Identification of Long Non-Coding RNAs Deregulated in Multiple Myeloma Cells Resistant to Proteasome Inhibitors

While the clinical benefit of proteasome inhibitors (PIs) for multiple myeloma (MM) treatment remains unchallenged, dose-limiting toxicities and the inevitable emergence of drug resistance limit their long-term utility. Disease eradication is compromised by drug resistance that is either present de novo or therapy-induced, which accounts for the majority of tumor relapses and MM-related deaths. Non-coding RNAs (ncRNAs) are a broad class of RNA molecules, including long non-coding RNAs (lncRNAs), that do not encode proteins but play a major role in regulating the fundamental cellular processes that control cancer initiation, metastasis, and therapeutic resistance. While lncRNAs have recently attracted significant attention as therapeutic targets to potentially improve cancer treatment, identification of lncRNAs that are deregulated in cells resistant to PIs has not been previously addressed. We have modeled drug resistance by generating three MM cell lines with acquired resistance to either bortezomib, carfilzomib, or ixazomib. Genome-wide profiling identified lncRNAs that were significantly deregulated in all three PI-resistant cell lines relative to the drug-sensitive parental cell line. Strikingly, certain lncRNAs deregulated in the three PI-resistant cell lines were also deregulated in MM plasma cells isolated from newly diagnosed patients compared to healthy plasma cells. Taken together, these preliminary studies strongly suggest that lncRNAs represent potential therapeutic targets to prevent or overcome drug resistance. More investigations are ongoing to expand these initial studies in a greater number of MM patients to better define lncRNAs signatures that contribute to PI resistance in MM.


Introduction
Multiple myeloma (MM) is characterized by the clonal proliferation of malignant plasma cells (PCs) within the bone marrow (BM) microenvironment [1,2]. MM is the second most common hematologic malignancy, with an incidence of 24,000 cases per year in the USA, and accounts for 2% of deaths from all cancers and ≈20% of deaths from hematologic cancers [3]. In Western countries, in the near future both the incidence and prevalence of this disease will increase as the result of an aging population and increased survival of those with the disease. As the number of patients surviving with MM increases, concern for the development of therapeutic resistance rises. Although the introduction significantly deregulated in MM cells resistant to the three FDA-approved PIs, bortezomib, ixazomib, and carfilzomib.

Cell Lines and Reagents
MM cell lines (MMCLs) were obtained from the National Cancer Institute, Bethesda, MD, USA and cultured in complete Roswell Park Memorial Institute (RPMI) media supplemented with 10% fetal calf serum and penicillin-streptomycin. Bortezomib, carfilzomib, and ixazomib were from ActiveBiochem (Maplewood, NJ, USA). All other chemicals used were reagent grade (Sigma Chemical, St. Louis, MO, USA).

Generation of PI-Resistant Cells
RPMI8226 cells were exposed to successively increased concentrations (3 nM, 5 nM, 10 nM, 20 nM, 50 nM, and 100 nM) of bortezomib, ixazomib, or carfilzomib to generate resistant cells. Parental cells were cultured under the same algorithm in vehicle (0.05% dimethyl sulfoxide, DMSO) alone.

Cell Growth and Proliferation
The effect of proteasome inhibitors on myeloma growth and proliferation was assessed by measuring XTT (Sigma) dye absorbance. First, 5 × 10 4 cells were plated in 96-well plates and incubated in media that lacked phenol red. Cells were then treated with drugs at the indicated concentration (10 nM) and incubated for 72 h. XTT-phenazine methosulfate (PMS) mixture (50 µL, 1 mg/mL XTT; 20 µM PMS) was prepared and added to plates that were then incubated for 4 h. Absorbance was then measured using a BMG Labtech FLUOstar OPTIMA plate reader (Ortenberg, Germany).

Detection of Apoptosis
First, 1 × 10 6 myeloma cells were cultured for 24 h in a medium with or without PIs. Cells were harvested, washed, and stained with annexin V/propidium iodide (PI). Annexin V + /PI − apoptotic cells were enumerated using the Epics flow cytometer (Beckman Coulter, Indianapolis, IN, USA). The percentage of cells undergoing apoptosis was defined as the sum of early apoptosis (annexin V + ) and late apoptosis (annexin V + and PI + ) cells. Annexin V fluorescein isothiocyanate (FITC) conjugate was added and the sample was analyzed using a Coulter ® epics ® XL-MCL system (Beckman Coulter, Indianapolis, IN, USA).

Gene Expression Microarray
Total RNA that contained both mRNA and ncRNA was isolated from patient samples using the RNeasy kit (Qiagen Inc., Germantown, MD, USA). The quality of the total RNA was confirmed using the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and the RNA6000 Nano assay (Agilent). For each sample, the 3 in vitro translation (IVT) express kit (Affymetrix, Santa Clara, CA, USA) synthesized biotin-labeled RNA target from 100 ng of the total RNA sample. The 3 IVT kit contains hybrid primers that bind polyA (polydT) as well as random hexamer primers that bind ncRNA sequence. The kit generates cDNA to both polyA (coding) and non-polyA (non-coding) RNAs. A hybridization cocktail that included 10 µg of target was created for each sample. Samples were hybridized to the Genechip Primeview Human Gene Expression probe array cartridge (Affymetrix). The PrimeView Human Gene Expression Array provides comprehensive coverage of the human genome in a cartridge array format. The array is comprised of >530,000 probes covering >36,000 transcripts and variants, which represent >20,000 genes. Arrays contain probes for >20,000 total mature miRNAs, snoRNAs, ncRNAs, and pre-miRNAs. Those probes that demonstrated a cutoff greater or less than 2-fold from normal PCs were further analyzed.

Biostatistical Analysis
ncRNA profiles from drug-naïve and drug-resistant myeloma cells were performed and the statistical significance of the differences was determined using the Student t test with a minimal level of significance of p < 0.05.

Generation of Myeloma Cells Resistant to Proteasome Inhibitors
RPMI8226 myeloma cells were treated with either vehicle (0.05% DMSO) or the proteasome inhibitors (PIs) bortezomib, carfilzomib, or ixazomib ( Figure 1). Over a period of six months, RPMI8226 cells were exposed to the PIs at successively increased concentrations that ranged from 1 nM up to 100 nM. Each of the three drug-resistant cell lines exhibited a reduced growth rate, as shown by trypan blue staining relative to the drug-naïve parental RPMI8226 cells (Figure 2).

Biostatistical Analysis
ncRNA profiles from drug-naïve and drug-resistant myeloma cells were performed and the statistical significance of the differences was determined using the Student t test with a minimal level of significance of p < 0.05.

Generation of Myeloma Cells Resistant to Proteasome Inhibitors
RPMI8226 myeloma cells were treated with either vehicle (0.05% DMSO) or the proteasome inhibitors (PIs) bortezomib, carfilzomib, or ixazomib ( Figure 1). Over a period of six months, RPMI8226 cells were exposed to the PIs at successively increased concentrations that ranged from 1 nM up to 100 nM. Each of the three drug-resistant cell lines exhibited a reduced growth rate, as shown by trypan blue staining relative to the drug-naïve parental RPMI8226 cells (Figure 2). Scheme to generate myeloma cell lines resistant to proteasome inhibitors. Drug-naïve parental RPMI8226 cells were exposed to either vehicle (dimethyl sulfoxide (DMSO) 0.05%) or bortezomib, carfilzomib, or ixazomib at indicated concentrations. Cells were exposed to the vehicle or drugs for three days, pelleted, washed, grown in fresh media for three weeks, and then exposed to the vehicle or drug at the higher concentration. Figure 1. Scheme to generate myeloma cell lines resistant to proteasome inhibitors. Drug-naïve parental RPMI8226 cells were exposed to either vehicle (dimethyl sulfoxide (DMSO) 0.05%) or bortezomib, carfilzomib, or ixazomib at indicated concentrations. Cells were exposed to the vehicle or drugs for three days, pelleted, washed, grown in fresh media for three weeks, and then exposed to the vehicle or drug at the higher concentration.

Drug-Resistant Cells Are Less Sensitive to Proteasome Inhibitor Effects on Cell Viability and Apoptosis
Parental and PI-resistant cells were treated with bortezomib, carfilzomib, or ixazomib and the effect on cell growth and proliferation was determined using the XTT assay ( Figure 3A). Importantly, each drug-resistant cell line was also resistant to the other two PIs, while the parental cells were sensitive to all PIs. The viability of drug-resistant cells was not affected by the PIs (10 nM), while the growth of parental cells was reduced by ≈80%. The PIs also induced apoptosis in parental cells, as determined by flow cytometry to detect annexin-positive cells ( Figure 3B). We determined that ≈22-28% of parental cells were annexin-positive after treatment with the three PIs but only 4-6% of the

Drug-Resistant Cells Are Less Sensitive to Proteasome Inhibitor Effects on Cell Viability and Apoptosis
Parental and PI-resistant cells were treated with bortezomib, carfilzomib, or ixazomib and the effect on cell growth and proliferation was determined using the XTT assay ( Figure 3A). Importantly, each drug-resistant cell line was also resistant to the other two PIs, while the parental cells were sensitive to all PIs. The viability of drug-resistant cells was not affected by the PIs (10 nM), while the growth of parental cells was reduced by ≈80%. The PIs also induced apoptosis in parental cells, as determined by flow cytometry to detect annexin-positive cells ( Figure 3B). We determined that ≈22%-28% of parental cells were annexin-positive after treatment with the three PIs but only 4%-6% of the drug-resistant cells were annexin-positive ( Figure 3B).

Figure 2.
Growth rate of drug-resistant myeloma cells. The growth rate of parental and drug-resistant cells was determined by counting live cells by trypan blue staining. Shown is the average of triplicate measurements.

Drug-Resistant Cells Are Less Sensitive to Proteasome Inhibitor Effects on Cell Viability and Apoptosis
Parental and PI-resistant cells were treated with bortezomib, carfilzomib, or ixazomib and the effect on cell growth and proliferation was determined using the XTT assay ( Figure 3A). Importantly, each drug-resistant cell line was also resistant to the other two PIs, while the parental cells were sensitive to all PIs. The viability of drug-resistant cells was not affected by the PIs (10 nM), while the growth of parental cells was reduced by ≈80%. The PIs also induced apoptosis in parental cells, as determined by flow cytometry to detect annexin-positive cells ( Figure 3B). We determined that ≈22-28% of parental cells were annexin-positive after treatment with the three PIs but only 4-6% of the drug-resistant cells were annexin-positive ( Figure 3B).  (A) Effect of PIs on parental and drug-resistant cell viability. Parental, bortezomib (BTZ)-, carfilzomib (CFZ)-, or ixazomib (IXZ)-resistant cells were exposed to bortezomib, carfilzomib, or ixazomib (10 nM). The XTT assay was used to measure the effect of drugs on MM growth and proliferation. (B) Effect of PIs on the induction of apoptosis in the parental (drug-sensitive) and drug-resistant RPMI8226 cells. Cells were exposed to each PI (10 nM) for 18 h and the percentage of annexin-positive cells was determined by flow cytometry. Shown is the average of triplicate measurements.

Discussion
The identification of lncRNAs deregulated in myeloma cells resistant to PIs provides exciting new information to help unravel drug resistance mechanisms and identifies potential new therapeutic targets to improve cancer treatment. In healthy cells under physiologic conditions, multiple lncRNAs converge to maintain a proper balance of proliferation, differentiation, and death (Table 3). In pathologies such as human cancers, lncRNAs are deregulated and have profound consequences on viability and therapeutic response. Importantly, a single lncRNA can regulate multiple targets, e.g., miRNAs, mRNAs, and genes. In cancer, the loss of a tumor-suppressive lncRNA may enhance the expression of target oncogenes, whereas increased expression of an oncogenic lncRNA may repress tumor suppressors. This realization has resulted in studies to understand the pathways regulated by lncRNAs using cell-based pre-clinical model systems and to comprehend the feasibility of restoring tumor-suppressive lncRNAs for cancer therapy.
A recent report investigated lncRNAs as biomarkers for predicting survival in MM patients (55). In addition, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been found to be overexpressed in MM and may represent a marker to predict MM progression (53). Finally, a recent study identified deregulated lncRNAs putatively associated with MM pathogenesis and possibly linked to the accepted multi-step process underlining the different stages of the disease (59). The present study was focused on the uniformly fatal plasma cell dyscrasia MM as a model to study the role of lncRNAs in cancer and drug resistance. Through such an approach, microarray-based profiling revealed a panel of lncRNAs differentially regulated in myeloma cells. The molecular basis of resistance to proteasome inhibitors is a fundamental biologic problem that impedes translational

Discussion
The identification of lncRNAs deregulated in myeloma cells resistant to PIs provides exciting new information to help unravel drug resistance mechanisms and identifies potential new therapeutic targets to improve cancer treatment. In healthy cells under physiologic conditions, multiple lncRNAs converge to maintain a proper balance of proliferation, differentiation, and death (Table 3). In pathologies such as human cancers, lncRNAs are deregulated and have profound consequences on viability and therapeutic response. Importantly, a single lncRNA can regulate multiple targets, e.g., miRNAs, mRNAs, and genes. In cancer, the loss of a tumor-suppressive lncRNA may enhance the expression of target oncogenes, whereas increased expression of an oncogenic lncRNA may repress tumor suppressors. This realization has resulted in studies to understand the pathways regulated by lncRNAs using cell-based pre-clinical model systems and to comprehend the feasibility of restoring tumor-suppressive lncRNAs for cancer therapy.
A recent report investigated lncRNAs as biomarkers for predicting survival in MM patients (55). In addition, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been found to be overexpressed in MM and may represent a marker to predict MM progression (53). Finally, a recent study identified deregulated lncRNAs putatively associated with MM pathogenesis and possibly linked to the accepted multi-step process underlining the different stages of the disease (59). The present study was focused on the uniformly fatal plasma cell dyscrasia MM as a model to study the role of lncRNAs in cancer and drug resistance. Through such an approach, microarray-based profiling revealed a panel of lncRNAs differentially regulated in myeloma cells. The molecular basis of resistance to proteasome inhibitors is a fundamental biologic problem that impedes translational capacity and therapeutic efficacy. Through correlative clinical and cytogenetic study of this disease, it is clear that what is called MM is a constellation of clinico-pathological findings characterized by genomic abnormalities, molecular pathogenesis, and clinical behavior. Ongoing studies are determining the role of individual ncRNAs and lncRNAs on protein ubiquitination pathways and the catalytic activity of the proteasome. However, currently there is no evidence that links lncRNAs to the UPS and provides a mechanistic link to drug resistance.  To the best of our knowledge, this is the first report describing the global expression profiles of lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identified 8 lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significantly downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRNAs, as well as the consequences of their deregulation, remain to be fully clarified, many of the individual lncRNAs identified here that are deregulated in MM CD138 + cells are also deregulated in PI-resistant MM cells. Therefore, PI-resistant MM cell lines represent a reasonable model system to investigate the role of individual lncRNAs in the disease process and drug-resistance. Many of the prior studies  To the best of our knowledge, this is the first report describing the global expression profile lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identifie lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD1 cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significan downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant c relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRN as well as the consequences of their deregulation, remain to be fully clarified, many of the individ lncRNAs identified here that are deregulated in MM CD138 + cells are also deregulated in PI-resist MM cells. Therefore, PI-resistant MM cell lines represent a reasonable model system to investig the role of individual lncRNAs in the disease process and drug-resistance. Many of the prior stud Genes 2016, 7, 84 9 of 13 genomic abnormalities, molecular pathogenesis, and clinical behavior. Ongoing studies are determining the role of individual ncRNAs and lncRNAs on protein ubiquitination pathways and the catalytic activity of the proteasome. However, currently there is no evidence that links lncRNAs to the UPS and provides a mechanistic link to drug resistance. To the best of our knowledge, this is the first report describing the global expression profiles of lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identified 8 lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significantly downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRNAs, as well as the consequences of their deregulation, remain to be fully clarified, many of the individual lncRNAs identified here that are deregulated in MM CD138 + cells are also deregulated in PI-resistant MM cells. Therefore, PI-resistant MM cell lines represent a reasonable model system to investigate  To the best of our knowledge, this is the first report describing the global expression profile lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identifie lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD1 cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significan downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant c relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRN as well as the consequences of their deregulation, remain to be fully clarified, many of the individ lncRNAs identified here that are deregulated in MM CD138 + cells are also deregulated in PI-resist MM cells. Therefore, PI-resistant MM cell lines represent a reasonable model system to investig genomic abnormalities, molecular pathogenesis, and clinical behavior. Ongoing studies are determining the role of individual ncRNAs and lncRNAs on protein ubiquitination pathways and the catalytic activity of the proteasome. However, currently there is no evidence that links lncRNAs to the UPS and provides a mechanistic link to drug resistance. To the best of our knowledge, this is the first report describing the global expression profiles of lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identified 8 lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significantly downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRNAs, as well as the consequences of their deregulation, remain to be fully clarified, many of the individual + Genes 2016, 7, 84 9 o genomic abnormalities, molecular pathogenesis, and clinical behavior. Ongoing studies determining the role of individual ncRNAs and lncRNAs on protein ubiquitination pathways the catalytic activity of the proteasome. However, currently there is no evidence that links lncRN to the UPS and provides a mechanistic link to drug resistance. To the best of our knowledge, this is the first report describing the global expression profile lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identifie lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD1 cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significan downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant c relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRN as well as the consequences of their deregulation, remain to be fully clarified, many of the individ Genes 2016, 7, 84 9 of 13 genomic abnormalities, molecular pathogenesis, and clinical behavior. Ongoing studies are determining the role of individual ncRNAs and lncRNAs on protein ubiquitination pathways and the catalytic activity of the proteasome. However, currently there is no evidence that links lncRNAs to the UPS and provides a mechanistic link to drug resistance. To the best of our knowledge, this is the first report describing the global expression profiles of lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identified 8 lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significantly downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRNAs,  To the best of our knowledge, this is the first report describing the global expression profile lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identifie lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD1 cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significan downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant c relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRN genomic abnormalities, molecular pathogenesis, and clinical behavior. Ongoing studies are determining the role of individual ncRNAs and lncRNAs on protein ubiquitination pathways and the catalytic activity of the proteasome. However, currently there is no evidence that links lncRNAs to the UPS and provides a mechanistic link to drug resistance.   To the best of our knowledge, this is the first report describing the global expression profiles of lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identified 8 lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significantly  To the best of our knowledge, this is the first report describing the global expression profile lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identifie lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD1 cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significan   genomic abnormalities, molecular pathogenesis, and clinical behavior. Ongoing studies are determining the role of individual ncRNAs and lncRNAs on protein ubiquitination pathways and the catalytic activity of the proteasome. However, currently there is no evidence that links lncRNAs to the UPS and provides a mechanistic link to drug resistance. To the best of our knowledge, this is the first report describing the global expression profiles of  To the best of our knowledge, this is the first report describing the global expression profile 6q13(+) miR-4436a lncRNA DEFB115-7 Genes 2016, 7, 84 9 of 13 genomic abnormalities, molecular pathogenesis, and clinical behavior. Ongoing studies are determining the role of individual ncRNAs and lncRNAs on protein ubiquitination pathways and the catalytic activity of the proteasome. However, currently there is no evidence that links lncRNAs to the UPS and provides a mechanistic link to drug resistance.  genomic abnormalities, molecular pathogenesis, and clinical behavior. Ongoing studies are determining the role of individual ncRNAs and lncRNAs on protein ubiquitination pathways and the catalytic activity of the proteasome. However, currently there is no evidence that links lncRNAs to the UPS and provides a mechanistic link to drug resistance. To the best of our knowledge, this is the first report describing the global expression profiles of lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identified 8 lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significantly downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRNAs, as well as the consequences of their deregulation, remain to be fully clarified, many of the individual lncRNAs identified here that are deregulated in MM CD138 + cells are also deregulated in PI-resistant MM cells. Therefore, PI-resistant MM cell lines represent a reasonable model system to investigate the role of individual lncRNAs in the disease process and drug-resistance. Many of the prior studies that investigated ncRNAs in MM, have not looked at individual patients but rather present the collective average of up-and downregulated ncRNAs in all patients.
ncRNA replacement therapy is an innovative strategy that could save patients from ineffective treatment, improve QOL, enhance OS, and reduce the devastating impact of cancer therapy on the  To the best of our knowledge, this is the first report describing the global expression profile lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identifie lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD1 cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significan downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant c relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRN as well as the consequences of their deregulation, remain to be fully clarified, many of the individ lncRNAs identified here that are deregulated in MM CD138 + cells are also deregulated in PI-resist MM cells. Therefore, PI-resistant MM cell lines represent a reasonable model system to investig the role of individual lncRNAs in the disease process and drug-resistance. Many of the prior stud that investigated ncRNAs in MM, have not looked at individual patients but rather present collective average of up-and downregulated ncRNAs in all patients.
ncRNA replacement therapy is an innovative strategy that could save patients from ineffec treatment, improve QOL, enhance OS, and reduce the devastating impact of cancer therapy on  To the best of our knowledge, this is the first report describing the global expression profiles of lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identified 8 lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significantly downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRNAs, as well as the consequences of their deregulation, remain to be fully clarified, many of the individual lncRNAs identified here that are deregulated in MM CD138 + cells are also deregulated in PI-resistant MM cells. Therefore, PI-resistant MM cell lines represent a reasonable model system to investigate the role of individual lncRNAs in the disease process and drug-resistance. Many of the prior studies that investigated ncRNAs in MM, have not looked at individual patients but rather present the collective average of up-and downregulated ncRNAs in all patients.  To the best of our knowledge, this is the first report describing the global expression profile lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identifie lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD1 cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significan downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant c relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRN as well as the consequences of their deregulation, remain to be fully clarified, many of the individ lncRNAs identified here that are deregulated in MM CD138 + cells are also deregulated in PI-resist MM cells. Therefore, PI-resistant MM cell lines represent a reasonable model system to investig the role of individual lncRNAs in the disease process and drug-resistance. Many of the prior stud that investigated ncRNAs in MM, have not looked at individual patients but rather present collective average of up-and downregulated ncRNAs in all patients.  To the best of our knowledge, this is the first report describing the global expression profiles of lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identified 8 lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significantly downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRNAs, as well as the consequences of their deregulation, remain to be fully clarified, many of the individual lncRNAs identified here that are deregulated in MM CD138 + cells are also deregulated in PI-resistant MM cells. Therefore, PI-resistant MM cell lines represent a reasonable model system to investigate the role of individual lncRNAs in the disease process and drug-resistance. Many of the prior studies that investigated ncRNAs in MM, have not looked at individual patients but rather present the Genes 2016, 7, 84 9 o genomic abnormalities, molecular pathogenesis, and clinical behavior. Ongoing studies determining the role of individual ncRNAs and lncRNAs on protein ubiquitination pathways a the catalytic activity of the proteasome. However, currently there is no evidence that links lncRN to the UPS and provides a mechanistic link to drug resistance.  To the best of our knowledge, this is the first report describing the global expression profile lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identifie lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD1 cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significan downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant c relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRN as well as the consequences of their deregulation, remain to be fully clarified, many of the individ lncRNAs identified here that are deregulated in MM CD138 + cells are also deregulated in PI-resist MM cells. Therefore, PI-resistant MM cell lines represent a reasonable model system to investig the role of individual lncRNAs in the disease process and drug-resistance. Many of the prior stud that investigated ncRNAs in MM, have not looked at individual patients but rather present genomic abnormalities, molecular pathogenesis, and clinical behavior. Ongoing studies are determining the role of individual ncRNAs and lncRNAs on protein ubiquitination pathways and the catalytic activity of the proteasome. However, currently there is no evidence that links lncRNAs to the UPS and provides a mechanistic link to drug resistance.  To the best of our knowledge, this is the first report describing the global expression profiles of lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identified 8 lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significantly downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRNAs, as well as the consequences of their deregulation, remain to be fully clarified, many of the individual lncRNAs identified here that are deregulated in MM CD138 + cells are also deregulated in PI-resistant MM cells. Therefore, PI-resistant MM cell lines represent a reasonable model system to investigate the role of individual lncRNAs in the disease process and drug-resistance. Many of the prior studies   To the best of our knowledge, this is the first report describing the global expression profile lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identifie lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD1 cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significan downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant c relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRN as well as the consequences of their deregulation, remain to be fully clarified, many of the individ lncRNAs identified here that are deregulated in MM CD138 + cells are also deregulated in PI-resist MM cells. Therefore, PI-resistant MM cell lines represent a reasonable model system to investig the role of individual lncRNAs in the disease process and drug-resistance. Many of the prior stud genomic abnormalities, molecular pathogenesis, and clinical behavior. Ongoing studies are determining the role of individual ncRNAs and lncRNAs on protein ubiquitination pathways and the catalytic activity of the proteasome. However, currently there is no evidence that links lncRNAs to the UPS and provides a mechanistic link to drug resistance. To the best of our knowledge, this is the first report describing the global expression profiles of lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identified 8 lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significantly downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRNAs, as well as the consequences of their deregulation, remain to be fully clarified, many of the individual lncRNAs identified here that are deregulated in MM CD138 + cells are also deregulated in PI-resistant  To the best of our knowledge, this is the first report describing the global expression profile lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identifie lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD1 cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significan downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant c relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRN as well as the consequences of their deregulation, remain to be fully clarified, many of the individ lncRNAs identified here that are deregulated in MM CD138 + cells are also deregulated in PI-resist 5q31(+) Unknown lncRNA DNAJB11-6 Genes 2016, 7, 84 9 of 13 genomic abnormalities, molecular pathogenesis, and clinical behavior. Ongoing studies are determining the role of individual ncRNAs and lncRNAs on protein ubiquitination pathways and the catalytic activity of the proteasome. However, currently there is no evidence that links lncRNAs to the UPS and provides a mechanistic link to drug resistance. To the best of our knowledge, this is the first report describing the global expression profiles of lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identified 8 lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significantly downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRNAs, as well as the consequences of their deregulation, remain to be fully clarified, many of the individual  To the best of our knowledge, this is the first report describing the global expression profile lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identifie lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD1 cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significan downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant c relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRN as well as the consequences of their deregulation, remain to be fully clarified, many of the individ  To the best of our knowledge, this is the first report describing the global expression profiles of lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identified 8 lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significantly downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRNAs,  To the best of our knowledge, this is the first report describing the global expression profile lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identifie lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD1 cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significan downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant c relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRN genomic abnormalities, molecular pathogenesis, and clinical behavior. Ongoing studies are determining the role of individual ncRNAs and lncRNAs on protein ubiquitination pathways and the catalytic activity of the proteasome. However, currently there is no evidence that links lncRNAs to the UPS and provides a mechanistic link to drug resistance.    To the best of our knowledge, this is the first report describing the global expression profile lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identifie lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD1 13q34(+) miR-485-5p To the best of our knowledge, this is the first report describing the global expression profiles of lncRNAs in samples of MM from patients and PI-resistant MM cells. Notably, our study identified 8 lncRNAs that were specifically upregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells. Another nine lncRNAs were significantly downregulated in both MM CD138 + cells compared to healthy CD138 + cells and PI-resistant cells relative to parental cells ( Figure 5). Despite the fact that the mechanism and the function of lncRNAs, as well as the consequences of their deregulation, remain to be fully clarified, many of the individual lncRNAs identified here that are deregulated in MM CD138 + cells are also deregulated in PI-resistant MM cells. Therefore, PI-resistant MM cell lines represent a reasonable model system to investigate the role of individual lncRNAs in the disease process and drug-resistance. Many of the prior studies that investigated ncRNAs in MM, have not looked at individual patients but rather present the collective average of up-and downregulated ncRNAs in all patients.
ncRNA replacement therapy is an innovative strategy that could save patients from ineffective treatment, improve QOL, enhance OS, and reduce the devastating impact of cancer therapy on the healthcare system [55][56][57][58] (Figure 6). Certain ncRNAs function as bona fide tumor suppressors and synthetic versions of these ncRNAs robustly interfere with tumor growth in animal models to strongly support the investigation and clinically development of ncRNA replacements. There are two main therapeutic strategies related to targeting ncRNAs. ncRNAs that acquire a gain-of-function in the diseased tissue, e.g., oncogenic ncRNAs, can be inhibited by using ncRNA antagonists, such as antagomirs. Alternatively, ncRNAs that show a loss of function, e.g., tumor suppressors, can be restored by using ncRNA mimics or replacements. ncRNA replacement therapy involves the re-introduction of ncRNAs into diseased tissues to reactivate pathways that drive a therapeutic response. Reactivation of these ncRNA-regulated pathways can interfere with the oncogenic properties of cancer cells, blocking uncontrolled proliferation and inducing apoptosis. Based upon our pre-clinical studies, we propose to investigate these ncRNAs as attractive targets for therapeutic intervention. Ultimately, our goal is to understand the roles of ncRNAs in cancer and the potential for manipulating miRNAs for cancer therapy as these molecules make their way towards the clinic. the diseased tissue, e.g., oncogenic ncRNAs, can be inhibited by using ncRNA antagonists, such as antagomirs. Alternatively, ncRNAs that show a loss of function, e.g., tumor suppressors, can be restored by using ncRNA mimics or replacements. ncRNA replacement therapy involves the reintroduction of ncRNAs into diseased tissues to reactivate pathways that drive a therapeutic response. Reactivation of these ncRNA-regulated pathways can interfere with the oncogenic properties of cancer cells, blocking uncontrolled proliferation and inducing apoptosis. Based upon our pre-clinical studies, we propose to investigate these ncRNAs as attractive targets for therapeutic intervention. Ultimately, our goal is to understand the roles of ncRNAs in cancer and the potential for manipulating miRNAs for cancer therapy as these molecules make their way towards the clinic. Theragnostics (a portmanteau of therapeutics and diagnostics) incorporate multiple disciplines, e.g., bioinformatics, pharmacogenomics, proteomics, and metabolomics, to design accurate diagnostic assays with a targeted therapy that is selected based upon testing results [62,63]. The strategy offers for an advanced molecular understanding of cancer, to develop more effective molecular targets and to design therapeutic agents based upon patient-specific biology of disease. As a result, patients should receive personalized, targeted therapy tailored to the specific biological and molecular features of their tumor. Accurate diagnostic testing is also designed to avoid the unnecessary treatment of patients that are not likely to demonstrate a significant clinical response or for whom a specific therapy is not appropriate. The wide-scale use of theragnostics requires further bioinformatic and genomic advances as standardized tools in predictive medicine. These are key to the development and implementation of personalized therapies that eventually will improve the OS of cancer patients. In oncology, the approach is aimed at a more accurate diagnosis of cancer and optimization of patient selection to identify those most likely to overcome drug resistance. ncRNAs are readily detected in body fluids, for example, serum, plasma or urine, as well as circulating tumor cells to demonstrate their potential as readily accessible, non-invasive diagnostic and prognostic biomarkers and potential therapeutics. Specific ncRNAs are aberrantly expressed early in myelomagenesis and may therefore more readily detect high-risk disease than current methods. Although only recently discovered, ncRNAs have rapidly advanced from preclinical studies to Theragnostics (a portmanteau of therapeutics and diagnostics) incorporate multiple disciplines, e.g., bioinformatics, pharmacogenomics, proteomics, and metabolomics, to design accurate diagnostic assays with a targeted therapy that is selected based upon testing results [62,63]. The strategy offers for an advanced molecular understanding of cancer, to develop more effective molecular targets and to design therapeutic agents based upon patient-specific biology of disease. As a result, patients should receive personalized, targeted therapy tailored to the specific biological and molecular features of their tumor. Accurate diagnostic testing is also designed to avoid the unnecessary treatment of patients that are not likely to demonstrate a significant clinical response or for whom a specific therapy is not appropriate. The wide-scale use of theragnostics requires further bioinformatic and genomic advances as standardized tools in predictive medicine. These are key to the development and implementation of personalized therapies that eventually will improve the OS of cancer patients. In oncology, the approach is aimed at a more accurate diagnosis of cancer and optimization of patient selection to identify those most likely to overcome drug resistance. ncRNAs are readily detected in body fluids, for example, serum, plasma or urine, as well as circulating tumor cells to demonstrate their potential as readily accessible, non-invasive diagnostic and prognostic biomarkers and potential therapeutics. Specific ncRNAs are aberrantly expressed early in myelomagenesis and may therefore more readily detect high-risk disease than current methods. Although only recently discovered, ncRNAs have rapidly advanced from preclinical studies to evaluation in human clinical trials. The development of ncRNA theragnostics should provide widely applicable tools for the targeted delivery of personalized medicines to improve the outcome of patients with MM.

Conclusions
We have identified a curated panel of deregulated lncRNAs in common within myeloma cells generated with acquired resistant to three different clinically-relevant PIs and MM patients. Our study has shown the value of PI-resistant cell lines as a preliminary model system to address the mechanistic role of individual lncRNAs in MM and the development of drug resistance. The results prioritize specific lncRNAs to be studied in terms of defining their role in myelomagenesis and the development of drug resistance. Further investigation is warranted to shed light on the precise mechanism of action of these lncRNAs and identify their biological functions and targets.