Role In Multiple Myeloma Research Articles

Atpase Family AAA Domain-Containing Protein 2 (ATAD2) As a Novel Target in Multiple Myeloma

Multiple myeloma (MM) is a genomically heterogenous malignancy characterized by a number of copy number alterations (CNA). Moreover, there is a clear evolution of genomic changes that may affect prognosis. To identify the drivers of this inherent genomic instability, we applied an integrated genomics approach utilizing genomic data for copy number changes and transcriptomic profile. We first identified genes whose expression correlated with total copy number events in a patient dataset (gse26863, n=246). We then applied those genes to identify the genes whose elevated expression correlated with both overall and event free survival in two different datasets (IFM70, n=170; gse2408, n=559). Elevated expression of this 30 gene signature also correlated with poor overall as well as event free survival in a third myeloma dataset (MMRF; P<0.0005 for both EFS and OS). We have begun to validate these genes for impact on genomic instability and on MM cell growth and survival. Here, we present the functional validation of AAA domain containing protein 2 (ATAD2), which is one of the top-most genes in our 30 gene signature in MM and has also been part of a chromosomal instability signature representing six different cancer types (Nature Genetics, 38: 9, 2006) and a mitotic chromosomal instability signature identified in breast cancer [Sci Transl Med, 2013]. Using The Cancer Genome Atlas data we have also correlated elevated ATAD2 expression with poor OS in pulmonary and pancreatic cancers (P<0.02). ATAD2 is a member of the AAA ATPase family of proteins containing two conserved ATPase domains and a bromodomain. Bromodomain containing proteins are involved in the regulation of gene expression and are frequently dysregulated in MM as well as other cancers. ATAD2 bromodomain selectively recognizes acetylated histone 4 (acetylated at K5 and K12) and has been shown to regulate many cellular processes including cell proliferation. ATAD2 has been shown to interact with and stimulate transcriptional activity of MYC and has also been shown to contribute to invasion and migration in several cancers. We have confirmed elevated ATAD2 expression relative to normal PBMC by Western blotting in all twelve myeloma cell lines tested. To further delineate its role in MM, we evaluated the impact of its knockdown on various parameters of growth and genome maintenance using shRNAs. Relative to control shRNA, knockdown with multiple different shRNAs resulted in near complete cell death in 3 MM cell lines (JJN3, H929 and RPMI) in five days. Reduced cell viability was accompanied by increased apoptosis as seen by annexin V/PI staining. Relative to control, ATAD2 knockdown in RPMI cells led to increase in the apoptotic cell fraction by 56% and in H929 cells by 42%. To investigate genomic impact of elevated ATAD2 expression, the live cell fraction from control and ATAD2-knockdown cells was evaluated, right after selection, for impact on the expression of γ-H2AX (a DNA break marker), pRPA32 (a marker of DNA end resection, a distinct step in the initiation of homologous recombination; HR) and recombinase RAD51 (a key player in HR). ATAD2-knockdown in H929 cells inhibited spontaneous DNA breaks, DNA end resection as well as RAD51 expression, suggesting that elevated ATAD2 contributes to increased spontaneous DNA damage and HR activity observed in MM cells. To further confirm its impact on genome stability, the live cell fraction from control and knockdown cells was evaluated for micronuclei (a marker of genomic instability). Relative to control shRNA-transduced cells, knockdown of ATAD2 cells reduced micronuclei by 58% in H929 and 53% in JJN3 cells. In summary, we demonstrate that elevated ATAD2 contributes to dysregulation of DNA repair and genome stability and is required for MM cell survival, indicating that it is a promising target to inhibit growth and reduce genomic evolution in myeloma. Disclosures Munshi: BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; C4: Current equity holder in private company; Janssen: Consultancy; Adaptive: Consultancy; Legend: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy.

Dihydroartemisinin Modulates Apoptosis and Autophagy in Multiple Myeloma through the P38/MAPK and Wnt/β-Catenin Signaling Pathways.

Dihydroartemisinin (DHA), an active metabolite and derivative of artemisinin, is the most effective antimalarial drug and has strong antitumor activity in various tumor types. It has recently been reported that DHA can induce autophagy and has significant effects on multiple myeloma (MM), but the mechanisms and the relationship between the autophagy and apoptosis induced by DHA remain to be elucidated. Herein, we demonstrated that DHA significantly induces cell death in a dose- and time-dependent manner via the extrinsic and intrinsic apoptosis pathways. Moreover, DHA-induced autophagy, which plays a prodeath role in MM, can regulate canonical apoptosis and vice versa. Furthermore, the P38/MAPK signaling pathway is responsible for decreased autophagy and increased apoptosis. DHA induces autophagy and apoptosis also through the inhibition of the Wnt/β-catenin signaling pathway. In addition, DHA shows a strong effect in a xenograft mouse model. Collectively, these findings reveal that DHA, as an artemisinin-based drug, could be an effective and safe therapeutic agent for MM.

The Possible Role of Point Mutations and Activation of the CDC27 Gene in Progression of Multiple Myeloma

Multiple myeloma (MM) is characterized by malignancy of terminally differentiated B-lymphocytes. Identification of mutations in genes and pathways in MM is crucial to elucidate the pathophysiology of this cancer. Genomic profiling of malignant myeloma cells may give unprecedented results, which may eventually lead to the development of novel drugs. MM is a complex disease with a complex interactive system that consists of dysregulation of different pathways that require precise investigation for designing more effective drugs. The recent advances in high throughput sequencing (HTS) and a better understanding of myeloma's genetic architecture provide yet another approach to uncovering the driver mutations in myeloma. In the present study, we aimed to study whether cell division cycle 27 (CDC27) has a role in MM's malignancy and performed Whole Exome Sequencing (WES) in four clinical cases. Given the importance of the CDC27 gene in cell division, various studies demonstrate in different cancers that the CDC27 gene is associated with tumor proliferation and progression. Analysis of our data in the coding region of MM cases revealed that all four cases under study carrying point mutations in multiple sites of the CDC27 gene.Interestingly, CDC27 plays a pivotal role in the anaphase-promoting complex during cell division. Therefore, we opine that CDC27 may also have a role in MM. We concluded that the effective investigation of translational genomics of CDC27 should be screened from the gene level to the functional level of the protein (bench to bedside research) and studying its possible role in MM cell progression, which might be a novel therapeutic approach.

Serum brain‐derived neurotrophic factor (BDNF) concentration predicts polyneuropathy and overall survival in multiple myeloma patients

Brain-derived neurotrophic factor (BDNF) is a protein with a potent influence on several aspects of neuronal and blood vessel functions. However, its prognostic potential and functional role in multiple myeloma (MM) remain largely unknown. In this study, we investigated the influence of BDNF on the risk of chemotherapy-induced peripheral neuropathy (CIPN) and clinical outcome. Study group consisted of 91 newly-diagnosed MM patients treated with bortezomib and/or thalidomide-based chemotherapy. Detection of BDNF in serum was performed using ELISA. Polyneuropathy was assessed according to the CTCAE Criteria v5. We observed that BDNF concentration correlated with the severity of polyneuropathy (P=0·0463). Higher BDNF values were noted in patients who responded to treatment (P=0·0326), and BDNF proved to be a useful marker to predict lack of response after eight cycles of treatment (sensitivity - 100%, specificity - 61·5%, P=0·0142). Moreover this marker showed significant diagnostic usefulness in diagnosis of CIPN (sensitivity - 76%, specificity - 71·43%; area under the curve (AUC)=0·77, 95%, confidence interval (CI): 0·64-0·88; P<0·0001). Low BDNF was an independent, unfavourable prognostic factor associated with reduced overall survival (OS) (hazard ratio (HR)=2·79, P=0·0470). In conclusion, BDNF level may play a prognostic role and constitute a useful biomarker in predicting CIPN in MM patients.

Expression Pattern and Biological Significance of the lncRNA ST3GAL6-AS1 in Multiple Myeloma.

The biological impact of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) is becoming an important aspect of investigation, which may contribute to the understanding of the complex pathobiology of the disease whilst also providing novel potential therapeutic targets. Herein, we investigated the expression pattern and the biological significance of the lncRNA ST3 beta-galactoside alpha-2,3 sialyltransferase 6 antisense RNA 1 (ST3GAL6-AS1) in MM. We documented a high ST3GAL6-AS1 expression level in MM compared to normal plasma cells (PCs) or other hematological malignancies. Transcriptome analyses of MM PCs from patients included in the CoMMpass database indicated a potential involvement of ST3GAL6-AS1 in MAPK signaling and ubiquitin-mediated proteolysis pathways. ST3GAL6-AS1 silencing by LNA-gapmeR antisense oligonucleotides inhibits cell proliferation and triggers apoptosis in MM cell line. Notably, ST3GAL6-AS1 silencing in vitro displayed the down-regulation of the MAPK pathway and protein ubiquitination. These data suggest that ST3GAL6-AS1 deregulation may play a pathogenetic role in MM by affecting both proliferation pathways and circuits fundamental for PC survival. However, ST3GAL6-AS1 expression levels seem not to be significantly associated with clinical outcome and its targeting appears to exert antagonistic effects with proteasome inhibitors used in MM. These findings strongly urge the need for further studies investigating the relevance of ST3GAL6-AS1 in MM.

The EGFL7-ITGB3-KLF2 axis enhances survival of multiple myeloma in preclinical models

AbstractAngiogenic factors play a key role in multiple myeloma (MM) growth, relapse, and drug resistance. Here we show that malignant plasma cells (cell lines and patient-derived MM cells) express angiocrine factor EGF like-7 (EGFL7) mRNA and protein. MM cells both produced EGFL7 and expressed the functional EGFL7 receptor integrin β 3 (ITGB3), resulting in ITGB3 phosphorylation and focal adhesion kinase activation. Overexpression of ITGB3 or EGFL7 enhanced MM cell adhesion and proliferation. Intriguingly, ITGB3 overexpression upregulated the transcription factor Krüppel-like factor 2 (KLF2), which further enhanced EGFL7 transcription in MM cells, thereby establishing an EGFL7-ITGB3-KLF2-EGFL7 amplification loop that supports MM cell survival and proliferation. EGFL7 expression was found in certain plasma cells of patients with refractory MM and of patients at primary diagnosis. NOD.CB17-Prkdc<scid>/J mice transplanted with MM cells showed elevated human plasma EGFL7 levels. EGFL7 knockdown in patient-derived MM cells and treatment with neutralizing antibodies against EGFL7 inhibited MM cell growth in vitro and in vivo. We demonstrate that the standard-of-care MM drug bortezomib upregulates EGFL7, ITGB3, and KLF2 expression in MM cells. Inhibition of EGFL7 signaling in synergy with BTZ may provide a novel strategy for inhibiting MM cell proliferation.

Multipl miyelomda N6-metiladenozin modifiye edici enzimlerin düzensizliği

Aim: N6-methyladenosine (m6A) mRNA modification plays essential roles in various cellular processes including regulation of gene expression. However, its role in Multiple Myeloma (MM) biology remains largely unknown. Therefore, the aim of this study is to investigate the expression levels of m6A-regulating enzymes in MM. Material and Methods: The expression of m6A-regulating enzymes was investigated in monoclonal gammopathy of undetermined significance (MGUS), smouldering MM (SMM), MM and in their normal counterparts using publicly available Gene Expression Omnibus datasets, GSE6477 and GSE47552.Results: By analyzing GEO DataSets GSE6477 and GSE47552, the expression levels of METTL3, METTL14 and FTO were found to be downregulated in MM, whereas no significant change was found for WTAP, ALKBH5, YTHDF1 and YTHDF2. YTHDF3 expression showed a reduction in MM based on GSE6477 data. Conclusion: The expression of m6A-modifying genes is deregulated in MM plasma cells compared to normal plasma cells. Altered expression of m6A-modifying genes seems to play a role in the promotion of MM.

Multiple Myeloma DREAM Challenge reveals epigenetic regulator PHF19 as marker of aggressive disease

While the past decade has seen meaningful improvements in clinical outcomes for multiple myeloma patients, a subset of patients does not benefit from current therapeutics for unclear reasons. Many gene expression-based models of risk have been developed, but each model uses a different combination of genes and often involves assaying many genes making them difficult to implement. We organized the Multiple Myeloma DREAM Challenge, a crowdsourced effort to develop models of rapid progression in newly diagnosed myeloma patients and to benchmark these against previously published models. This effort lead to more robust predictors and found that incorporating specific demographic and clinical features improved gene expression-based models of high risk. Furthermore, post-challenge analysis identified a novel expression-based risk marker, PHF19, which has recently been found to have an important biological role in multiple myeloma. Lastly, we show that a simple four feature predictor composed of age, ISS, and expression of PHF19 and MMSET performs similarly to more complex models with many more gene expression features included.

Knockdown of lncRNA SNHG16 suppresses multiple myeloma cell proliferation by sponging miR-342-3p

BackgroundAberrant expression of long non-coding RNAs (lncRNAs) is closely associated with development and prognosis of human cancers. LncRNA SNHG16 is reportedly involved in human cancer; however, its roles in multiple myeloma (MM) remain unclear.MethodsIn this study, we investigated the function and molecular mechanisms of SNHG16 in MM. MM cells were transfected with si-SNHG16 or si-NC. SNHG16 expression levels was measured by qRT-PCR. Cell proliferation was monitored using the MTS. Flow cytometry assay was performed to measure the cell cycle and apoptosis. Luciferase reporter assay were performed to confirm the sponged miRNAs of SNHG16.ResultsSNHG16 expression was up-regulated in MM tissues. SNHG16 knockdown suppressed cell proliferation, arrested cell cycle transition from G1 to S phase, and promoted the apoptosis of MM cells. Moreover, SNHG16 knockdown promoted cleaved-Caspase-3, cleaved-Caspase-9, Foxa3a, and Bax expression, while markedly inhibiting CCND1, Bcl-2, Cyclin D1, PI3K, and p-AKT expression in MM cells. miR-342-3p was a direct target of SNHG16. SNHG16 knockdown significantly increased miR-342-3p expression in MM cells. Overexpression miR-342-3p markedly suppressed cell proliferation, arrested cell cycle transition from G1 to S phase, and promoted apoptosis of MM cells. Overexpression of miR-342-3p markedly promoted cleaved-Caspase-3/-9, Foxa3a, and Bax expression, and inhibited CCND1, Bcl-2, Cyclin D1, PI3K, and p-AKT expression in MM cells. Additionally, repression of miR-342-3p could rescue the effect of SNHG16 knockdown on MM cell proliferation, cycle arrest, apoptosis, and related protein expression.ConclusionKnockdown of lncRNA SNHG16 suppresses MM cell proliferation by sponging miR-342-3p, implicating SNHG16 as a novel therapeutic target for MM.

MicroRNA-146b overexpression associates with deteriorated clinical characteristics, increased International Staging System stage, cacoethic chromosome abnormality, and unfavorable prognosis in multiple myeloma patients.

BackgroundMicroRNA‐146b (miR‐146b) is a critical regulator and prognosis biomarker in several hematological malignancies, whereas its role in multiple myeloma (MM) was unclear. Therefore, this study aimed to investigate the significance of miR‐146b in MM patients.MethodsThe plasma cells were separated from bone marrow samples of 180 symptomatic MM patients (before treatment) and 50 healthy controls (HCs), and subsequently detected by reverse transcription‐quantitative polymerase chain reaction for miR‐146b expression.ResultsMiR‐146b was increased in MM patients compared with HCs (P < .001), and it predicted increased MM risk (area under curve (AUC): 0.879, 95% confidence interval (CI): 0.822‐0.936). For clinical parameters, miR‐146b was positively correlated with serum creatinine (P = .047), beta‐2‐microglobulin (P < .001), lactate dehydrogenase (P < .001), bone lesion (P = .027), International Staging System (ISS) stage (P < .001), and t (4; 14; P = .006), while negatively correlated with albumin (P = .004) in MM patients. For prognosis, miR‐146b was decreased in complete response (CR) patients compared with non‐CR patients (P = .025), as well as in overall response rate (ORR) patients compared with non‐ORR patients (P = .036), and it discriminated CR patients from non‐CR patients (AUC: 0.610, 95% CI: 0.523‐0.698) and distinguished ORR patients from non‐ORR patients (AUC: 0.602, 95% CI: 0.501‐0.703) in MM patients. Moreover, miR‐146b was correlated with worse progression‐free survival (P = .007) and overall survival (P = .014) in MM patients.ConclusionMiR‐146b was overexpressed in MM patients and predicted increased MM risk; meanwhile, it correlated with deteriorated clinical properties, increased ISS stage, cacoethic chromosome abnormality, and worse prognosis in MM patients.

Down-regulation of RFWD3 inhibits cancer cells proliferation and migration in gastric carcinoma.

The E3 ligase RING finger and WD repeat domain 3 (RFWD3) can stabilize p53 in response to DNA damage, participate in replication checkpoint, and have an important role in multiple myeloma, testicular germ cell tumor and lung carcinogenesis. Its expression and molecular mechanisms have never been explored in gastric cancer. In present study, the RFWD3 was over-expressed in both AGS and HGC-27 gastric cancer cells are found. Knockdown of RFWD3 suppressed cells proliferation activity of gastric cancer cells. And further study showed down-regulation of RFWD3 promotes cell apoptosis, suppresses cell migration and invasion and blocks G2/M cell cycle progression, which may be related with AKT, ERK/P38 and Slug pathways. Collectively, the results of the present study showed that RFWD3 might be a candidate oncogene for gastric cell proliferation and may have an important role in gastric carcinogenesis.

Blockade of PLD1 potentiates the antitumor effects of bortezomib in multiple myeloma cells by inhibiting the mTOR/NF-κB signal pathway

ABSTRACT Objective Phospholipase D1 (PLD1) is an enzyme of the phospholipase D (PLD) superfamily. It is involved in the occurrence of various tumors. However, its role in multiple myeloma (MM) remained undefined. This study aimed to investigate the mechanism of PLD1 in the therapy of myeloma disease. Material and Methods Cell lines U266 and H929 were treated with PLD1 specific inhibitor VU0359595 combined bortezomib, a proteasome inhibitor. Their effects on MM cell proliferation, apoptosis, and relevant signal pathways of apoptosis were determined by cell counting kit-8 (CCK-8), real-time polymerase reaction chain (RT–PCR), ATP assay, and western blot. Results PLD1 was highly expressed in U266 and H929 cells. VU0359595 didn’t affect the proliferation and apoptosis of MM cells. However, VU0359595 could enhance growth inhibition, decreasing mitochondrial membrane potentials (MMPs) and ATP levels of bortezomib treated MM cells. VU0359595 also strengthened bortezomib-induced apoptosis via activating caspase-8, caspase-9, caspase-3; and down-regulating the expressions of anti-apoptosis proteins BCL-2. In addition, the bortezomib-induced cytotoxicity on MM cells was significantly augmented by VU0359595 through efficient suppression of the mTOR/NF-κB signal pathway. Conclusion PLD1 inhibition can remarkably exert antitumor effects with bortezomib on MM, which is a novel potentially targeting therapeutic agent, especially for drug-resistant MM patients.

Multiple myeloma-derived exosomes inhibit osteoblastic differentiation and improve IL-6 secretion of BMSCs from multiple myeloma

Bone marrow stromal cells (BMSCs) play a critical role in multiple myeloma (MM) pathogenesis by cell contact, and secretion of cytokines, growth factors and extracellular vesicles. Exosomes are secreted by...

APOBEC3B Induction Following DNA Damage Response Modulates the Survival and Treatment Response in Human Multiple Myeloma

Apolipoprotein B mRNA editing catalytic polypeptide-like 3B (APOBEC3B, A3B) is one of 7-membered DNA cytosine deaminase family, causing cytosine-to-uracil (C-to-U) deamination in single-stranded DNA and promoting mutations in multiple human cancers including multiple myeloma (MM). High APOBEC3B expression is found in a significant portion of MM patients with MAF overexpression among t(14;16) and t(14;20). A3B upregulation is further associated with poor prognosis in MM, suggesting its role in the MM pathophysiology. However, approximately 23% MM patients with high APOBEC3 activity are associated with MAF/MAFA/MAFB translocations, the remainder of patients with high APOBEC3 carry neither translocations nor overexpression of these genes. Besides, studies are lacking on how A3B is regulated and the role of A3B in drug responses in MM. We here defined new mechanisms controlling A3B expression and further characterized its impact on treatment responses to current anti-MM therapies. Using qRT-PCR, A3B transcript is significantly higher than other members of the APOBEC3 gene family in MM cell lines (n=19) and MM patients, indicating that A3B may play a major role in MM. Using immunoblotting analysis, A3B protein expression was further confirmed in MM cell lines with various levels (n=10). Importantly, A3B mRNA upregulation by 1.34-42.64 folds was observed in CD138-purified cells from majorities of MM patients (83.3%) when compared to PBMC from the same individual (n=12). In MM cell lines without MAF/MAFA/MAFB translocation as a study model, higher A3B protein expression is associated with higher DNA damage levels as evidenced by higher γ-H2AX. These results suggest that A3B expression might be influenced by DNA damage levels in MM cells. Following a short time treatment of gamma-irradiation to cause DNA damages, A3B expression in viable MM cells was enhanced in a dose-dependent manner. We next treated MM cells (n=5) with common anti-MM drugs such as Melphalan (Mel) and Bortizomib (btz), both of which induce DNA damages, followed by examination of changes in A3B and γ-H2AX. Under sublethal treatment conditions of Mel or btz, A3B was consistently induced at both mRNA and protein levels in multiple MM cell lines regardless of the baseline A3B expression. Significantly, A3B was upregulated and associated with increased γ-H2AX in patient MM cells treated with Mel or btz under sub-lethal doses. Since DNA damages activate the ATR/ATM pathway, we next investigated whether these kinases mediate A3B induction following treatments with these compounds in MM cells. The presence of ATM or ATR inhibitors blocked A3B upregulated by these DNA damage-inducing treatments in MM cell lines (n=3), indicating an ATM/ATR-dependent pathway for A3B changes. Next, gene-specific CRISPR knock out (KO) and inducible-shRNA knockdown (KD) were used to determine the functional impact of perturbation of A3B in proliferation and survival of MM cells. Both KO and KD of A3B decreased growth and viability of MM cell lines regardless of sensitive or resistant to dexamethasone or lenalidomide. Using LIVE/DEAD fixable Aqua Stain and annexin V-based flow cytometric analysis, A3B inhibition enhanced growth arrest followed by apoptosis in MM cells. Significantly, A3B KD by its shRNA in RPMI8226 MM cells enhanced sensitivity to pomalidomide. Taken together, these data indicate that increased A3B level plays a critical role in MM cell survival and drug responses. DNA damages triggered by IR, Mel, or btz further enhance A3B expression via ATM/ATR pathway, which in turn increases subclonal diversity leading to drug resistance. The role of A3B in disease pathophysiology and progression, coupled with its function in mediating treatment response, suggest potential utility of targeting A3B in MM. Disclosures Munshi: Celgene: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Takeda: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

The Role of PHF19 As a Promoter of Tumorigenicity and Therapeutic Target in Multiple Myeloma

Introduction - Multiple Myeloma (MM) is a hematologic malignancy characterized by clonal growth of differentiated plasma cells (PCs). Despite improvement in MM therapy, the disease remains mostly incurable and is characterized by recurrent relapses with development of resistant clones that eventually lead to patient death. The pathways that lead to resistant and aggressive MM are not fully understood highlighting the need to improve our understanding of MM biology to identify potential new pathways and therapeutical targets. PHD Finger Protein 19 (PHF19) is a regulator of Polycomb Repressive Complex 2 (PRC2), the sole methyltransferase complex capable of catalyzing H3K27me3 to induce and enforce gene repression. PRC2 employs enhancer of zeste homolog 1 and 2 (EZH1/EZH2) as enzymatic subunits to hypermethylate H3K27. While overexpression and gain of function mutations of EZH1/2 have been observed in many cancers the role of this particular pathway in MM remains poorly understood. In the present study, we report on PHF19 as a new candidate gene to play a potential crucial role in MM oncogenesis. Methods- Gene expression profiling (GEP; Affymetrix U133 Plus 2.0) was performed on 739 MM patients (from total therapy trials [TT] 3-5; low risk MM n=636, high risk MM n=103), 42 patients with monoclonal gammopathy of undetermined significance (MGUS), 73 smoldering MM patients, 42 patients with primary plasma cell leukemia and 34 healthy donors. Myeloma risk was determined by the GEP 70 signature as previously defined. To test the implications of functional PHF19 knock down (KD) we used TRIPZ inducible PHF19 shRNA vs. scrambled control (Dharmacon) in two MM cell lines (JJN3 and ARP1). Real time PCR as well as western blotting was used to confirm PHF19 KD as well as to elucidate the effect on H3K27me3 (Cell Signaling). Functional in vitro studies included proliferation (Promega), clonogenic assays (StemCell), cell cycle and apoptosis assays (both Invitrogen). In vivo studies were performed using SCID mice that were subjected to tail vain injection with PHF19 KD JJN3 cells (n=10) or scrambled shRNA control (n=10). Weekly ELISA (Bethyl) and in vivo imaging (Xenogen) were performed and survival was recorded. Results- GEP of the previously mentioned patient populations and healthy controls identified PHF19 (chr9q33.2) as a candidate gene that was consistently dysregulated in MM patients. Mean expression levels at different MM stages correlated with disease aggressiveness (ANOVA, p&amp;lt;0.0001), Figure 1. High expression of PHF19 (log2&amp;gt;10.46) at diagnosis correlated significantly with adverse clinical parameters, including ISS III, anemia and elevated LDH, as well as worse overall survival (5 yr OS = 29% for patients with high PHF19 expression vs 77% for patients with low PHF19 expression [log2&amp;lt;10.46], p&amp;lt; 0.0001). These results led us to test the implications of functional PHF19 KD using TRIPZ inducible PHF19 shRNA vs. scrambled control in the JJN3 and ARP1 MM cell lines. PHF19 KD led to a drastic reduction of H3K27me3 thereby resulting in significantly reduced proliferation via cell cycle arrest, while apoptosis was not substantially altered. Clonogenic assays showed a significant reduction in colony numbers and size of MM cells with PHF19 KD compared to the control (&amp;gt;75% reduction in both cell lines, p&amp;lt;0.05). Xenograft studies showed consistently less tumor burden in the mice injected with PHF19 KD cells compared to scrambled control, evident through ELISA testing for IgG Kappa (Median =180 mg/ml for scrambled control vs 80 mg/ml for PHF19 KD at week 8, p=0.07) and bioimaging (Median bioilumisence 2.1x108 p/s for scrambled control vs. 0.8x108 p/s for PHF19 KD at week 8, non-significant). Median OS in mice injected with PHF19 KD cell was substantially longer (66 days) compared to mice subjected to scrambled control cells (54 days), p=0.052. Conclusion- In summary we show that PHF19 is upregulated in malignant plasma cells of MM patients and that PHF19 expression levels increase with advanced MM stages. High PHF19 expression was a marker of adverse prognosis in our total therapy (TT 3-5) cohort. Most importantly, in-vitro and in-vivo functional studies showed that PHF19 has important biological functions in MM. These results suggest that epigenetic regulation through histone methylation, in particular, H3K27 trimethylation, plays a crucial role in MM and the affected downstream pathways should be further elucidated. Disclosures Boyle: Janssen: Honoraria, Other: Travel; Abbvie: Honoraria; Amgen: Honoraria, Other: travel; Takeda: Honoraria, Other: travel; Celgene Corporation: Honoraria, Other: Travel. van Rhee:Kite Pharma: Consultancy; Adicet Bio: Consultancy; Karyopharm Therapeutics: Consultancy; Takeda: Consultancy; Sanofi Genzyme: Consultancy; Castleman Disease Collaborative Network: Consultancy; EUSA: Consultancy. Walker:Celgene: Research Funding.

Restoring Innate and Adaptive Immune Repertoire in Multiple Myeloma for Therapeutic Application

Despite advances and improvements in survival, majority of multiple myeloma (MM) patients ultimately relapses. Extensive analysis on the properties of tumor cells has provided interesting insights into the disease biology allowing for the identification of novel targets and development of related therapeutics. However, the key microenvironmental influences, especially immune microenvironment, that drive the disease and impact outcome remain to be fully characterized. We have now performed both single cell RNA-sequencing and high-dimensional CyTOF analysis of both peripheral blood (PBMC) and bone marrow mononuclear cells (BMMC) from MM patients and healthy donors (HD) and evaluated both phenotypic and functional state of the MM immune repertoire. In addition to humoral immunodeficiency impacting aberrations in B-cell subsets (B1a, B1b, B2 and Breg cells) and the disfunction in the adaptive immune system including an increase in the immunosuppressive cells (Tregs or myeloid-derived suppressor cells), a significant impairment of the innate immunity was identified in MM patients with a progressive decline in functional state of natural killer (NK) cells. Additionally, a lower expression of the activating receptors NKG2D and NKp46 as opposed to a higher expression of certain inhibitory KIR receptors was observed in NK cells from MM patients compared to HD. This immunosuppressive microenvironment allows tumor immune escape and ultimately myeloma cell growth. To overcome some of the immune dysfunction observed in MM, we have evaluated the role of IL15, a key player in both innate and adaptive immunity, with antitumor activity via enhancing both NK and memory T cell functions. Using extensive flow cytometry-based analysis, we evaluated impact of recombinant IL15 on PBMC from HD and MM patients at different stages of disease. Treatment with recombinant IL15 rescued the immune effector cell decline observed in MM patients. Specifically, a 2.9-fold-increase in CD8+ CD45RO+ CCR7- effector memory T cells and a 1.5-fold-increase in NK populations in both HD and MM PBMC was observed. For a subset of MM patients, we have also confirmed these positive effects on BMMC. Importantly, cytotoxicity tests revealed an improvement in NK cell effector functions leading to increased tumor cell recognition and killing, while we did not observe any direct effect of IL15 on growth and viability of MM cells. Integration of IL15 in the immunotherapeutic arsenal is limited by the unfavorable pharmacokinetic properties. Therefore, for clinical application and to restore both innate and adaptive immunity, we evaluated impact of NKTR-255, a polymer-conjugated IL15 receptor agonist designed to engage the IL-15 pathway to stimulate and expand natural killer (NK) cells and promote the survival and expansion of central memory CD8+ T cells without inducing suppressive regulatory T cells. Treatment with NKTR-255 enhanced the number and function of both NK and CD8+ effector memory T cell populations in PBMC from HD and MM patients in a dose dependent manner. Interestingly, the natural killer T (NKT) cells, heterogeneous group of T cells that share properties of both T cells and NK cells with important role in MM, were also increased in number by NKTR-255. To evaluate if NKTR-255 increases NK recognition of MM cells, cytotoxicity tests were performed using several MM cell lines and primary MM cells as targets, and both NK cells purified from PBMC of HD and MM patients as effectors. NKTR-255 was able to revert the inhibitory status of NK cells from MM patients (via induction of NKG2D expression), and significantly increase NK susceptibility of the MM cells in a dose dependent manner. Taken together, our data suggest a significant impact of NKTR-255 on the activation of effector cell function to efficiently target MM cells. This study has important translational implications and highlights the importance of restoring the balance between innate and adaptive immunity in MM. Disclosures Miyazaki: Nektar Therapeutics: Employment, Equity Ownership. Madakamutil:Nektar Therapeutics: Employment, Equity Ownership. Munshi:Amgen: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Oncopep: Consultancy.

Combined Targeting of Distinct c-Myc and JunB Transcriptional Programs for Multiple Myelioma Therapy

c-Myc plays a pivotal role in multiple myeloma (MM) pathogenesis; and the BET protein BRD4 is a key regulator of c-Myc transcription. Recently, a pathophysiologic role in MM has also been attributed to the AP-1 family member JunB. Approaches to target transcription factors (TFs), such as c-Myc and JunB, currently emerge among the most promising novel anti-MM strategies, with a potentially high therapeutic index. However, redundancy phenomena represent a major challenge for targeting c-Myc- or JunB-mediated tumor proliferation programs. MZ-1 is a novel proteolysis-targeting chimera combining the recognition sequence for the E3-ligase Von-Hippel-Lindau with JQ1, a moiety that targets BRD4. Indeed, beyond direct inhibition by JQ1, MZ-1 significantly decreased BRD4 as well as c-Myc protein levels in MM cell lines and primary cells. Consequently, MZ-1 inhibited MM cell growth through G0/G1 arrest; and induced tumor cell death. Moreover, patient-derived BMSC- or exogenous IL-6- induced BRD4/c-Myc upregulation in MM cells was inhibited by MZ-1 treatment, indicating that targeting BRD4, at least in part, overcomes the protective effect of the microenvironment. Of note, MZ-1 did not have an impact on BMSCs/IL-6- induced upregulation of JunB RNA or protein levels. Conversely, dox-induced knockdown of BMSC/IL-6-triggered JunB upregulation in TetshJunB/MM.1S cells did not decrease BRD4/c-Myc RNA or protein levels. Similar data were obtained in other MM cell lines and primary cells. RNAseq, unbiased GSEA, and luciferase reporter assays of representative target genes further supported the co-existence of c-Myc- and JunB-mediated proliferative programs, which are initiated by the same stimuli. Importantly, MZ-1 in combination with dox-induced knockdown of BMSC/IL-6-triggered JunB upregulation in TetshJunB/MM.1S cells significantly decreased both BRD4/c-Myc as well as JunB protein levels; synergistically inhibited tumor cell proliferation and induced cell death. Similar results were obtained in other MM cell line and primary cells upon treatment with MZ-1 and transient siJunB. Finally, our preliminary results show the synergistic in vivo activity of MZ-1 in BMSC:TetshJunB/MM.1S versus BMSC:TetshSCR/MM.1S-carrying doxycyclin- treated NSG mice. Taken together, these data for the first time delineate the existence of distinct c-Myc and JunB transcriptional programs that contribute to MM proliferation. They thereby provide the preclinical rationale for the development of therapeutic strategies that combine targeting of distinct transcriptional programs to improve outcome in MM. Disclosures Vallet: Roche Pharmaceuticals: Consultancy; Pfizer: Honoraria; MSD: Honoraria. Podar:Celgene: Consultancy, Honoraria; Amgen Inc.: Honoraria; Roche Pharmaceuticals: Research Funding; Takeda: Consultancy; Janssen Pharmaceuticals: Consultancy, Honoraria.

RFWD2 Induces Cellular Proliferation and Proteasome Inhibitor Resistance By Mediating p27 Ubiqutinaiton in Multiple Myeloma

Purpose: In recent years, with the emergence of targeted proteasome inhibitors (PIs), the treatment of multiple myeloma (MM) has made great progress and significantly improves the survival rate of patients. However, MM remains an incurable disease, mainly due to the recurrence of drug resistance. The constitutive photomorphogenic 1 (RFWD2, also known as COP1), is closely related to the occurrence and development of tumors, but its role in MM is largely unknown. This study was aimed to explore the mechanism of RFWD2 on cell proliferation and resistance to proteasome inhibitor in MM. Experimental Design: Using gene expression profiling (GEP) samples, we verified the relation of RFWD2 to MM patients' survival and drug-resistance. The effect of RFWD2 on cell proliferation was confirmed by MTT and cell cycle analysis in RFWD2-overexpressed and RFWD2-knockdown MM cells. MTT and apoptosis experiments were performed to evaluate whether RFWD2 influenced the sensitivity of MM cells to several chemotherapy drugs. MM xenografts were established in immunodeficient NOD/SCID mice by injecting wild-type or RFWD2 over-expression MM cells with drug intervention. The mechanism of drug resistance was elucidated by analyzing the association of RFWD2 with E3 ligase of p27. Bortezomib-resistant RPMI 8226 cells were used to construct RFWD2 knockdown cells, which were injected into NOD/SCID mice to assess the effect of RFWD2 on bortezomib resistance in vivo. Results: RFWD2 expression was closely related to poor outcome, relapse and bortezomib resistance in MM patients' GEP cohorts. Elevated RFWD2 induced cell proliferation, while decreased RFWD2 inhibited cell proliferation and induced apoptosis in MM cells. RFWD2-overexpression MM cells resulted in PIs resistance, however, no chemotherapy resistance to adriamycin and dexamethasone was observed in vitro. In addition, overexpressing RFWD2 in MM cells led to bortezomib resistance rather than adriamycin resistance in myeloma xenograft mouse model. RFWD2 regulated the ubiquitination degradation of P27 by interacting with RCHY1 ubiquitin ligase. The knockdown of RFWD2 in bortezomib-resistant RPMI 8226 cells overcame bortezomib resistance in vivo. Conclusions: Our data demonstrate that elevated RFWD2 induces MM cell proliferation and resistance to PIs, but not to adriamycin and dexamethasone both in vitro and in vivo through mediating the ubiquitination of p27. Collectively, RFWD2 is a novel promising therapeutic target in MM. Disclosures No relevant conflicts of interest to declare.

Targeting the p62-ZZ/N-End Rule Pathway in Multiple Myeloma Overcomes Proteasome Inhibitor-Resistance Via Induction of Necroptosis and Enhances the Bone Anabolic Effects of Proteasome Inhibitors

Multiple myeloma (MM) is incurable and 80% of MM patients develop MM bone disease (MMBD). MMBD lesions do not heal due to the persistent suppression of bone formation, which markedly increases mortality and contributes to MM drug resistance. Current treatments for MMBD, such as bisphosphonates and denosumab, target bone destruction but do not result in new bone formation. Although proteasome inhibitors (PIs) have greatly improved survival of MM patients, they only have transient bone anabolic effects. Further, development of drug resistance to PIs remains a major clinical problem. We previously showed that the ZZ domain of p62 (sequestosome-1),plays an important role in both MM growth and suppression of osteoblast (OB) differentiation in the MM microenvironment, by regulating multiple signaling pathways and acting as a cargo-receptor for autophagy. Recently, our collaborators showed that the p62-ZZ is a high-affinity N-recognin of the N-end rule pathway (NERP). p62-ZZ also serves as the molecular switch for necroptotic versus apoptotic cell death pathways. We previously reported that MM cells or TNFα prevent OB differentiation by inducing persistent epigenetic repression of the Runx2-P1 promoter in MM patient bone marrow stromal cells (BMSCs), via the transcriptional repressor Gfi1. We found that blocking p62-ZZ by saturating it with a novel synthetic p62-ZZ/NERP competing ligand, XRK3F2, prevented and reversed MM-induced Gfi1 occupancy at the Runx2-P1 promoter, allowing BMSCs to increase OB marker gene expression and to mineralize. These results suggest that targeting the p62-ZZ/N-end rule pathway would enhance the bone anabolic effects of PIs. To test this hypothesis, we first exposed normal OBs to different doses of bortezomib (Btz) or XRK3F2 or their combination. The combination significantly increased OB differentiation markers Runx2 (60%), Osterix (20%) and ATF4 (60%), and induced mineral deposition compared with either drug alone. The combination also blocked TNFα up-regulation of Gfi1 and suppression of OB differentiation. Interestingly, none of the concentrations tested decreased OB viability. Studies with MM patient-BMSCs showed that XRK3F2 reversed suppression of OB differentiation induced by MM cells, allowing them to mineralize. Importantly, our preliminary in vivo data showed that administration of XRK3F2 to mice with established MM induced dramatic cortical bone formation in MM-containing bones but had no effect on tumor burden. We and others previously showed that MM cells subjected to sustained proteasomal inhibition, rely on p62-mediated autophagic degradation to reduce the proteotoxic load caused by excessive immunoglobulin synthesis. We recently found that targeting the p62-ZZ domain in human MM cells, increases Btz-induced MM cell death, independently of their p53 status. The combination also significantly reduced cell viability in Btz resistant cells although no caspase 3 activation was observed, suggesting a caspase-3 independent cell death. To determine the mechanism(s) responsible for MM cell death induced by the combination, we pretreated MM cell lines and primary CD138+ MM cells with Z-DEVD (20μM), bafilomycin (Baf, 40nM), or necrostatin1 (NEC1, 50μM). The anti-MM effects of XRK3F2 or Btz+XRK3F2 were fully blocked by NEC1, an inhibitor of necroptosis, but not by inhibitors of caspase-dependent apoptosis (Z-DEVD) or autophagy (Baf), supporting that p62-ZZ regulates necroptosis in MM cells. RNAseq analysis of the additive effect of Btz+XRK3F2 on gene expression showed a total of 583 differentially regulated genes, including 374 significantly down-regulated and 209 significantly upregulated. GO term analysis of up-regulated DEGs identified an enrichment in the endoplasmic reticulum (ER) stress and ER unfolded protein response, and regulation of transcription in response to stress and autophagy. In summary, our results demonstrate that targeting the p62-ZZ/N-end rule pathway in combination with PIs in MM significantly reduces MM cell viability by activating multiple death pathway and overcomes PI-resistance of MM cells. In addition, targeting the p62-ZZ in OBs potentiates the bone anabolic action of PIs and reverses the persistent OB suppression induced by MM cells to allow bone formation. Thus, p62-ZZ plays a critical role in MM and bone cells and identifies p62-ZZ as an important molecular target for the treatment of MMBD. Disclosures Xie: Oxis Biotech: Consultancy; ID4Pharma: Other: Founder. Roodman:Amgen: Membership on an entity's Board of Directors or advisory committees.

Genome Wide Transcriptomic Analysis Identifies Dysregulated Splicing Factor Profile with Molecular and Functional Role in Multiple Myeloma

Alternative splicing (AS) is a critical post-transcriptional event, which affects the number of cellular processes. Aberrant splicing of some genes has been reported in multiple myeloma (MM). However, to date, whole-transcriptome-wide AS study has not been performed. We used deep RNA-sequencing data from 16 normal plasma cells (NPC) and 360 newly-diagnosed MM patients to describe the landscape of the alternative splicing events and the molecular mechanisms driving aberrant AS in MM. Global splicing analysis showed that mutually exclusive exon (MXE) (n=510) and Skipped Exon (SE) (n=417) are the most frequent splicing events in MM compared to NPC. Among these events, ~54% were observed in genes which are not differentially expressed between MM and NPC and 46% of the AS events (SE, MXE, retained intron, alternative 3'/5' sites) were observed in differentially expressed genes targeting 203 unique genes. AS affected RNA transcription regulation genes such as IKZF1, IKZF3, and key regulatory elements in MM including, IRF3, IRF4, or key transcription factors such as MEF2C, XPB1, STAT2, and ILF3. In general, AS targetted DNA replication, cell cycle, and apoptosis pathways. MM subgroups showed a heterogeneity for AS events. Monosomy 14, t(4;14), del1p and del17p had the highest number of unique (not observed in other subgroups) AS events compared to NPC.To understanding the molecular mechanisms driving aberrant alternate splicing we next investigated115 splicing factors (SF) in MM and associated them with AS events. We observed that ~40% of SF were dysregulated (dysregulated expression and/or copy number alteration) in MM compared to NPC, including SRSF, PCBP and RBM families. To understand the key binding regions, we have performed SF binding motif enrichment analysis around AS events and found that SRFS1, SRSF9, and PCB1 motifs to be enriched among the splicing events. Importantly, SRSF1 expression was linked with survival in two independent MM datasets.We therefore explored functional role of SRSF1 in MM with perturbation studies. While upregulation of SRSF1 expression significantly increased the cell growth and survival, conversely downregulation of SRSF1 inhibited the both. To dissect the mechanisms of SRSF1-mediated MM growth induction, we utilized SRSF1 mutants lacking either of the 2 RNA-recognition motifs or the serine/argine-rich C-terminal domain involved in protein-protein interactions, and recruitment of spliceosome components. We also utilized a C-terminal fusion of SRSF1 with the nuclear-retention signal of SRSF2 (NRS1 mutant), to force SRSF1 retention in the nucleus and assess the role of its nuclear versus cytoplasmic functions. These studies suggested that SRSF1-regulated AS effects MM cell proliferation. We surprisingly also found that even NRS1 mutant failed to promote MM growth, suggesting an important role of cytoplasmic SRSF1 in promoting MM cells proliferation.We next investigated alternative splicing pattern changes induced by SRSF1 knock down.When analyzing cellular functions of SRSF1-regulated splicing events, we found that SRSF1 knock down affect's genes in the RNA processing pathway as well as genes involved in cancer-related functions such as mTOR, E2F and MYC-related pathways. Splicing analysis was corroborated with immunoprecipitation (IP) followed by mass spectrometry (MS) analysis of T7-tagged SRSF1 MM cells.Finally, using genome wide chromatin and transcription landscape mapping techniques, we have found SRSF1 to be under the transcriptional control of oncogenic E2F1 in MM cells. Consistent with these findings, we observed greater in vitro loss of viability in a large panel of MM cell lines compared with PBMCs from healthy volunteers, following exposure to the splicing modulator pladeniolide. In summary, this study for the first time reports a detailed splicing landscape in myeloma and highlights the biological and clinical importance of alternative splicing events. Moreover, these results indicate a functional role and clinical significance of a gene involved in regulation of alternate splicing in MM, highlighting the need to further understand the splicing pattern in myeloma initiation and progression. Disclosures Anderson: Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Oncopep: Other: Scientific Founder; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder. Avet-Loiseau:takeda: Consultancy, Other: travel fees, lecture fees, Research Funding; celgene: Consultancy, Other: travel fees, lecture fees, Research Funding. Munshi:Adaptive: Consultancy; Abbvie: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Oncopep: Consultancy; Amgen: Consultancy; Celgene: Consultancy.