substrate receptor of CUL4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase, is a direct target for thalidomide teratogenicity and antitumor activity of IMiDs (now known as Cereblon E3 ligase modulators: CELMoDs). Despite recent advances in developing potent CELMoDs and CRBN-based proteolysis-targeting chimeras (PROTACs), many questions apart from clinical efficacy remain unanswered. CRBN is required for the action of IMiDs, but its protein expression levels do not correlate with intrinsic resistance to IMiDs in MM cells, suggesting other factors involved in regulating resistance to IMiDs. Our recent work revealed that the CRL4CRBN-p97 pathway is required for degradation of natural substrate glutamine synthetase (GS) and neosubstrates. Here, I show that USP15 is a key regulator of the CRL4CRBN-p97 pathway to control stability of GS and neosubstrates IKZF1, IKZF3, CK1-α, RNF166, GSPT1, and BRD4, all of which are crucial drug targets in different types of cancer. USP15 antagonizes ubiquitylation of CRL4CRBN target proteins, thereby preventing their degradation. Notably, USP15 is highly expressed in IMiD-resistant cells, and depletion of USP15 sensitizes these cells to lenalidomide. Inhibition of USP15 represents a valuable therapeutic opportunity to potentiate CELMoD and CRBN-based PROTAC therapies for the treatment of cancer.

Effectiveness of Long-Term Treatment of Multiple Myeloma in Regular Care: Comparison of a Longitudinal and a Cross-Sectional Analysis Approach

Introduction: New drugs for multiple myeloma have considerably increased the options for consecutive treatment lines in regular care. Official treatment guidelines still discuss several regimens per line, and therefore, current practice is of topical interest. Large cross-sectional studies revealed a greater than linear loss of patients reaching consecutive treatment lines of ever decreasing effectiveness. Methods: In a longitudinal approach, we analyzed data of all 145 multiple myeloma patients treated in our outpatient clinic in Germany between January 1, 2012, and December 31, 2019, using a time-to-event analysis with death as competitive risk. Results: The estimated incidences of reaching the 2nd, 3rd, 4th, and 5th lines of therapy were 88, 66, 44, and 30%, respectively. Median times to subsequent treatment lines were 34, 18, 14, 13, and 15 months, respectively. Discussion: Percentages of patients reaching later therapy lines were considerably greater than predicted by cross-sectional studies and median times after the 1st line did not suggest a further decrease in effectiveness, while use of new drug regimens was similar to that reported in cross-sectional studies. Conclusion: Effectiveness of later therapy lines appears to be underestimated by cross-sectional analyses, and the conveyed focus on 1st-line treatment for multiple myeloma needs to be scrutinized.

Place in therapy of innovative drugs in multiple myeloma in 2021 and 2023 according to an expert panel Delphi consensus.

The objective of this study was to understand the potential use of single agents and drug combinations in multiple myeloma (MM) across treatment lines in the years 2021 and 2023. The method used was Delphi Panel Method survey, administered to European Myeloma Network (EMN) Italy Working Group centres. Future treatments were identified assessing all available web-based information sources, including therapies (single drugs or combinations) with strong evidence of efficacy, likely to be on the Italian market in 2021 and 2023. Participants were asked to report on the likelihood of prescription for MM therapies, across treatment lines. Across the 15 centres taking part in the survey, about 890 patients per year are forecasted to receive a new diagnosis of MM. In 2021, the Panel forecasted 66% of 1L-TE (transplant eligible) patients will be treated with bortezomib-thalidomide-dexamethasone (VTD) and 32% of patients with daratumumab-bortezomib-thalidomide-dexamethasone (DVTd), with a substantial decrease of VTD (15%) and a marked increase of DVTd (81%) forecasted for 2023. The 2L and 3L R(lenalidomide)-based combination treatments are expected to drop and will likely be substituted by a steep increase in P(pomalidomide)-based regimes (from 7% to 23%). On the contrary, in 3L treatment, all combination therapies (with the exception of IsaPd - isatuximab-pomalidomide-dexamethasone) are expected to lose market share in favour of the most recent new therapies. Expert Panel agrees that many different new drugs and combinations will be used in MM, with different mechanisms of action, both at diagnosis and in subsequent phases of the disease, with a corresponding decline of the drugs currently used.

Abstract LB217: ROBO1 promotes homing, dissemination, and survival of multiple myeloma within the bone marrow microenvironment

Abstract Introduction: The bone marrow (BM) niche promotes multiple myeloma (MM) growth, survival and drug resistance. Therapies targeting both cancer cells and the microenvironment are highly effective. We were interested in identifying novel signaling pathways supporting MM pathogenesis through MM-BM crosstalk. The transmembrane receptor Roundabout 1 (ROBO1) plays a role in growth and dissemination of solid tumors, however its function in MM is unknown. Material and Methods: We analyzed ROBO1 expression in cell lines and primary samples via western blot, immunohistochemistry (IHC) and gene expression profiling. We used short hairpin RNA and CRISPR-Cas9 for ROBO1 knock down (KD) and knock out (KO), respectively. For protein structure-function and rescue studies, we stably expressed full-length (FL) or mutant ROBO1 devoid of extracellular (Cyt) or intracellular domain (DeltaCyt), including patient-derived G674* truncation, with a C-terminus FLAG tag. We used a 3D hydrogel encapsulation system to study proliferation; FLAG immunoprecipitation (IP) followed by mass spectrometry to identify ROBO1 interacting partners; and immunofluorescence to detect ROBO1 localization. To study tumor growth in vivo, we performed PET-CT of mice inoculated subcutaneously or intramedullary with WT or ROBO1 KO MM cells and retrieved tumors for RNA sequencing. To study dissemination and homing, KO and FL addback MM cells were injected intravenously in SCID mice. Mice were monitored for development of tumors or hindlimb paralysis and femora/tumors harvested once mice reached endpoint. Results: ROBO1 is highly expressed in MM cell lines and primary cells but low/absent in normal plasma cells and other hematologic cancer cell lines. ROBO1 KD is specifically cytotoxic for MM cells and ROBO1 KO decreases proliferation, a phenotype fully rescued by FL ROBO1. Compared to WT, ROBO1 KO significantly decreases intramedullary (mean tumor volume (MTV): 1323 vs 457 mm3, p value= 0.02) and extramedullary (MTV: 2684 vs 823 mm3, p value= 0.001) tumor growth in vivo. We further discovered that ROBO1 KO decreases adhesion of MM to BM endothelial and BMSC, which is fully rescued by FL ROBO1. In a disseminated mouse model, ROBO1 KO cells generate bone plasmacytoma with reduced BM invasion, as compared to the extensive BM infiltration observed with ROBO1 FL cells. Consistently, in primary samples from patients, we detected ROBO1 expression only in 1 out of 10 solitary plasmacytoma (dim staining) as compared to 14 out of 14 MM bone marrow biopsy samples tested (11 strong, 3 dim, p value= 0.0001). Mechanistically, we show for the first time that ROBO1 C-terminus is cleaved in a ligand-independent fashion; translocates to the nucleus; and is necessary and sufficient to rescue ROBO1 KO proliferative defect. Viceversa, mutants lacking the cytoplasmic domain, including the G674* truncation, act dominantly negative. Interactomic and RNA sequencing studies point to a previously unknown function of ROBO1 in RNA processing, setting the bases for future studies. Conclusions: We show for the first time that ROBO1 is necessary for MM growth and homing to the BM. Cleaved ROBO1 cytosolic domain translocates to the nucleus and is necessary and sufficient to rescue ROBO1 KO proliferative defect, possibly by participating in RNA processing. These data suggest that ROBO1 C-terminus may be a novel molecular target in MM. Citation Format: Giada Bianchi, Peter G. Czarnecki, Matthew Ho, Aldo M. Roccaro, Antonio Sacco, Yawara Kawano, Annamaria Gulla, Anil Aktas Samur, Tianzeng Cheng, Kenneth Wen, Yu-Tzu Tai, Maria Moscvin, Xinchen Wu, Gulden Camci-Unal, Matteo Claudio Da VIa, Niccolo Bolli, Ruben D. Carrasco, Irene M. Ghobrial, Kenneth C. Anderson. ROBO1 promotes homing, dissemination, and survival of multiple myeloma within the bone marrow microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB217.

The Novel Subtype-Selective Histone Deacetylase (HDAC) Inhibitor, Chidamide, Exerts Dual Anti-Myeloma and Bone Protective Effect in Vitro and In Vivo

Background and Purpose: Despite the advance in multiple myeloma treatment, drug resistance is still inevitable and the prognosis of most patients remains poor. Chidamide, a new subtype-selective histone deacetylase (HDAC) inhibitor, which is produced independently in China, is designed to inhibit the activity of HDAC 1, 2, 3 and 10. In the present work, we investigated the effect of chidamide on multiple myeloma and its associated osteolytic bone disease with different models.Experiment Design: In vitro cytotoxicity of chidamide was tested on myeloma cell lines (MM.1s, CAG, ARP-1, 8226, U266, H929, LP-1, ANBL-6, OPM2) with CCK-8 and flow cytometry. Synergistic combinations with standard drugs in 8226 and CAG cell lines were analyzed by compusyn software. The effect on osteoclast formation was assessed in osteoclastic cultures. The mechanisms of chidamide were explored by western blot and immunofluorescence. Murine models were employed for in vivo works, IVIS in vivo imaging system, ELISA (human Ig lambda, CTX-I, PINP) and micro-CT were used to surveillance the tumor burden and examine the bone destruction.Results: The cytotoxic effect of chidamide on myeloma cells is due to induction of cell apoptosis and cell cycle disruption (G1 phase arrest) in a dose dependent manner (IC50 varies from 2 to 5 uM in different cell lines) which mediated by increasing Cleaved Caspase-3, Caspase-7, Caspase-8, Caspase-9, p21 and p27, decreasing C-Myc, Mcl-1, Bcl-xL, CDK4 and CDK6. SensitiveSOCS3/JAK2/STAT3 cell signaling was involved in regulating these proteins by increasing the expression of SOCS3, reducing the expression of phospho-JAK2 and phospho-STAT3, thus inhibiting the proliferation and inducing cell cycle disruption. After treating with chidamide, the expression levels of HDAC1, 2, 3, 10 were not changed significantly, which indicated chidamide suppressed the function of these targets while not affected their expression. Notably, chidamide exerted synergistic anti-myeloma effect with dexamethasone, carfilzomib or pomalidomide (combination index < 1). Also, chidamide showed effective cytotoxicity in the co-culture of myeloma cells with bone marrow stromal cells and osteoclasts. Importantly, chidamide suppressed in vitro osteoclast formation and resorption, inhibited the expression of the key molecules (Cathepsin K, c-fos, NFATc1) and de-phosphorylation of p-AKT and p-JNK, and disrupted the F-acting ring formation. Finally, chidamide significantly reduced the tumor volume in the subcutaneous xenografts models, also the level of monoclonal protein Ig lambda secreted by myeloma cells decreased in disseminated myeloma murine models after treatment. Moreover, chidamide not only prevented tumor-associated bone loss in the disseminated murine model which was partially related with reducing the tumor burden but also had a direct bone protective effect on RANKL-induced rapid bone loss in non-tumor bearing mouse model with serum PINP (bone formation) and trabecular numbers increased while serum CTX-I (bone resorption) and trabecular space diminished compared with the vehicle group.Conclusion: Our work demonstrates dual anti-myeloma and bone protective effect of chidamide in vitro and in vivo, and shows its synergistic anti-myeloma effect with dexamethasone, carfilzomib and pomalidomide. These findings strongly suggest the potential clinical use of this drug in multiple myeloma in the near future. DisclosuresNo relevant conflicts of interest to declare.

The Mechanisms of PIM Kinase Inhibitor, SGI-1776 Free Base, in Treating Myeloma-Associated Bone Disease

Background and Purpose: The PIM kinase family, which includes PIM1, 2 and 3 and is expressed in a variety of solid and hematological malignancies, has an essential role in cell growth and survival. Multiple myeloma remains an incurable hematologic cancer which always companions with osteolytic bone disease. In this work, we investigated the mechanisms of a PIM kinase inhibitor, SGI-1776 free base, in anti-myeloma-associated osteolytic bone disease in vitro and in vivo.Experiment Design: CCK-8 and flow cytometry were used to assess the cytotoxicity of SGI-1776 free base on myeloma cell lines (MM.1s, CAG, ARP-1, 8226, H929, LP-1, OPM2) . The effect on RANKL-induced osteoclasts was assessed in osteoclastogenic and osteoblastic cultures. Western blot and immunofluorescence were used to investigate the mechanisms of SGI-1776 free base. SiRNAs were used to mimic the inhibition of PIM kinase by the drug. IVIS in vivo imaging system, ELISA (human Igk, CTX-I, PINP) and micro-CT (parameters such as trabecular numbers and trabecular space) were used to surveillance the tumor burden and examine the bone destruction in murine models.Results: SGI-1776 free base induced apoptosis and cell cycle arrest in the G1 phase in myeloma cells mediated by decreasing the phosphorylation of PIM kinase targets C-Myc, Bad, 4EBP1 and H3. Also, the expression of Mcl-1, C-Myc, cyclin D2 and cyclin E1 were reduced, and the inhibition of NF-kB and STAT3 pathways also observed. The IC50 varies from 2 to 6 uM in different cells. Expression of apoptosis related proteins Cleaved Caspase-3, 7, 8, 9 and PARP-1 were increased. Importantly, SGI-1776 free base could overcome the protective effect of bone marrow stromal cells and osteoclasts on myeloma cells. Knocking down PIM kinase (PIM-1, 2, and 3) by SiRNAs slowed down the proliferation rates of myeloma cells (8226 and MM.1s), indicating the pro-growth impact of PIM kinase on myeloma. Notably, SGI-1776 free base inhibited osteoclast formation and resorption though decreasing PIM-1, c-fos, NFATc1, Cathepsin K and DUSP-1. Calcium coated slide assay, TRAP staining and F-acting ring formation also proved the inhibition effect. Also, phosphorylation levels of Erk, p-38, p-65, AKT and AMPK were reduced in the cells treated with SGI-1776 free base. Knocking down PIM-1 impaired the F-acting ring formation and reduced OC numbers compared with controls, and STAT3 pathway was inhibited as well. ALP staining, ALP activity and alizarin red staining showed SGI-1776 would not suppress osteoblasts formation and its mineralization. Finally, SGI-1776 free base reduced the tumor burden in subcutaneous and disseminated myeloma murine models and prevented myeloma associated bone loss with serum PINP (bone formation) and trabecular numbers increased while serum monoclonal Igk (tumor burden), CTX-I (bone resorption) and trabecular space diminished compared with the vehicle group. Moreover, SGI-1776 free base could directly reduce the RANLK-induced rapid bone loss in non-tumor bearing mouse model.Conclusion: Our investigation reveals the mechanisms of SGI-1776 free base in treating myeloma associated bone disease. The drug has a dual effect of anti-myeloma and bone protection. These results support the clinical use of this drug in multiple myeloma. DisclosuresNo relevant conflicts of interest to declare.

Myeloma-Associated Adipocytes Exhibit Reduced Adipogenic Gene Expression and Delipidation

Multiple Myeloma (MM) progression and drug resistance depend on bidirectional interactions with the local microenvironment that results both in a destruction of the marrow and survival of the tumor. Similarly to how MM cells both depend on and destroy (or render dysfunctional) BM mesenchymal stromal cells (MSCs), we hypothesized that MM cells also affect and are affected by BM adipocytes (BMAs), contributing to disease progression. Obesity, which is a risk factor for MM development with overweight individuals being 1.5-2 times more likely to develop the disease, also correlates to increased bone marrow adipose tissue (BMAT). Similarly, correlations between aging and BMAT, as well as aging and MM exist. These correlations suggest a role for BMAs in the development and progression of MM and make elucidating the signals and cross-talk between MM and BMAT extremely relevant in the understanding of the disease. BMAT-derived signaling molecules may promote myelomagenesis and enhance tumor growth, but this has been largely unexplored. In addition, while the effect of myeloma on bone cells has been widely characterized within the past few years, the direct effect of MM cells on BMAs is unknown and potentially revolutionary for our understanding and treatment of MM.In the present study, we explored the direct effects of MM cells on adipocytes and BMAs in 2D and novel 3D tissue engineered cultures. 3T3-L1 pre-adipocytes or primary mouse or human BM-MSCs were differentiated into lipid-laden adipocytes using previously published methods, and co-cultured with MM cell lines (MM.1S, MM.1R, 5TGM1, and OPM2). For mature adipocytes, cells were differentiated for approximately 9 days (mouse) or 21 days (human) prior to myeloma co-culture. To investigate the effect of myeloma cells on MSC adipogenic differentiation capacity, pre-adipocytes were grown to confluency and then myeloma cells were added for a 3-day exposure period prior to adipogenic differentiation. Direct and indirect (MM conditioned media (CM) and transwell cultures) effects of myeloma cells on MSC adipogenesis were assessed with oil red-o functional assays for lipid accumulation and/or qRT-PCR of total adipocyte RNA. Statistical significance was determined via Student's T-test or ANOVA. We also developed a novel, tissue-engineered (TE) 3-D in vitro MAT model using silk scaffolds seeded with MSCs grown in adipogenic media. TE-MAT was validated to represent normal, healthy MAT using confocal imaging and microarray. MM cells (MM.1S, 5TGM1 and OPM2) were cultured on TE-MAT for up to 2 weeks and examined with confocal microscopy; lipid accumulation was detected via Oil Red-O staining in these cultures.Indirect co-culture of 3T3-L1 adipocytes with myeloma cells severely and significantly reduced both adipogenic transcription factors (Fig. 1A- Ppar γ, OPM2 p<0.01; Cebp α MM.1S p<0.01, OPM2 p<0.0001, 5TGM1 p<0.001) as well as mature adipocyte markers (Fig. 1B- Adipoq, MM.1S p<0.001, 5TGM1 p<0.001, and OPM2 p<0.01). This effect was also observed in BM-MSCs differentiated into adipocytes from mouse (Fig. 1C- Ppar γ, MM.1S p<0.0001, 5TGM1 p<0.05, MM.1R p<0.0001; Fig. 1D- Adipoq, MM.1S p<0.0001, MM.1R p<0.0001), and human donors (Fig. 1E- PPAR γ, MM.1S p<0.05; Fig. 1F- FABP4, OPM2 p<0.0001), suggesting a common mechanism of MM-induced inhibition between peripheral adipocytes and BMAs. Lipid accumulation was also reduced in 3T3-L1 adipocytes in direct co-culture with MM.1S cells for 7-14 days. The trend for this effect was apparent beginning at 5 days and significant at later time points (Fig. G- 1 week p<0.01; 2 weeks p<0.05). Similarly, preliminary results showed decreased adipogenic potential from MM patient-derived MSCs vs normal controls in oil red-o staining. Co-culture with myeloma cells reduced the expression of genes involved in lipogenesis and fatty acid oxidation in both adipocyte cell types including: Srebf1 (mMAT, MM.1S p<0.0001, 5TGM1 p<0.001), Cpt1 (3T3-L1, MM.1S p<0.001), Fasn (3T3-L1, MM.1S p<0.0001) suggesting a suppression of adipogenic function as well as phenotype. Importantly, similar delipidation was also observed in TE-MAT co-cultures with myeloma cells (Fig. 1H). These combined results indicate an adverse effect of myeloma cells on adipocytes, and illuminate the BMA as a new member of the complex microenvironmental milieu in which the myeloma cell thrives. [Display omitted] DisclosuresFalank:BioPact Ventures, LLC: Research Funding. Reagan:BioPact Ventures, LLC: Research Funding.

The Exosomes-Derived EGFR Ligand Amphiregulin (AREG) Is a New Pro-Osteoclastogenic Factor in Multiple Myeloma

Growing evidence indicates the critical role of the microvesicles to sustain multiple myeloma (MM) cell viability, survival and drug resistance into the bone marrow (BM) microenvironment. Recently it has also been suggested that MM cell-derived exosomes have a role in the induction of osteoclasts (OCs) differentiation and activity. The mechanism by which exosomes might be involved in MM-induced osteoclastogenesis is not known and was investigated in this study focusing on the possible role of Epidermal growth factor receptor (EGFR) ligands because it has been previously published their involvement in osteolytic bone metastasis by breast and lung cancer.Thus the aim of the study was to investigate the presence of EGFR ligands in exosomes derived from different MM cell lines and primary CD138+ cells and to determine whether exosome-enclosed EGFR ligands are able to modulate the tumor microenvironment by affecting the function of OCs.We firstly analyzed the expression of EGFR ligands at mRNA level on plasmacells (PCs) from patients with different monoclonal gammopathies (11 monoclonal gammopathy of undetermined significance, 133 MM patients at the diagnosis, 9 primary plasma cell leukemia) in a published dataset (accession number GSE16122) and we found that CD138+ cells expressed Amphiregulin (AREG) mRNA at high but variable levels among MM patients. Consistently, in a different dataset (GSE2658) of PCs, it has been previously reported that, among the EGFR ligands, AREG is significantly over-expressed by MM cells as compared to normal PCs (GSE5900).Secondly we isolated exosomes by ultracentrifugation, from MM1S cell line and from BM plasma samples of 4 patients with active MM, and were characterized by western blot analysis of the exosome marker TSG101. We found that AREG was specifically enriched in exosomes derived from both MM1S and plasma samples of 3 patients, as confirmed by its absence in the exosome-deprived conditioned medium of MM1S cell line.In order to test whether MM-exosomes could potentiate MM-induced osteoclastogenesis through the activation of the EGFR pathway, primary CD14+ monocytes purified from peripheral blood (PB) of 3 healthy donors and a murine cell line (RAW264.7) were used as OC models. Cells were treated with (i) exosomes (25 mg/ml) from both MM1S and plasma samples, pre-treated or not with anti-AREG neutralizing antibodies (Abs) (20 mg/ml) or (ii) with recombinant human (rh) AREG (20 mg/ml). Interestingly we found that MM1S exosome treatment induced the activation of EGFR as showed by the increase EGFR phosphorylation in RAW 264.7 cell line; this effect was reverted in the presence of anti-AREG neutralizing Ab. In addition we found that the six days treatment with MM1S exosomes induced a significant increase in the mRNA expression level of SNAIL, a downstream target of EGFR, both in RAW 264.7 and PB CD14+. The presence of anti-AREG neutralizing reverted this effect. Finally we showed a significant increased of the expression of OC specifics marker such as Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant Acid Phosphatase (TRAP), both in RAW 264.7 and PB CD14+ cells after treatment with MM-derived exosomes as compared to the control. The pro-osteoclastogenic effect of MM-derived exosomes was significantly abrogated by exosome pre-treatment with anti-AREG neutralizing Ab suggesting a possible direct effect of MM-exosome derived AREG on OC differentiation. However we also performed treatment of human pre-osteoblasts (PreOBs), obtained from mesenchymal stromal cells, with MM-derived exosomes in the presence or the absence of anti AREG Ab hypothesizing an indirect pro-osteoclastogenic effect mediated by the inhibition of osteoprotegerin (OPG) production by PreOBs.In conclusion, our data indicate that the EGFR ligands are packed into MM-derived exosomes and involved in OCs differentiation thus highlighting the role of AREG as a potential new player in MM-induced bone resorption. DisclosuresNo relevant conflicts of interest to declare.

Costs Associated with Productivity Loss Among U.S. Patients Newly Diagnosed with Multiple Myeloma Receiving Oral Versus Injectable Chemotherapy

BackgroundThe use of novel Multiple Myeloma (MM) drug agents has been associated with improved therapeutic outcomes and survival; however, MM continues to pose a significant economic burden on both patients and healthcare systems. Evaluating the economic implications and outcomes of available treatments is important to improve efficiency of care.Patients with MM may experience productivity loss, including lost days from work or inability to work due to MM symptoms or to undergoing treatment. Although direct costs of illness have been well described in the literature, indirect costs associated with MM are understudied. Here, we examine the extent of workplace productivity loss among U.S. adult patients with MM and associated costs. These outcomes were compared among patients who received injectable versus oral MM therapy.MethodsAnalyses were conducted using the Truven Health Analytics MarketScan Commercial Claims and Encounters with Medicare Supplemental Coordination of Benefits and Health and Productivity Management Databases. Subjects at least 18 years of age diagnosed with MM between January 1, 2008 and December 31, 2014 were included in our study and followed until December 31, 2015 (Figure 1). Subjects were analyzed one year prior to, and one year following first MM diagnosis. Patients receiving only oral therapy were compared with those who received injectable therapy.Productivity loss was evaluated using several key variables found within workplace absenteeism (WAB), short-term disability (STD), and long-term disability (LTD) claim files. Workplace productivity loss (WPL), an aggregate of absenteeism days, was calculated using claims for WAB, STD, and LTD benefits. Cost of productivity loss was calculated for each patient by multiplying the number of absenteeism days (as indicated in the aggregate WPL measure) by the average daily wage for all occupations, as reported by the U.S. Bureau of Labor Statistics (Table 1).Absenteeism days and costs were compared among study groups using multi-variable zero-inflated Poisson regression. The following variables were used as covariates in the model: age, index year, gender, Charlson comorbidity index, and plan type. An interrupted time-series analysis was also conducted to evaluate changes in productivity losses in the pre- vs. post-diagnosis periods, and to compare these among the treatment groups.ResultsThe final study cohort included 299 patients with newly diagnosed MM, of whom 73 received oral therapy and 226 received injectable therapy. Baseline characteristics of the study groups are depicted in Table 2. In the zero-inflated Poisson regression, treatment type was a significant predictor of productivity loss (Figure 2). Patients who received injectable therapy missed an average of 110 work days in the one year post-diagnosis, compared with 88 for patients receiving only oral therapy (difference of 22 days, 95% CI: 19 - 25, p<0.001). Treatment type was also a significant predictor of cost of lost productivity in this model (Figure 3). Patients who received injectable therapy experienced productivity loss valued at $18,329, compared with patients who only received oral drug therapy ($14,467). The difference in valuated productivity losses between study cohort groups was also statistically significant ($3,862, 95% CI: $3,518 - $4,205, p<0.001).An interrupted time-series analysis showed a significant increase in missed work days per month following the initial diagnosis of MM for both treatment groups. Patients receiving injectable therapy experienced an immediate increase of 6.9 lost productivity days per month (95% CI: 5.1, 8.7, p<0.001), versus patients receiving oral therapy only (4.4 days per month; 95% CI: 2.7, 6.2, p<0.001). The difference in these values was not statistically significant, although a strong trend was observed (2.5 day per month; 95% CI: -0.1, 5.0, p=0.057).ConclusionsIn conclusion, patients newly diagnosed with MM face significant losses in productivity. Injectable MM therapy users experience significantly more utilization of disability benefits and incur higher productivity costs, compared to oral MM therapy users. Further studies elucidating the nature of the differences between parenteral and non-parenteral chemotherapy users are needed. [Display omitted] DisclosuresMerola:Millennium Pharmaceuticals, Inc.: Consultancy. Yong:Millennium Pharmaceuticals, Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Noga:Millennium Pharmaceuticals, Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Shermock:Millenium Pharmaceuticals: Consultancy.

Drug and Solute Transporters in Mediating Resistance to Novel Therapeutics in Multiple Myeloma.

Multiple myeloma remains an incurable malignancy of plasma cells. Novel therapies, notably proteasome inhibitors and immunomodulatory drugs, have improved the survival of multiple myeloma patients; however, patients either present with, or develop resistance to, these therapies. Resistance to traditional chemotherapeutic agents can be caused by cellular drug efflux via adenosine triphosphate (ATP)-binding cassette (ABC) transporters, but it is still not clear whether these transporters mediate resistance to proteasome inhibitors and immunomodulatory drugs in multiple myeloma. Solute carrier (SLC) transporters also play a role in cancer drug resistance due to changes in cell homeostasis caused by their abnormal expression and changes in the solutes they transport. In this review, we evaluate resistance to novel therapies used to treat multiple myeloma, as mediated by drug and solute transporters.

Hsp90/C terminal Hsc70-interacting protein regulates the stability of Ikaros in acute myeloid leukemia cells.

The stability of Ikaros family zinc finger protein 1 (Ikaros), a critical hematopoietic transcription factor, can be regulated by cereblon (CRBN) ubiquitin ligase stimulated by immunomodulatory drugs in multiple myeloma. However, other stabilization mechanisms of Ikaros have yet to be elucidated. In this study, we show that the pharmacologic inhibition or knockdown of Hsp90 downregulates Ikaros in acute myeloid leukemia (AML) cells. Proteasome inhibitor MG132 but not autophagy inhibitor chloroquine could suppress the Hsp90 inhibitor STA-9090-induced reduction of Ikaros, which is accompanied with the increased ubiquitination of Ikaros. Moreover, Ikaros interacts with E3 ubiquitin-ligase C terminal Hsc70 binding protein (CHIP), which mediates the STA-9090-induced ubiquitination of Ikaros. In addition, the knockdown of Ikaros effectively inhibits the proliferation of leukemia cells, but this phenomenon could be rescued by Ikaros overexpression. Collectively, our findings indicate that the interplay between HSP90 and CHIP regulates the stability of Ikaros in AML cells, which provides a novel strategy for AML treatment through targeting the HSP90/Ikaros/CHIP axis.

Design, Synthesis and in vitro Evaluation of 2-(Quinoline-8-sulfonamido)pentanedioic Acid Analogues as Antiangiogenic and Antitumor Agents on Multiple Myeloma

Thalidomide is presently approved as antiangiogenic and anticancer drug in multiple myeloma. The authors present a number of analogue-based designs of N-(o-carboxybenzoyl)-DL-glutamic acid, a major thalidomide metabolite. The compounds were synthesized and biologically tested in multiple myeloma as anticancer agents. Three compounds inhibited HUVEC proliferation at low micromolar concentrations, indicating that they are antiangiogenic and cytotoxic to human multiple myeloma RPMI8226. The active compounds were tested for antiproliferative activity on HUVECs using the dye exclusion method with trypan blue. Dimethyl-2-(quinoline-8-sulfonamido)pentanedioate (2c), in particular, inhibits VEGFR-2 phosphorylation at the Tyr-1175 residue, as determined by SDS PAGE. The binding mode of (2c) was predicted in silico in order to better understand how it interacts with essential amino acid residues in the VEGFR-2 active site. The binding energy was calculated as -161.41kcal/mol. in vitro Study of the compounds on the Vero cell line shows less toxicity towards the normal endothelial cells than the cancer cells.

Ubiquitination and Ubiquitin-Like Modifications in Multiple Myeloma: Biology and Therapy

Simple SummaryMultiple myeloma is a cancer of plasma cells causing bone fractures, anemia, renal insufficiency and hypercalcemia. Despite the introduction of new drugs in the past years, it still remains incurable and most patients die from the disease. Multiple myeloma cells are characterized by the production of high amounts of monoclonal antibodies. Therefore, maintaining protein homeostasis from synthesis through folding to degradation is crucial for multiple myeloma cells. While protein ubiquitination and organized degradation are typically considered critical for cellular health, an emerging strategy is to block these processes to induce cell death in disease-state cells characterized by protein over-production. Recent development of compounds that alter the ubiquitin proteasome pathway and drugs that affect ubiquitin-like modifications appear promising in both preclinically and in clinical trials. This review summarizes the impact of protein modifications such as ubiquitination and ubiquitin-like modifications in the biology of multiple myeloma and how it can be exploited to develop new effective therapies for multiple myeloma.Multiple myeloma is a genetically heterogeneous plasma cell malignancy characterized by organ damage and a massive production of (in-)complete monoclonal antibodies. Coping with protein homeostasis and post-translational regulation is therefore essential for multiple myeloma cells to survive. Furthermore, post-translational modifications such as ubiquitination and SUMOylation play key roles in essential pathways in multiple myeloma, including NFκB signaling, epigenetic regulation, as well as DNA damage repair. Drugs modulating the ubiquitin–proteasome system, such as proteasome inhibitors and thalidomide analogs, are approved and highly effective drugs in multiple myeloma. In this review, we focus on ubiquitin and ubiquitin-like modifications in the biology and current developments of new treatments for multiple myeloma.

Immunomodulatory drugs in multiple myeloma: Impact of the SCARMET (Self CARe and MEdication Toxicity) educational intervention on outpatients’ knowledge to manage adverse effects

Long-term multiple myeloma therapy by immunomodulatory drugs (IMiDs) raises the question of management of adverse effects. The aim of this study is to assess the impact of an educational session for patients on the acquisition of knowledge to manage hematologic and thromboembolic adverse effects of IMiDs. In this prospective single-center study, patients attended an educational session with a hospital clinical pharmacist and a nurse. The primary endpoint was the patient’s level of knowledge for the management of IMiDs adverse effects, assess with a dedicated questionnaire administered before the session then 1 and 6 months after. Assessment of knowledge was combined with self-assessment of certainty. The secondary endpoints were adherence and IMiD treatment satisfaction. 50 patients were included. Patient knowledge increased at 1 month (p<0.001) despite a loss of knowledge at 6 months (p<0.05). Six months after the educational intervention, the number of patients with skills considered satisfactory by the pharmacist and nurse increased (p<0.01). Most patients showed satisfactory adherence, with medication possession ratio ≥ 80%. The Self CARe and MEdication Toxicity (SCARMET) study highlighted the impact of multidisciplinary follow-up in multiple myeloma patients to improve knowledge of toxicity self-management.

Dynamic Epigenetic Landscapes Define Multiple Myeloma Progression and Drug Resistance

Multiple myeloma (MM) is an incurable cancer of bone marrow-resident plasma cells, which evolves from a premalignant state, MGUS, to a form of active disease characterized by an initial response to therapy, followed by cycles of therapeutic successes and failures, culminating in a fatal multi-drug resistant cancer. The molecular mechanisms leading to disease progression and refractory disease in MM remain poorly understood. To address this question, we have generated a new database, consisting of 1,123 MM biopsies from patients treated at the H. Lee Moffitt Cancer Center. These samples ranged from MGUS to late relapsed/refractory (LR) disease, and were comprehensively characterized genetically (844 RNAseq, 870 WES, 7 scRNAseq), epigenetically (10 single-cell chromatin accessibility, scATAC-seq) and phenotypically (537 samples assessed for ex vivo drug resistance). Mutational analysis identified putative driver genes (e.g. NRAS, KRAS) among the highest frequent mutations, as well as a steady increase in mutational load across progression from MGUS to LR samples. However, with the exception of KRAS, these genes did not reach statistical significance according to FISHER's exact test between different disease stages, suggesting that no single mutation is necessary or sufficient to drive MM progression or refractory disease, but rather a common "driver" biology is critical. Pathway analysis of differentially expressed genes identified cell adhesion, inflammatory cytokines and hematopoietic cell identify as under-expressed in active MM vs. MGUS, while cell cycle, metabolism, DNA repair, protein/RNA synthesis and degradation were over-expressed in LR. Using an unsupervised systems biology approach, we reconstructed a gene expression map to identify transcriptomic reprogramming events associated with disease progression and evolution of drug resistance. At an epigenetic regulatory level, these genes were enriched for histone modifications (e.g. H3k27me3 and H3k27ac). Furthermore, scATAC-seq confirmed genome-wide alterations in chromatin accessibility across MM progression, involving shifts in chromatin accessibility of the binding motifs of epigenetic regulator complexes, known to mediate formation of 3D structures (CTCF/YY1) of super enhancers (SE) and cell identity reprograming (POU5F1/SOX2). Additionally, we have identified SE-regulated genes under- (EBF1, RB1, SPI1, KLF6) and over-expressed (PRDM1, IRF4) in MM progression, as well as over-expressed in LR (RFX5, YY1, NBN, CTCF, BCOR). We have found a correlation between cytogenetic abnormalities and mutations with differential gene expression observed in MM progression, suggesting groups of genetic events with equivalent transcriptomic effect: e.g. NRAS, KRAS, DIS3 and del13q are associated with transcriptomic changes observed during MGUS/SMOL=&amp;gt;active MM transition (Figure 1). Taken together, our preliminary data suggests that multiple independent combinations of genetic and epigenetic events (e.g. mutations, cytogenetics, SE dysregulation) alter the balance of master epigenetic regulatory circuitry, leading to genome-wide transcriptional reprogramming, facilitating disease progression and emergence of drug resistance. Figure 1: Topology of transcriptional regulation in MM depicts 16,738 genes whose expression is increased (red) or decreased (green) in presence of genetic abnormality. Differential expression associated with (A) hotspot mutations and (B) cytogenetic abnormalities confirms equivalence of expected pairs (e.g. NRAS and KRAS, BRAF and RAF1), but also proposes novel transcriptomic dysregulation effect of clinically relevant cytogenetic abnormalities, with yet uncharacterized molecular role in MM. Figure 1 Disclosures Kulkarni: M2GEN: Current Employment. Zhang:M2GEN: Current Employment. Hampton:M2GEN: Current Employment. Shain:GlaxoSmithKline: Speakers Bureau; Amgen: Speakers Bureau; Karyopharm: Research Funding, Speakers Bureau; AbbVie: Research Funding; Takeda: Honoraria, Speakers Bureau; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive: Consultancy, Honoraria; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Siqueira Silva:AbbVie: Research Funding; Karyopharm: Research Funding; NIH/NCI: Research Funding.

CHEK1 and circCHEK1_246aa Promote Multiple Myeloma Malignancy By Evoking Chromosomal Instability and Bone Lesion

Key findings CHEK1 and circCHEK1_246aa induce multiple myeloma cell proliferation, drug resistance, and bone lesion formation CHEK1 and circCHEK1_246aa evoke myeloma chromosomal instability, partially through CEP170 activation Abstract Multiple myeloma (MM) is characterized by clonal expansion of plasma cells in the bone marrow (BM). Therefore, effective therapeutic interventions must target both myeloma cells and the BM niche. In the present study, we first demonstrated that CHEK1 expression was significantly increased in human MM samples relative to normal plasma cells, and that in MM patients, high CHEK1 expression was associated with poor outcomes. CHEK1 overexpression increased cellular proliferation in MM cells and evoked drug resistance in vitro, while CHEK1 knockdown abrogated this effect. Moreover, CHEK1 was a high-risk gene for poor outcome in MM patients, and, in paired samples from MM patients taken from newly diagnosed and relapsed MM, CHEK1 expression was upregulated. CHEK1-mediated increases in cell proliferation and drug resistance were due in part to CHEK1-induced chromosomal instability (CIN), as demonstrated by Giemsa staining, exon sequencing, and immunofluorescence. CHEK1 activated CIN, partly by phosphorylating CEP170. Interestingly, CHEK1 promoted osteoclast differentiation by direct phosphorylation and activation of NFATc1, indicating that CHEK1 inhibition could target both MM cell proliferation and macrophage osteoclast differentiation in the BM niche. Intriguingly, we also discovered that MM cells expressed circCHEK1_246aa, a circular CHEK1 RNA, which encoded and was translated to the CHEK1 kinase catalytic center. Transfection of circCHEK1_246aa increased MM CIN and osteoclast differentiation similarly to CHEK1 overexpression, suggesting that MM cells could secrete circCHEK1_246aa in the BM niche to increase the invasive potential of MM cells and promote osteoclast differentiation. Finally, we demonstrated in vivo in xenograft models that CHEK1 overexpression prompted MM proliferation and drug resistance, while CHEK1 knockdown conversely inhibited MM growth. Together, these findings suggest that targeting the enzymatic catalytic center encoded by CHEK1 mRNA and circCHEK1_246aa is a promising therapeutic modality to target both MM cells and the BM niche. Figure Disclosures No relevant conflicts of interest to declare.

Impact of the Availability of Novel Drugs on Overall Survival of Multiple Myeloma Patients: A Real-World Study

Introduction: Multiple myeloma is an incurable hematologic malignancy. However, many novel drugs (lenalidomide, carfilzomib, daratumumab, elotuzumab and ixazomib) have recently been approved in Brazil for this disease. Proving that the availability of these drugs increases survival is not straightforward, since pivotal trials with these drugs were designed to show benefit in progression-free survival and not in overall survival. Hypothesis: We have hypothesized that the availability of novel drugs would reduce the risk of death. Methods: This is a real-world, unicenter analysis that included patients with multiple myeloma treated between 1993 and 2017. The availability of novel drugs (lenalidomide, carfilzomib, daratumumab, elotuzumab and ixazomib) was treated as a time-dependent covariate and analyzed by Cox model. The analysis was controlled for the International Staging System (ISS) and for age. Results: With a median follow-up of 6 years, we included 151 patients with multiple myeloma. Fourty percent had ISS-I, 34% ISS-II and 26% ISS-III. Mean age was 60.5 y/o (SD: 12.2). There has been 51 deaths, and 5-y overall survival was 90% for ISS-I, 80% for ISS-II and 54% for ISS-III (p=0.0004). In multivariable analysis, age (HR=1.06, p&amp;lt;0.0001) and ISS-III (HR=4.51, p&amp;lt;0.0001) were risk factors for death, while the availability of two or more novel drugs was protective (HR=0.31, p=0.006). The availability of a single novel drug was also protective (HR=0.58), although it did not reach statistical significance (p=0.13). Adjusted Simon-Makuch survival plot is in figure 1 (adjusted for ISS III and 60 y/o). Discussion: Our results show that the availability of two or more novel drugs increases overall survival in multiple myeloma. This is an important finding, since the pivotal trials were designed to show advantages in progression-free survival and, once achieved, the experimental drug was offered to the control group, hampering overall survival analyses. Overall survival benefit, therefore, are more likely to be seen in real-world studies. We included the availability of novel drugs as a time-dependent, and the interpretation of our results is that patients alive when two novel drugs became available had benefit in overall survival. Our study has two weaknesses. Two or more novel drugs were more likely to be available for patients from a more recent time. This difference could be due to changes in supportive care, but there has been no major change in supportive care for multiple myeloma patients. Another weakness is that patient alive in the more recent years may have not received novel drug-based treatment. Considering that multiple myeloma is an incurable disease, this is unlikely for the majority of patients. In conclusion, the availability of two or more novel drugs for multiple myeloma improves survival. Figure Disclosures No relevant conflicts of interest to declare.

Proteasome Subunits Differentially Control Myeloma Cell Viability and Proteasome Inhibitor Sensitivity.

We generated eight multiple myeloma cell lines resistant to bortezomib; five acquired PSMB5 mutations. In 1,500 patients such mutations were rare clinically. To better understand disruption of proteasomes on multiple myeloma viability and drug sensitivity, we systematically deleted the major proteasome catalytic subunits. Multiple myeloma cells without PSMB5 were viable. Drug-resistant, PSMB5-mutated cell lines were resensitized to bortezomib by PSMB5 deletion, implying PSMB5 mutation is activating in its drug resistance function. In contrast, PSMB6 knockout was lethal to multiple myeloma cell lines. Depleting PSMB6 prevented splicing of the major catalytic subunits PSMB5, PSMB7, PSMB8, and PSMB10; however, PSMB6 engineered without splicing function or catalytic activity, also restored viability, inferring the contribution of PSMB6 to proteasome structure to be more important than functional activity. Supporting this, bortezomib sensitivity was restored in drug-resistant multiple myeloma cell lines by low level expression of mutated PSMB6 lacking splicing function. Loss of PSMB8 and PSMB9 was neither lethal nor restored bortezomib sensitivity. Significant codependency of PSMB5, PSMB6, and PSMB7 expression was observed. We demonstrated elevated levels of PSMB6 and 7, but not 8 and 9, in some, but not all, serial patient samples exposed to proteasome inhibitors. In summary, we show PSMB6 and PSMB7, but not PSMB5, to be essential for multiple myeloma cell survival, this dependency is structural and that upregulation or activating mutation of PSMB5, 6, and 7 confers proteasome inhibitor resistance, while depletion confers sensitivity. IMPLICATIONS: These findings support modulation of PSMB5, PSMB6, or PSMB7 expression as a new therapeutic strategy.

Meta-analysis of clinical trials to assess denosumab over zoledronic acid in bone metastasis.

Background Bone metastases-induced skeletal complications result in reduced patient survival, lower quality of life, and an increase in healthcare costs. Previously, zoledronic acid (ZA) was the standard choice of treatment for bone metastases, but another drug, denosumab, has also shown promise. However, the clinical utility of these two drugs requires further exploration. Aim of the review Due to the lack of direct comparisons regarding the efficacy of these drugs in both solid tumors and multiple myeloma (MM), we herein tried to conduct a meta-analysis to compare their efficacy in parallel for bone metastases treatment in both solid tumor and MM patients. Methods Multiple databases including Cochrane Library, MEDLINE, EMBASE, and Web of Science were searched to identify randomized controlled trials (RCTs) reported up to March 2019 directly comparing denosumab with ZA in solid tumors and MM. Information about the following events was primarily searched: time to first on-study skeletal-related event (SRE), time to first and subsequent SREs, and overall survival. Information about secondary outcomes including disease progression, pain, health-related quality of life, and adverse events was also recorded. Results Overall, we analyzed data from four distinct RCTs including 7441 patients, and our analysis revealed that patients in the denosumab group had a significantly delayed incidence to the first and subsequent SREs. In addition, denosumab resulted in a higher incidence of hypocalcemia and osteonecrosis of the jaw (ONJ), and a lower incidence of renal toxicity and acute phase reactions, in comparison to ZA. Conclusion Overall, denosumab showed superiority in delaying the first and subsequent SREs, and hence seems to be a promising choice for managing bone metastases in both solid tumors and MM. However, it can induce a higher incidence of ONJ and hypocalcaemia, but these are preventable and manageable effects.