Implications In Multiple Myeloma Research Articles

Alterations in chromosome 1q in multiple myeloma randomized clinical trials: a systematic review

Extra copies of chromosome 1q21 (+1q: gain = 3 copies, amp >= 4 copies) are associated with worse outcomes in multiple myeloma (MM). This systematic review assesses the current reporting trends of +1q, the efficacy of existing regimens on +1q, and its prognostic implications in MM randomized controlled trials (RCTs). Pubmed, Embase and Cochrane Registry of RCTs were searched from January 2012 to December 2022. Only MM RCTs were included. A total of 124 RCTs were included, of which 29 (23%) studies reported on +1q. Among them, 10% defined thresholds for +1q, 14% reported survival data separately for gain and amp, and 79% considered +1q a high-risk cytogenetic abnormality. Amongst RCTs that met the primary endpoint showing improvement in progression free survival (PFS), lenalidomide maintenance (Myeloma XI), selinexor (BOSTON), and isatuximab (IKEMA and ICARIA) were shown to improve PFS for patients with evidence of +1q. Some additional RCT’s such as Myeloma XI+ (carfilzomib), ELOQUENT-3 (elotuzumab), and HOVON-65/GMMG-HD4 (bortezomib) met their endpoint showing improvement in PFS and also showed improvement in PFS in the +1q cohort, although the confidence interval crossed 1. All six studies that reported HR for +1q patients vs. without (across both arms) showed worse OS and PFS for +1q. There is considerable heterogeneity in the reporting of +1q. All interventions that have shown to be successful in RCTs and have clearly reported on the +1q subgroup have shown concordant direction of results and benefit of the applied intervention. A more standardized approach to reporting this abnormality is needed.

The Metabolic Features of Osteoblasts: Implications for Multiple Myeloma (MM) Bone Disease.

The study of osteoblast (OB) metabolism has recently received increased attention due to the considerable amount of energy used during the bone remodeling process. In addition to glucose, the main nutrient for the osteoblast lineages, recent data highlight the importance of amino acid and fatty acid metabolism in providing the fuel necessary for the proper functioning of OBs. Among the amino acids, it has been reported that OBs are largely dependent on glutamine (Gln) for their differentiation and activity. In this review, we describe the main metabolic pathways governing OBs' fate and functions, both in physiological and pathological malignant conditions. In particular, we focus on multiple myeloma (MM) bone disease, which is characterized by a severe imbalance in OB differentiation due to the presence of malignant plasma cells into the bone microenvironment. Here, we describe the most important metabolic alterations involved in the inhibition of OB formation and activity in MM patients.

Longitudinal Assessment and Prognostic Importance of Minimal Residual Disease (MRD) By Multiparametric Flow Cytometry in Patients with Systemic Light Chain (AL) Amyloidosis

Background:Minimal residual disease (MRD) assessment can provide an index of robust disease control, particularly if MRD negativity is sustained over time. Whereas MRD status has well-established prognostic implications in multiple myeloma, its role in light chain (AL) amyloidosis-a related but biologically distinct plasma cell disorder-is presently under investigation.Aims:The aims of our study were to evaluate (1) major organ deterioration-progression-free survival (MOD-PFS) based on MRD status and (2) MRD evolution patterns during disease surveillance for patients with AL amyloidosis in hematologic complete response (hemCR) after therapy.Methods:We established MRD status using multiparametric 10-color flow cytometry of bone marrow aspirates. At least 2 x 10 6 events were measured using a Beckman Coulter Navios Flow Cytometer and analyzed by Kaluza Software (sensitivity level of ≤10 -5). Sequential MRD testing was performed (≥12 months apart) for patients returning for follow-up. We evaluated hematologic and organ responses or progression based on consensus guidelines (Palladini, 2012). MOD-PFS was defined as time from hemCR achievement to major organ deterioration (i.e., the kidney or heart), hematologic progression or death from any cause (whichever occurred first). Presence of MRD was not used to guide treatment decisions.Results:A total of 96 patients with hemCR after therapy were tested, of whom 46 (48%) were MRD negative and 50 (52%) MRD positive on first assessment. The median estimated clone size for those with MRD positivity was 3.4 x 10 -4 (range 1.4 x 10 -5 to 5.7 x 10 -3). Baseline characteristics were similar between groups, except for greater use of daratumumab and ≥2 lines of therapy to achieve hemCR in the MRD-negative group. MRD positivity correlated with a higher level of involved free light chains (iFLC) in the serum at the time of MRD assessment (23.4 mg/L vs 17.3 mg/L, P = .011). In regard to organ responses, patients with MRD negativity had a significantly higher rate of renal response (91% vs 67%, P = .017) but a similar rate of cardiac response (74% vs 68%, P = .668).After a median follow-up of 50 months from hemCR achievement, the MRD-negative group demonstrated a superior MOD-PFS (HR 0.38, 95% CI 0.16-0.88, P = .034). Progression or organ deterioration events in the MRD-negative group occurred early after hemCR achievement, while in the MRD-positive group these events continued to occur over time. A total of 8 patients had died, including 4 in each group.Sequential MRD testing (median 12 months apart, range 12-28 months) was performed for 43 patients, of whom 16/19 (84%) had sustained MRD negativity; 21/24 (88%) had persistent MRD positivity; and 6/43 (14%) had MRD conversion. No patient received therapy between MRD assessments. Among those with sustained MRD negativity, there were 2 major organ deterioration events. The 3 patients who experienced loss of MRD negativity (which occurred at 12, 107 and 114 months after last treatment) had no progression or organ deterioration events. Of those with persistent MRD positivity, 13 (62%) had durable hemCR and organ responses; moreover, 4/14 (29%) and 1/11 (9%) of these patients attained even deeper renal and cardiac responses, respectively, in the interval between sequential MRD testing. Clone progression (defined as >1-log growth in MRD clone size) was observed in 3/21 (14%) patients with persistent MRD positivity, none of whom demonstrated hematologic relapse.Conclusions:We evaluated the dynamics of MRD status in relation to clinical outcomes and found that patients with MRD negativity had longer survival free from hematologic progression, major organ deterioration or death. Longer prospective follow-up is needed to determine whether this translates into an overall survival benefit. MRD conversion occurred in a small proportion (14%) of patients, but was not linked to adverse outcomes. Understanding the role of cross-sectional MRD testing for clinical disease tracking in AL amyloidosis requires further analysis with larger sample size. [Display omitted] DisclosuresSanchorawala: Prothena: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Research Funding; Pfizer: Honoraria; Karyopharm: Research Funding; Sorrento: Research Funding; Celgene: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Caelum: Membership on an entity's Board of Directors or advisory committees, Research Funding; Regeneron: Membership on an entity's Board of Directors or advisory committees; Proclara: Membership on an entity's Board of Directors or advisory committees; Oncopeptide: Research Funding.

Abnormalities in Mitochondrial DNA Copy Number Have Pathogenetic and Prognostic Implications in Multiple Myeloma

Mitochondria are bioenergetic and biosynthetic organelles which control crucial biological pathways such as cell growth, proliferation and apoptosis among others. Over the last two decades, many studies have examined the role of mitochondrial DNA (mtDNA) alterations in carcinogenesis, via inactivating genetic mutations or dysregulation of the mtDNA copy number in the cell. However, the status of the mtDNA copy number and its implication in the pathogenesis of Multiple Myeloma (MM) remains unknown. Here, we have analyzed the mtDNA copy number in asymptomatic and symptomatic stages of MM and evaluated its clinical impact.We have estimated the mtDNA copy number in purified plasma cells from 139 patients across different stages of the disease (4 healthy donor, 19 MGUS, 14 Smoldering MM (SMM), 78 newly diagnosed MM (ndMM) and 24 relapse (rMM). The mtDNA copy number quantification was performed by RT-PCR using 2 Taqman probes which targeted the mitochondrial gene MT-RNR1 and the nuclear gene RPPH1. We have also used whole-genome or whole-exome sequencing data to examine changes in mtDNA copy number in newly-diagnosed or relapsed MM (n=64 samples) including paired sample from SMM patients progressing to ndMM (n=10). FastMitocalc and Samtools were used to extract reads aligning to mitochondrial genome from bam files and calculate de mtDNA copy number by comparing to the number of reads aligning to nuclear genome. Differential gene expression analysis was done by using canEvolve.org ocogenomics web portal (Multiple Myeloma, GSE6477 study).We observed a significant increase in the average mtDNA copy number in myeloma cells compared to healthy plasma cells (39 vs 228 copies; p-value = 0.02). Plasma cells from MGUS cells showed lower mtDNA content than more advanced stages of the disease (129 vs 242 mtDNA copies). Interestingly, amongst 10 paired samples from SMM progressing to MM an increase in the mtDNA copy number was observed in 8 of the 10 patients, being more evident when the time to progression was longer. Additionally, we also observed an increase in the mitochondrial content in 17 of the 21 patient samples examined pre and post relapse, including those patients with three consecutive relapsed samples available. We have evaluated the impact on survival of the mtDNA copy number in 69 newly diagnosed patients and we have found that patients with greater than 400 mtDNA copy number had significantly shorter progression free survival compared to the rest (p-value = 0.024). The differential expression analysis of plasma cells from MGUS, SMM, ndMM and rMM patients compared to normal plasma cells confirmed an overexpression of the mitochondrial biogenesis regulators (TFAM, TUFM, MYC and HNRNPK).We describe for the first time that mtDNA copy number increase across different stages of MM. Our data provides evidence that the malignant transformation of the myeloma cells from asymptomatic stages implies activation of mitochondrial biogenesis resulting in increased mtDNA levels. Functional experiments are ongoing to confirm the metabolic activation of myeloma cells over the time. These results highlight mitochondria as an important component of pathogenesis and progression of MM and as a potential target for therapy. DisclosuresNo relevant conflicts of interest to declare.

P0139RETROSPECTIVE ANALYSIS OF RENAL PATHOLOGY AND CYTOGENETIC EXAMINATION IN PATIENTS WITH MULTIPLE MYELOMA WITH RENAL IMPAIRMENT

Abstract Background and Aims Renal impairment is one of the common implications in multiple myeloma. But the relationships between the renal pathology and cytogenetic features are not fully understood. To explore the renal pathology and cytogenetic features in patients of multiple myeloma with renal impairment. Method Retrospective of our hospital from January 2009 to January 2019, newly diagnosed multiple myeloma patients with renal impairment. The relationship between the results of Fluorescence in situ hybridization (FISH) and renal pathological findings was analyzed. Statistical analysis was performed using SPSS 20.0. Results A total of 20 patients underwent renal biopsy, included 12 males and 8 females. FISH result showed that there were 7 cases of interstitial nephritis, 3 of them were negative for FISH, and the remaining that IgH rearrangement, 1q21 amplification, RB1 deletion, D13S319 deletion, and P53 deletion detection rates were 42.86%, 28.57%, 28.57%, 28.57% and 14.29%. The incidence was lower, which was statistically significant (P<0.01). There were 6 cases of cast nephropathy, IgH rearrangement, 1q21 amplification, RB1 deletion, D13S319 deletion, and P53 deletion detection rates were 66.67%, 50%, 66.67%, 50% and 0%. Compared with the total probe positive rate, there was no statistical significance (P>0.05). There were 4 cases of acute tubular necrosis, IgH rearrangement, 1q21 amplification, RB1 deletion, D13S319 deletion, and P53 deletion detection rates were 100%, 50%, 50%, 25% and 25%, respectively. Compared with the total probe positive rate, there was no statistical significance (P>0.05). There are 1 of amyloidosis and 1 of tubular nephropathy with amyloidosis were positive for 5 probes. One case of light chain deposition disease was positive for RB1 gene deletion + D13S319 gene deletion. Conclusion FISH in patients with different renal pathological changes is characterized by heterogeneity, which can be used to predict the risk of renal damage and speculate on possible renal pathological types to guide prognosis.

Extramedullary plasmacytomas and prognostic implications in multiple myeloma

Extramedullary plasmacytomas and prognostic implications in multiple myeloma

Targeting Myeloma Cell Metabolism Via Disruption of the Lnc-17-92 Transcriptional Program: Druggable New Vulnerability in Multiple Myeloma

Long noncoding RNAs (lncRNA) are major regulators of chromatin dynamics and gene expression. We have recently performed deep RNA sequencing of CD138+ cells from 360 uniformly-treated, newly-diagnosed multiple myeloma (MM) patients (IFM/DFCI 2009) and described the lncRNA landscape and their role as independent risk predictors for clinical outcome in MM. Moreover, we have identified one of these lncRNAs - lnc-17-92 - as an independent risk predictor highly correlating with EFS and OS in newly-diagnosed MM providing rationale to define its molecular role in MM. Lnc-17-92 is generated at MIR17HG gene locus and is known for being involved in the biogenesis of miR-17-92 cluster of microRNAs. We here establish, for the first time, role of this transcript as a lncRNA with microRNA-independent function and molecular and biological implications in MM. Having confirmed its expression in MM cell lines and primary MM cells, we have utilized antisense oligonucleotides (n=3) to suppress lnc-17-92 expression in large panel of human MM cell lines (HMCLs) (n=12) and primary patient MM cells (n=13). Lnc-17-92 inhibition impaired MM cell proliferation leading to apoptotic cell death. This inhibitory effect was not rescued by ectopic expression of miR-17-92 microRNAs, confirming independent activity of lnc-17-92 on MM cell growth and viability. The microRNA-independent role of lnc-17-92 in transcriptional control was further confirmed using DROSHAKOcells. Analysis of transcriptomic changes after lnc-17-92 modulation in HMCLs and primary MM cells identified bona fide transcriptional targets of lnc-17-92. Using two independent MM RNA-seq datasets, we observed high correlation (R> 0.4) between lnc-17-92 expression and the expression of 12 of the transcriptional targets identified above. Interestingly, these genes were significantly enriched within metabolic pathways, suggesting an unexplored role for lnc-17-92 in MM cell metabolism. Further analysis using an RNAi-based loss-of-function screening in 3 HMCLs revealed Acetyl-CoA Carboxylase Alpha (ACC1) as a novel myeloma vulnerability. ACC1 encodes the limiting enzyme in the de novo lipogenesis pathway. Analysis of incorporation of C14-radiolabeled glucose into lipids in MM cells revealed that inhibition of ACC1 or lnc-17-92 strongly inhibited de novo lipogenesis in HMCLs and in primary MM cells. We have used ACC1 conditional KD MM cells expressing IPTG-inducible ACC1 shRNAs and confirmed significant role of ACC1 in MM cell growth and survival, both in vitro and in vivo in SCID mice model. Importantly, supplementation of palmitate, the main downstream product of ACC1 activity, significantly reverses the growth inhibitory effect of either ACC1 or lnc-17-92 suppression in MM cells. These data suggest an important role for lipogenesis pathway on lnc-17-92-promoted MM cell growth. We have further investigated mechanism by which lnc-17-92 may exert its transcriptional control. Protein-RNA pulldown assay established MYC as interacting partner of lnc-17-92. This interaction was confirmed by immunoprecipitation of MYC-bound RNA followed by qRT-PCR with specific primers for detection of lnc-17-92. ChIP-seq analysis revealed a direct binding of MYC at regulatory regions of ACC1 in MM.1S cells; these data were corroborated by the decreased ACC1 expression observed in MYC KD MM cells. Taken together, these data suggest that lnc-17-92 may function as a scaffold between MYC and the E-box motifs present on ACC1 intronic sequences, facilitating MYC binding and its transcriptional activity on ACC1. Finally, for translational application, we have pre-clinically investigated ND-646, a clinically applicable small molecule inhibitor of ACC1. Analysis of incorporation of C14-radiolabeled glucose into lipids confirmed its effect on lipogenesis in MM, which was associated with a significant in vitro growth inhibitory activity in large panel of HMCLs and primary patient MM cells. In vivo studies in murine model of human MM, using this oral agent, are ongoing and will be presented. In conclusion, we here report for the first time the microRNA-independent role of lnc-17-92 in MM pathobiology with direct impact on transcriptional control of lipogenesis. The availability of oral inhibitor of this pathway may allow the clinical application of this unique targeted therapy in MM. Disclosures Anderson: Janssen: Other: Advisory Board; Gilead Sciences: Other: Advisory Board; OncoPep: Other: Scientific founder ; Sanofi-Aventis: Other: Advisory Board; C4 Therapeutics: Other: Scientific founder . Munshi:Abbvie: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Adaptive: Consultancy; Oncopep: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Oncopep: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Abbvie: Consultancy.

Cross Talk Networks of Mammalian Target of Rapamycin Signaling With the Ubiquitin Proteasome System and Their Clinical Implications in Multiple Myeloma.

Multiple myeloma (MM) is the second most common hematological malignancy and results from the clonal amplification of plasma cells. Despite recent advances in treatment, MM remains incurable with a median survival time of only 5-6years, thus necessitating further insights into MM biology and exploitation of novel therapeutic approaches. Both the ubiquitin proteasome system (UPS) and the PI3K/Akt/mTOR signaling pathways have been implicated in the pathogenesis, and treatment of MM and different lines of evidence suggest a close cross talk between these central cell-regulatory signaling networks. In this review, we outline the interplay between the UPS and mTOR pathways and discuss their implications for the pathophysiology and therapy of MM.

Molecular Mechanisms of p53 Deregulation in Cancer: An Overview in Multiple Myeloma.

The p53 pathway is inactivated in the majority of human cancers. Although this perturbation frequently occurs through the mutation or deletion of p53 itself, there are other mechanisms that can attenuate the pathway and contribute to tumorigenesis. For example, overexpression of important p53 negative regulators, such as murine double minute 2 (MDM2) or murine double minute 4 (MDM4), epigenetic deregulation, or even alterations in TP53 mRNA splicing. In this work, we will review the different mechanisms of p53 pathway inhibition in cancer with special focus on multiple myeloma (MM), the second most common hematological malignancy, with low incidence of p53 mutations/deletions but growing evidence of indirect p53 pathway deregulation. Translational implications for MM and cancer prognosis and treatment are also reviewed.

Extracellular vesicle cross-talk in the bone marrow microenvironment: implications in multiple myeloma.

The bone marrow (BM) represents a complex microenvironment containing stromal cells, immune cells, osteoclasts, osteoblasts, and hematopoietic cells, which are crucial for the immune response, bone formation, and hematopoiesis. Apart from soluble factors and direct cell-cell contact, extracellular vesicles (EVs), including exosomes, were recently identified as a third mediator for cell communication. Solid evidence has already demonstrated the involvement of various BM-derived cells and soluble factors in the regulation of multiple biological processes whereas the EV-mediated message delivery system from the BM has just been explored in recent decades. These EVs not only perform physiological functions but can also play a role in cancer development, including in Multiple Myeloma (MM) which is a plasma cell malignancy predominantly localized in the BM. This review will therefore focus on the multiple functions of EVs derived from BM cells, the manipulation of the BM by cancer-derived EVs, and the role of BM EVs in MM progression.

Cross-talk between two antioxidants, thioredoxin reductase and heme oxygenase-1, and therapeutic implications for multiple myeloma

Multiple myeloma (MM) is characterized by an accumulation of abnormal clonal plasma cells in the bone marrow. Despite recent advancements in anti-myeloma therapies, MM remains an incurable disease. Antioxidant molecules are upregulated in many cancers, correlating with tumor proliferation, survival, and chemoresistance and therefore, have been suggested as potential therapeutic targets. This study investigated the cross-talk between two antioxidant molecules, thioredoxin reductase (TrxR) and heme oxygenase-1 (HO-1), and their therapeutic implications in MM. We found that although auranofin, a TrxR inhibitor, significantly inhibited TrxR activity by more than 50% at lower concentrations, myeloma cell proliferation was only inhibited at higher concentrations of auranofin. Inhibition of TrxR using lower auranofin concentrations induced HO-1 protein expression in myeloma cells. Using a sub-lethal concentration of auranofin to inhibit TrxR activity in conjunction with HO-1 inhibition significantly decreased myeloma cell growth and induced apoptosis. TrxR was shown to regulate HO-1 via the Nrf2 signaling pathway in a ROS-dependent manner. Increased HO-1 mRNA levels were observed in bortezomib-resistant myeloma cells compared to parent cells and HO-1 inhibition restored the sensitivity to bortezomib in bortezomib-resistant myeloma cells. These findings indicate that concurrent inhibition of HO-1 with either a TrxR inhibitor or with bortezomib would improve therapeutic outcomes in MM patients. Hence, our findings further support the need to target multiple antioxidant systems alone or in combination with other therapeutics to improve therapeutic outcomes in MM patients.

Genomic Landscape and Its Prognostic Implications in Multiple Myeloma Using a Targeted Sequencing Approach

▪Next-generation sequencing (NGS) studies have shown that multiple myeloma is a heterogeneous disease with a complex subclonal architecture and few recurrently mutated genes. Only a minority of variants are potentially actionable and studies on their prognostic value are lacking. Therefore, strategies to investigate the landscape of chromosomal and gene lesions of a large number of myeloma samples in a robust fashion is needed. In this study, we developed a target-enrichment strategy to streamline simultaneous analysis of gene mutations, copy number changes and IGH translocations in multiple myeloma in a high-throughput fashion using NGS.We designed Agilent SureSelect cRNA pull down baits to target 246 genes implicated in myeloma, in lymphoid malignancies or other cancers, 2538 single nucleotide polymorphisms to detect copy number and allelic ratio at the single-gene and whole-genome level, and we tiled the whole IGH locus to detect IGH translocations and V(D)J rearrangements. As a pilot, we sequenced 13 myeloma cell lines and 10 control haematopoietic cells lines and validated its sensitivity and specificity. We have next applied this baitset to unmatched DNA from CD138-purified plasma cells from 426 patients at diagnosis, including 51 matched samples from a previous whole exome sequencing dataset. We sequenced at an average depth of 329x with 77% of the target region covered at >30x using Hiseq2000 machines (Illumina Inc.). We applied algorithms developed in-house to detect point mutations, insertion-deletions and IGH translocations, filtering out potential artifacts and germline variants. We then annotated as "oncogenic" all variants previously reported as somatic in cancer by the COSMIC database.418/426 patients had at least one variant, and overall we identified 2207 variants of which 667 were oncogenic. 212/246 evaluated genes were mutated at least once, but only 102 had at least one oncogenic variant. Furthermore, 417/667 (63%) oncogenic variants were accounted for by the top 8 driver genes previously identified (KRAS, NRAS, TP53, FAM46C, BRAF, DIS3, TRAF3, SP140). Additional findings of interest included clustered mutations in SF3B1, EGR1, IRF4, but in <5% of patients each. We identified IGH translocations in 127/375 patients, including 70 t(11;14) and 35 t(4;14). FISH validation suggested our pulldown approach had 93% sensitivity and 98% specificity. Given the high number of variables and patients, we looked at pair-wise interactions between genes and with karyotypic features. We found that BRAF variants are not mutually exclusive with KRAS or NRAS mutations, a finding with implications for pathogenesis and treatment with BRAF inhibitors. Furthermore, samples with IGH translocations, and t(4;14) in particular, had fewer mutations overall, were significantly enriched for mutations in SF3B1 and depleted of mutations in FAM46C, suggesting that the genomic landscape of myeloma may in part vary with karyotypic features.We then looked at prognostic models. While TP53 mutations had the strongest effect both on overall survival (OS) and progression free survival (PFS), we found a favorable impact of TRAF3 mutations on PFS, and a negative one of NRAS and SP140. More importantly, pairwise interactions served as a starting ground for a rationale subgroup analysis. For example, while FAM46C mutations had no effect on OS across the whole cohort, they predicted better survival in patients without IGH translocations (median 94 months for WT FAM46C vs. not reached for MUT, p=0.042 log-rank test). Similarly, we found a different magnitude of the negative impact of TP53 mutations on OS based on karyotype. Survival was shortened more than 8-fold in cases with both an IGH translocation and a TP53 mutation (median 89 months for WT TP53 vs. 11 for MUT, p=2e-10), while this effect was much less in the absence of an IGH translocation (94 months for WT vs. 50 for MUT, p=0.02).In conclusion, the large sample size and extent of sequencing provides further insight into the genomic landscape of myeloma and how this impacts the clinical phenotype confirming the utility of the targeted sequencing to both understand the biology as well as its clinical application. DisclosuresCampbell:14M genomics: Other: Co-founder and consultant.

47 Genes Define Myeloma Cell Acquired Resistance to Bortezomib and Have Profound Prognostic Implications in Multiple Myeloma

▪The proteasome inhibitor Bortezomib (Bz), the first agent of a new class of drugs in Multiple Myeloma (MM), has shown remarkable activity and forms an integral part of modern MM treatment. Nevertheless, resistance to Bz eventually develops in a significant proportion of patients, with adverse effects on survival. Numerous publications have addressed this issue through in vitro developed models of acquired Bz resistance (BzR). However the results were quite different in each publication, none of the produced Bz myeloma cell lines was provably stable, no common mechanism of resistance could be demonstrated, and hence were of minimal relevance to the clinical setting.In order to address these issues an effort was made for the development of an in vitro model of acquired BzR that would resemble the clinical reality in the most accurate way. Two myeloma cell lines were used, one resembling a multisensitive (JJN3) and the other a multiresistant (U266) drug behavior, that were both sensitive to Bz. An at least 20 fold increase in the 48h Bz IC50 was noted for both cell lines. The increase in the IC50 was able to be verified a year after culturing the cell lines in normal medium thus ensuring a stable resistance phenotype.To delineate the molecular mechanisms that underlie the development of BzR a combined genetic/Gene Expression Profile (GEP) and functional/Proteomics approach was used with emphasis in the common elements of both cell lines. The hypothesis was that if certain pathways are activated in the cells that actually produce the phenotype of BzR they must fulfil two important criteria: 1) They must be present in all the levels of the BzR, 2) The gene changes have to be verified in the level of the gene encoded proteins thus securing their functional importance.GEP of the naïve cell lines along with the GEP of the Bz resistant cells at different levels of BzR (5-fold, 10-fold, 20-fold) were used. The statistical analysis revealed 100 gene probes common in both cell lines that achieved their highest change as soon as BzR was established and remained stable at that level for all later versions (P<0.1, q<0.1) and 115 gene probes common in both cell lines that their change was proportional to the level of BzR (P<0.001, q <0.005).The proteomics analysis of the Bz resistant cell lines at their latest level of resistance (20-fold) revealed 262 proteins common in both cell lines that were up-regulated and 263 common in both cell lines that were down-regulated (change >10% to be considered significant).The intersection of the list of the common genes with the list of the common proteins revealed 47 gene-proteins all but one novel in MM. They can be grouped in distinct biological categories with the most prominent ones being the ROS/Mitochondrial Factor category comprising of 10 gene-proteins, the E3 Ubiquitin Pathway 6 genes-proteins and Translation Regulation 5 genes-proteins.Even more importantly 30 of them have profound survival implications in MM -all of them novel in MM- both for Overall Survival (OS) and Progression Free Survival (PFS) in both Bz (TT3) and non Bz (TT2) containing protocols implying that myeloma cells apply both Bz specific and non-specific mechanisms to acquire BzR.Based on these 30 genes-proteins a GEP risk score (GEP-30) was constructed that was able to achieve remarkable statistical power in both Bz containing and non containing trials of both newly diagnosed (TT2 with and without thalidomide i.e. TT2+ and TT2-, TT3a, TT3b, HOVON, MRC IX, Figure 1A,B,C) and relapsed MM (TT6 , OS: NR vs 1.52 yr P<0.00001, PFS: NR vs 1.13 yr P<0.00001 for low and high risk) [Display omitted] [Display omitted] [Display omitted] DisclosuresStein:University of Arkansas for Medical Sciences: Employment. Barlogie:University of Arkansas for Medical Sciences: Employment. Epstein:University of Arkansas for Medical Sciences: Employment. Heuck:Janssen: Other: Advisory Board; Celgene: Consultancy; Millenium: Other: Advisory Board; Foundation Medicine: Honoraria; University of Arkansas for Medical Sciences: Employment.

147 - Cross-Talk between Two Antioxidants, Thioredoxin Reductase 1 and Heme Oxygenase-1 and Therapeutic Implications in Multiple Myeloma

The intensification of agricultural management has caused some weed species to become rare in arable farming systems. It is difficult to disentangle which management practices are the least harmful for the conservation of rare arable weeds because of their sparse presence. In this research, we overcame the limitations of previous analyses of rare weeds by analyzing them in a large number of plots (1957) at the edges of multiple organic fields (304), which maximized the probability of detecting these species. We evaluated the relationships between farming practices and local site conditions and the presence of rare arable species that are characteristic of cereal fields.We detected 46 of the 65 rare weeds that are known to inhabit the study area, but their frequency was very low. Cereal crops, either alone or in mixtures with legumes, enhanced the probability of finding rare weed species, while fertilization had a detrimental effect. Other management practices that were considered had no effect on the presence of rare arable weeds. However, selected rare species tended to fare better under particular local conditions and to be favored by specific management practices. In contrast, a significant amount of the variance of the rare weed presence was explained by farm-related and field-related random factors. Thus, the occurrence of rare arable species is apparently determined by stochastic factors that may be related to the local species pool that likely depends on the history of fields and farms. Therefore, conservation efforts should be focused on areas currently inhabited by rare arable species.

Abstract 3035: HDAC8 is recruited to DNA double strand breaks sites and affects the homologous recombination efficiency in multiple myeloma

Abstract Epigenomic changes have emerged as an important player of cellular regulation and understanding of pathogenesis of Multiple Myeloma (MM) as well as of other cancers. In recent years, both clinical and preclinical studies have confirmed that MM is vulnerable to epigenetic intervention, with histone deacetylases (HDACs) emerging as the most promising epigenetic targets. Although Pan-HDAC inhibitors are effective as therapeutic agents, there is increasing emphasis on understanding the biological and molecular roles of individual HDACs. Here we evaluated the role of HDAC8, a member of Class I HDAC isoenzymes in MM. First, we evaluated the expression of HDAC8 in 172 newly-diagnosed MM patients from the IFM myeloma dataset and observed HDAC8 overexpression as well as its significant correlation with poor survival outcome (P&amp;lt;0.0015). We further evaluated the expression of HDAC8 in HMCLs (probe ID_223909-s_at, 223345_at) and confirmed the high expression and its cytoplasmic and nuclear localization in all MM cells lines studied. The HDAC8 depletion (lentiviral-shRNA)in HMCLs resulted in significant inhibition of proliferation of MM cells as measured by 3[H]-thymidine assay, and as decrease in colony formation evaluated after 3 weeks post transfection (P&amp;lt;.001). We observed similar cell growth inhibition using PCI-34051, a small molecule HDAC8 inhibitor. Interestingly, the combination of HDAC8 inhibitor with melphalan or bendamustine enhanced the anti-MM effects of the DNA damaging agents (all p&amp;lt;0.01). Immunoblotting analysis using a panel of 15 antibodies for DNA damage response (DDR) pathway confirmed increased levels of DNA damage in OPM2 and MM1S cells lacking HDAC8. Consistent with this observation HDAC8 depletion led to decreased homologous recombination (HR) activity as measured by plasmid-based assay. We performed singe cell electrophoresis (Comet-assay) and observed decreased repair of DSBs after IR in OPM2-HDAC8 depleted cells as well as after pharmacologic inhibition of HDAC8. Importantly, using laser micro-irradiation in myeloma and U2OS cells, we observed HDAC8 recruitment to DSBs sites. The HDAC8 was co-localized and co-immunoprecipitated with Rad51 after IR, and with Scm3, member of cohesion complex suggesting its relation with cytoskeleton. In MM1s cells containing a stably integrated Rad51-luciferase reporter construct, the addition of HDAC8 inhibitor suppressed Rad51, confirming the immunoblotting findings. A mass spectromentry-based analysis identified the HDAC8-interacting complexes with cohesion- (cohesin subunit SA-2, Condensin-2) and DDR-key components (Mre11a, XRCC1, Rad50). In conclusion, our results demonstrate impact of epigenomic change on DNA integrity through connection between HDAC8 and DNA damage response pathway, and provide insights into the effect of HDAC8 on DNA stability and cell growth and survival that may have therapeutic implications in MM. Citation Format: Maria Gkotzamanidou, Masood Shammas, Jesús Martín Sánchez, Mehmet Kemal Samur, Stephane Minvielle, Florence Magrangeas, Herve Avet-Loiseau, Athanasios-Meletios Dimopoulos, Kenneth C. Anderson, Nikhil C. Munshi. HDAC8 is recruited to DNA double strand breaks sites and affects the homologous recombination efficiency in multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3035. doi:10.1158/1538-7445.AM2015-3035

The novel biomarker of alternative macrophage activation, soluble mannose receptor (sMR/sCD206): Implications in multiple myeloma

Tumor-associated macrophages (TAMs) play an important role in the pathophysiology of human malignancies. They support growth of cancer cells by promoting angiogenesis, and by inhibiting tumour cell apoptosis and anti-tumor immune reactions. Several membrane proteins are well-described markers of human TAMs, including the haemoglobin scavenger receptor CD163 and the macrophage mannose receptor (MR/CD206). Interestingly, both CD163 and MR exist as soluble serum proteins (sCD163 and sMR) that may reflect the activation state of tissue macrophages, including TAMs. Here, we report the first data on sMR as a biomarker in patients with a malignant disease. We have measured concentrations of sMR in peripheral blood serum (n=104) from patients with newly diagnosed multiple myeloma (MM) by an enzyme-linked immunosorbent assay, and examined associations with data from medical records. At diagnosis, sMR levels were elevated in 27% of patients, and decreased after treatment. Further, sMR levels were associated with prognostic markers in MM, and elevated sMR (>0.43mg/L) was an independent marker of overall survival in a multivariate analysis (HR=2.20, P=0.006). Levels of sMR in blood samples showed significant association with sCD163, which may indicate common origin from CD163+MR+TAMs.

Immunosurveillance and therapy of multiple myeloma are CD226 dependent.

Multiple myeloma (MM) is an age-dependent hematological malignancy. Evaluation of immune interactions that drive MM relies on in vitro experiments that do not reflect the complex cellular stroma involved in MM pathogenesis. Here we used Vk*MYC transgenic mice, which spontaneously develop MM, and demonstrated that the immune system plays a critical role in the control of MM progression and the response to treatment. We monitored Vk*MYC mice that had been crossed with Cd226 mutant mice over a period of 3 years and found that CD226 limits spontaneous MM development. The CD226-dependent anti-myeloma immune response against transplanted Vk*MYC MM cells was mediated both by NK and CD8+ T cells through perforin and IFN-γ pathways. Moreover, CD226 expression was required for optimal antimyeloma efficacy of cyclophosphamide (CTX) and bortezomib (Btz), which are both standardly used to manage MM in patients. Activation of costimulatory receptor CD137 with mAb (4-1BB) exerted strong antimyeloma activity, while inhibition of coinhibitory receptors PD-1 and CTLA-4 had no effect. Taken together, the results of this study provide in vivo evidence that CD226 is important for MM immunosurveillance and indicate that specific immune components should be targeted for optimal MM treatment efficacy. As progressive immunosuppression associates with MM development, strategies aimed to increase immune functions may have important therapeutic implications in MM.

HDAC8 Mediates Homologous Recombination and Cytoskeleton Integrity in Myeloma with Potential Impact on Cell Growth and Survival

Epigenomic changes have become an important component of cellular regulation and ultimately, of our understanding of oncogenomics in Multiple Myeloma (MM) as well as in other cancers. In recent years, both clinical and preclinical studies have confirmed that MM is vulnerable to epigenetic intervention, with histone deacetylases (HDACs) emerging as the most promising epigenetic targets. Although Pan-HDAC inhibitors are effective as therapeutic agents, there is increasing emphasis on understanding the biological and molecular roles of individual HDACs. Here we have evaluated the role of HDAC8, a member of Class I HDAC isoenzymes in MM.First, we evaluated the expression of HDAC8 in 172 newly-diagnosed MM patients from the IFM myeloma dataset and observed HDAC8 overexpression as well as its significant correlation with poor survival outcome (p<0.0015). We further evaluated the expression of HDAC8 in HMCLs (probe ID_223909-s_at, 223345_at) and confirmed the high expression and its cytoplasmic and nuclear localization in all six MM cells lines studied (MM1S, OPM2, RPMI8226, U266, MOLP8 and NCI-H929) and in primary bone marrow plasma cells (CD138+) from newly diagnosed MM patients (N=3).To address the functional role of HDAC8 in MM biology and to evaluate its potency as therapeutic target, we used a lentiviral-shRNA delivery system for HDAC8-knockdown in MM1S and OPM2 myeloma cells. The HDAC8 depletion in HMCLs resulted in significant inhibition of proliferation of MM at 1 week as measured by 3[H]-thymidine assay, and as decrease in colony formation evaluated after 3 weeks post transfection (p<.001). We observed similar cell growth inhibition using PCI-34051, a small molecule HDAC8 inhibitor. Interestingly, the combination of HDAC8 inhibitor with melphalan or bendamustine enhanced the anti-MM effects of the DNA damaging agents (all p<0.01) and was confirmed to be synergistic using Calcusyn software. Immunoblotting using a panel of 15 antibodies for DNA damage response (DDR) pathway proteins (including γH2Ax, pATM, pATR, pBRCA1, pBRCA2, pCHK2, pCHK1, ku70, RPA70, 53BP1, DNA-PKs, pP53) confirmed increased levels of DNA damage in OPM2 and MM1S cells with HDAC8 depletion. In consistence with this observation HDAC8 knockdown led to decreased homologous recombination (HR) activity as measured by a transient direct repeat DsRED-GFP/I-SceI plasmid-based assay. We performed singe cell electrophoresis under neutral conditions (comet-assay) in OPM2 and MM1S after HDAC8 depletion with or without exposure to gamma irradiation (γ-IR), and in OPM2 and MM1S cells treated and untreated with HDAC8 inhibitor in combination with γ-IR and observed decreased repair of DSBs after γ-IR measured following HDAC8 knockdown as well as following treatment of the cells with HDAC8 inhibitor. Importantly, using laser micro-irradiation in myeloma and U2OS cells, we observed HDAC8 recruitment to DSBs sites. Moreover, the HDAC8 protein was co-localized and co-immunoprecipitated with Rad51 after IR, and with Scm3, member of cohesion complex after mitotic sychronization, suggesting its relation with cytoskeleton. We confirmed the significant alteration in expression of cohesion complex members SMC1 and RAD21 after HDAC8 depletion and re-overexpression in MM cells. In MM1s cells containing a stably integrated Rad51-luciferase reporter construct, the addition of HDAC8 inhibitor resulted in a decrease in Rad51 promoter activity, confirming the immunoblotting findings. An ongoing mass spectromentry-based analysis is expected to identify thoroughly the HDAC8-interacting proteins.In conclusion, our results demonstrate an impact of aberrant epigenome on DNA integrity through connection between HDAC8 and DDR pathway, and provide insights into the effect of HDAC8 on cellular growth and survival with potent therapeutic implications in MM. DisclosuresAnderson:Celgene: Consultancy; Sanofi-Aventis: Consultancy; Onyx: Consultancy; Acetylon: Scientific Founder, Scientific Founder Other; Oncoprep: Scientific Founder Other; Gilead Sciences: Consultancy.

Bone Marrow Microenvironment Regulates Alternative Splicing Events in Myeloma Cells through Downregulation of RNA Binding Protein Fox2

Alternative splicing is a crucial mechanism for gene regulation, which enhances the diversity of transcriptome and proteome. Misregulation of alternative splicing has been implicated in number of disease processes including cancer. Our data utilizing exon array profile from 170 uniformly treated newly diagnosed patients with MM confirms clinical relevance of splicing as demonstrated by impact of level and extent of alternate splicing on both progression free and overall survival. Fox2, a RNA splicing factor, is one of the most important genes predicting clinical outcome in these patients. We confirmed Fox2 expression in 10 MM cell lines at both RNA and protein levels. Immunohistochemistry staining showed a predominant nuclear localization of Fox2. Importantly, we also observed that MM cell - bone marrow stromal cells (BMSC) interaction led to significant inhibition of Fox2 expression in MM cells. Similar response was also observed using BMSC supernatants. While IL6 treatment significantly downregulated the expression of Fox2 in MM1S and RPMI8226 cells in a dose-dependent manner, IGF-1 treatment had no significant impact on Fox2 expression in MM cell lines. Since Fox2 has been described to plays a role in the maintenance of cell cytoskeleton, we therefore evaluated whether Fox2 might influence the migration and adhesion in MM cells. Transwell migration assay showed enhanced migration rate of Fox2-knocking down- MM1S and RPMI8226 cells versus controls. We also observed the increased cell adhesion to fibronetin in both cell lines upon Fox2 knockdown. Actin polymerization evaluated by Alexa488-conjugated phalloidin staining and confocal microscope analysis showed Fox2 knocking down cells with increased actin polymerization in both MM1S and RPMI8226 cell lines. Interstingly, we observed that Fox2 knockdown in MM cell lines did not affect the cell proliferation and survival. As Fox-2 is a splicing factor, we further evaluated the molecular impact of Fox2 expression in multiple myeloma by RNA-seq analysis. Our data revealed that Fox2 functions in regulating both protein-coding and non-coding RNA alternative splicing. Knockdown of Fox2 resulted in significant isoform up-regulation (60 in MM1S and 151 in RPMI8226) and down-regulation (70 in MM1S and 69 in RPMI8226). Gene enrichment analysis showed these genes are clustered in cell cytoskeleton regulation, microtubule-based movement, ATP binding, amongst others. Our study then focused on Fox2 knockdown-induced significant isoform switch in MM cell lines. We designed the primers testing the spliced exons and confirm the isoform switch in MM cells by PCR analysis (e.g. Pyk2 and PFDN6). Importantly, our RNA seq data showed that Fox2 regulates the expression of a series of microRNAs and long-noncoding RNAs (e.g. MALAT1 RPMI8226 CNT (58) vs Fox2 knockdown (108)), which provides us a new insight into impact of Fox2 on non-protein coding RNAs. We have also validated the RNA-seq data by Q-PCR analysis.In summary, our results identify Fox2 as a biologically important RNA binding protein that is regulated by bone marrow microenvironment interaction and with essential function and potential clinical implications in multiple myeloma. DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow Microenvironment Affects The Pathogenesis Of Multiple Myeloma Through Downregulation Of Alternative Splicing Factor Fox2 In Myeloma Cells

Alternate splicing is an important post translational change that alters specificity of gene function. Misregulation of alternative splicing has been implicated in number of disease processes including cancer. We have analyzed alternate splicing in myeloma using high throughput GeneChip Human Exon 1.0 ST Arrays in 170 uniformly treated patients and identified pattern of splicing as well as their impact on both overall and event free survival in myeloma. We have now further analyzed this data and identified Fox2, a RNA alternative splicing regulator, as one of the most important genes predicting clinical outcome in these patients. We observe that the expression level of Fox2 correlates with the frequency of RNA splicing and disease prognosis in MM patients. We have now further investigated the molecular role of Fox2 in myeloma. Fox2 expression was detected in all 10 MM cell lines tested at both RNA and protein levels. Immunohistochemistry staining showed a predominant nuclear localization of Fox2. We next evaluated impact of IL-6 on Fox2 expression in MM1S and RPMI8226 MM cell lines and observed dose-dependent reduction in Fox2 expression. Importantly, MM cell - bone marrow stromal cells (BMSC) interaction also led to significant inhibition of Fox2 expression in MM1S and RPMI8226 cells. Similar response was also observed using BMSC supernatants. On the other hand, IGF-1 stimulation showed slight upregulation of Fox2 in MM cell lines. We have also evaluated impact of IL-6 on Fox2 and splicing using genomewide RNA-seq and confirmed the results. Fox2 was downregulated 33% in MM1S and 37% in RPMI8226 at gene expression level. To study its role in MM, we knocked down the expression of Fox2 in MM1S and RPMI8226 cell lines by using Fox2-directed siRNA. Compared to control cell lines, Fox2 knockdown in MM cell lines did not affect the cell proliferation and survival, as measured by cell titer glo luminescent cell viability assay and annexin V and PI staining respectively. Since Fox2 has been described to plays a role in the maintenance of cell cytoskeleton, we therefore evaluated whether Fox2 might influence the migration and adhesion in MM cells. Transwell migration assay showed enhanced migration rate of Fox2-knocking down- MM1S and RPMI8226 cells versus controls. We also observed the increased cell adhesion to fibronetin in both cell lines upon Fox2 knockdown. Actin polymerization evaluated by Alexa488-conjugated phalloidin staining and confocal microscope analysis showed Fox2 knocking down cells with increased actin polymerization in both MM1S and RPMI8226 cell lines. Currently, RNA seq data following Fox2 knock down in MM cell lines is being evaluated to define the molecular mechanisms of bone marrow microenvironment-mediated Fox2-regualted alternative splicing events in MM. In summary, our results identify Fox2 as a biologically important splicing factor with essential function and potential clinical implications in multiple myeloma. Disclosures:No relevant conflicts of interest to declare.