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

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

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Discovery and Optimisation of Bacterial Nitroreductases for Use in Anti-Cancer Gene Therapy

10.26686/wgtn.16985503 ◽

2021 ◽

Author(s):

◽

Gareth Adrian Prosser

Keyword(s):

Dna Damage ◽

Mammalian Cells ◽

Excision Repair ◽

Alkylating Agents ◽

Dna Lesions ◽

Site Directed Mutagenesis ◽

In Vitro Assays ◽

E Coli ◽

Anti Cancer

<p>Nitroaromatic prodrugs are biologically inert compounds that are attractive candidates for anti-cancer therapy by virtue of their ability to be converted to potent DNA alkylating agents by nitroreductase (NTR) enzymes. In gene-directed enzyme-prodrug therapy (GDEPT), NTR-encoding therapeutic transgenes are delivered specifically to tumour cells, whereupon their expression confers host cell sensitivity to subsequent systemic administration of a nitroaromatic prodrug. The most well studied NTR-GDEPT system involves reduction of the aziridinyl dinitrobenzamide prodrug CB1954 by the Escherichia coli NTR NfsB. However, low affinity of this enzyme for CB1954 has so far limited the clinical efficacy of this GDEPT combination. The research described in this thesis has primarily sought to address this limitation through identification and optimisation of novel NTR enzymes with improved nitroaromatic prodrug reductase activity. Efficient assessment of NTR activity from large libraries of candidate enzymes requires a rapid and reliable screening system. An E. coli-based assay was developed to permit indirect assessment of relative rates of prodrug reduction by over-expressed NTRs via measurement of SOS response induction resulting from reduced prodrug-induced DNA damage. Using this assay in concert with other in vitro and in vivo tests, more than 50 native bacterial NTRs of diverse sequence and origin were assessed for their ability to reduce a panel of clinically attractive nitroaromatic prodrugs. Significantly, a number of NTRs were identified, particularly in the family of enzymes homologous to the native E. coli NTR NfsA, which displayed substantially improved activity over NfsB with CB1954 and other nitroaromatic prodrugs as substrates. This work also examined the roles of E. coli DNA damage repair pathways in processing of adducts induced by various nitroaromatic prodrugs. Of particular interest, nucleotide excision repair was found to be important in the processing of DNA lesions caused by 4-, but not 2-nitro group reduction products of CB1954, which suggests that there are some parallels in the mechanisms of CB1954 adduct repair in E. coli and mammalian cells. Finally, a lead NTR candidate, YcnD from Bacillus subtilis, was selected for further activity improvement through site-directed mutagenesis of active site residues. Using SOS screening, a double-site mutant was identified with 2.5-fold improved activity over the wildtype enzyme in metabolism of the novel dinitrobenzamide mustard prodrug PR-104A. In conclusion, novel NTRs with substantially improved nitroaromatic prodrug reducing activity over previously documented enzymes were identified and characterised. These results hold significance not only for the field of NTR-GDEPT, but also for other biotechnological applications in which NTRs are becoming increasingly significant, including developmental studies, antibiotic discovery and bioremediation. Furthermore, the in vitro assays developed in this study have potential utility in the discovery and evolution of other GDEPT-relevant enzymes whose prodrug metabolism is associated with genotoxicity.</p>

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Discovery and Optimisation of Bacterial Nitroreductases for Use in Anti-Cancer Gene Therapy

10.26686/wgtn.16985503.v1 ◽

2021 ◽

Author(s):

◽

Gareth Adrian Prosser

Keyword(s):

Dna Damage ◽

Mammalian Cells ◽

Excision Repair ◽

Alkylating Agents ◽

Dna Lesions ◽

Site Directed Mutagenesis ◽

In Vitro Assays ◽

E Coli ◽

Anti Cancer

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Dysregulated Apurinic/Apyrimidinic Endonucleases (Ape1 and Ape2) Lead to Genetic Instability in Multiple Myeloma.

Blood ◽

10.1182/blood.v104.11.1418.1418 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1418-1418

Author(s):

Masood A. Shammas ◽

Hemant Koley ◽

Sima Shah ◽

Ramesh B. Batchu ◽

Pierfrancesco Tassone ◽

...

Keyword(s):

Multiple Myeloma ◽

Homologous Recombination ◽

Genomic Instability ◽

Cell Lines ◽

Plasma Cells ◽

Dna Recombination ◽

Endonuclease Activity ◽

Single Nucleotide ◽

Genetic Changes

Abstract Multiple myeloma (MM) is associated with significant genomic instability. Homologous recombination (HR), which is elevated in MM, is considered to be responsible for this instability. As endonucleases play an important role in mediating HR, here we have evaluated the role of endonuclease in biology and progression of MM. Gene expression profile using Affymetrix U133 array showed > 2 fold elevation of Ape1 or Ape2 or both in 5 of 6 MM cell lines and 12 of 15 patient samples. Immunocytochemistry confirmed upregulation of Ape1 protein in MM cell lines. A Plasmid degradation assay confirmed significantly elevated endonuclease activity in MM cells compared to normal plasma cells. To identify the pre-dominating endonuclease activity, the degradation assay was carried out in the presence of specific endonuclease inhibitors. Harmane and methoxyamine (MA), specific inhibitors of apurinic/apyrimidinic endonucleases effectively inhibited significant endonuclease activity, while other endonuclease inhibitors ACPD and FK506 had minimal effects, confirming predominant role of apurinic/apyrimidinic endonucleases (APE) in mediating increased endonuclease activity in MM. We investigated the role of elevated APE endonuclease activity on DNA recombination and subsequent genomic re-arrangements. Using a plasmid-based assay we have previously demonstrated significantly elevated homologous recombination (HR) in MM. Inhibition of endonuclease by methoxyamine suppressed HR activity by 85 ± 2% in MM cells. Next, we evaluated whether inhibition of HR by methoxyamine can affect the frequency of acquisition of new genetic changes in MM cells using single nucleotide polymorphism (SNP) arrays (Affymetrix) as indicator of genomic instability. In three independent experiments, methoxyamine reduced the acquisition of new loss of heterozygocity (LOH) loci by an average of 71%. These data suggest that the dysregulated APE endonucleases contribute significantly to the genomic instability, acquisition of new mutations and progression of MM and provides the rationale for targeting endonuclease activity to prevent disease progression including development of drug resistance.

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Microrna Expression Profile Identifies Distinct Clinically Relevant Sub-Groups in Multiple Myeloma: Novel Prognostic Markers and Potential Targets for Therapy

Blood ◽

10.1182/blood.v112.11.96.96 ◽

2008 ◽

Vol 112 (11) ◽

pp. 96-96 ◽

Cited By ~ 2

Author(s):

Sophia Adamia ◽

Herve AvetLoiseau ◽

Samirkumar B Amin ◽

Yu-Tzu Tai ◽

Steven P. Treon ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Differentiation ◽

Clinical Outcome ◽

Signaling Pathways ◽

Cell Lines ◽

Plasma Cells ◽

Normal Plasma ◽

Group A ◽

Group B

Abstract MicroRNA, an abundant class of small endogenous RNAs, regulate target genes through inducing translational inhibition and cleavage of targeted transcripts. To date, microRNAs have been implicated in normal biological processes, including development, cell differentiation, apoptosis and proliferation as well as in malignant transformation. However, their role in multiple myeloma (MM) remains unknown. Here we investigated role of microRNAs in myelomagenesis, and their influence on prognosis and clinical outcome. We evaluated profiles of 384 microRNAs in bone marrow derived CD138+ plasma cells (PC) from 79 uniformly treated MM patients, 11 MM cell lines and 9 healthy donors using qRT-PCR based microRNA array. The relative expression was calculated using comparative Ct method, and data was normalized using endogenous controls and analyzed using SDS, RQ manager, R and dChip softwares. MicroRNA expression profiles detected in MM patients were correlated with clinical outcome measures. We observed significant modulate expression of 61 microRNAs in myeloma cells compared to normal plasma cells. When more stringent criteria were used, we identified 24 differentially expressed microRNAs in patient samples. Further, unsupervised hierarchical clustering of filtered microRNAs, based on their DCt values, identified two major groups within the MM population (groups A and group B). Samples of Group A clusters with MM cell lines, indicating more proliferative nature of MM patient cells. Within B group, a second degree node group B2, clusters with normal plasma cells indicating more indolent course, while patients in an additional node B1 represented an assorted pattern. The unsupervised clustering of all MM samples showed consistent changes in miR-30b, -30c, -30d, -142-5p, -24, -191, -181d, -374, -146b, -140, -145, -125a, -151, -223, -155, let7b, indicative of a role of these microRNA in myelomagenesis; while supervised analysis of samples within groups A and B identified modulated expression of different sets of miRNAs. In group A miR-585 and let-7f were upregulated 8–12 fold, while miRs -125a, -126, -155, -223, -146a, -374 -19a, -20a, -26a, -30a -5p, -30b, and -30d were significantly downregulated; in group B, all differentially expressed microRNAs were downregulated (p<0.001) compared to normal plasma cells. These modulated miRNAs target critical signaling pathways including apoptosis, hematopoietic cell differentiation and proliferation, survival and angiogenesis by upregulating function of HOX9, c-myc, VCAM-1, Bcl-2, E2F1, SHP1, SHP2, VEGF, and DUSp6 molecules. We further analyzed the effect of microRNA on clinical outcome. We have observed significantly superior event free and overall survival of patients in group B2 compared to patients in group A (2 yr estimated EFS 79% versus 54% respectively; p=0.05; and 2 yr estimated OS 94% versus 70% respectively; p =0.017). Taken together this data identifies critical microRNAs as modulators of gene expression and signaling pathways and provides potential novel microRNA and gene targets in MM to both understand biological behavior and for therapeutic application.

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DNA Replication but Not Nucleotide Excision Repair Is Required for UVC-Induced Replication Protein A Phosphorylation in Mammalian Cells

Molecular and Cellular Biology ◽

10.1128/mcb.20.8.2696-2705.2000 ◽

2000 ◽

Vol 20 (8) ◽

pp. 2696-2705 ◽

Cited By ~ 23

Author(s):

Gregory Rodrigo ◽

Sophie Roumagnac ◽

Marc S. Wold ◽

Bernard Salles ◽

Patrick Calsou

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Nucleotide Excision Repair ◽

Mammalian Cells ◽

Excision Repair ◽

Protein A ◽

Replication Protein A ◽

Replication Protein ◽

Dependent Manner ◽

Nucleotide Excision

ABSTRACT Exposure of mammalian cells to short-wavelength light (UVC) triggers a global response which can either counteract the deleterious effect of DNA damage by enabling DNA repair or lead to apoptosis. Several stress-activated protein kinases participate in this response, making phosphorylation a strong candidate for being involved in regulating the cellular damage response. One factor that is phosphorylated in a UVC-dependent manner is the 32-kDa subunit of the single-stranded DNA-binding replication protein A (RPA32). RPA is required for major cellular processes like DNA replication, and removal of DNA damage by nucleotide excision repair (NER). In this study we examined the signal which triggers RPA32 hyperphosphorylation following UVC irradiation in human cells. Hyperphosphorylation of RPA was observed in cells from patients with either NER or transcription-coupled repair (TCR) deficiency (A, C, and G complementation groups of xeroderma pigmentosum and A and B groups of Cockayne syndrome, respectively). This exclude both NER intermediates and TCR as essential signals for RPA hyperphosphorylation. However, we have observed that UV-sensitive cells deficient in NER and TCR require lower doses of UV irradiation to induce RPA32 hyperphosphorylation than normal cells, indicating that persistent unrepaired lesions contribute to RPA phosphorylation. Finally, the results of UVC irradiation experiments on nonreplicating cells and S-phase-synchronized cells emphasize a major role for DNA replication arrest in the presence of UVC lesions in RPA UVC-induced hyperphosphorylation in mammalian cells.

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DNA Methyltransferase Inhibitors Increase Expression of CD38 and Enhance Daratumumab Efficacy in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-114206 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5618-5618 ◽

Cited By ~ 1

Author(s):

Priya Choudhry ◽

Margarette C. Mariano ◽

Arun P Wiita

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Cell Lines ◽

Research Funding ◽

Plasma Cells ◽

Cpg Island ◽

Ex Vivo ◽

Single Agent ◽

Cd38 Expression

Abstract Introduction: The anti-CD38 monoclonal antibody Daratumumab is highly effective against multiple myeloma, is well tolerated, and has high single agent activity as well as combination effects with lenalidomide-dexamethasone as well as bortezomib-dexamethasone. Patient response to daratumumab monotherapy is highly correlated with pretreatment levels of CD38 expression on MM plasma cells (Nijhof et al, Leukemia (2015) 29:2039) and CD38 loss is correlated with daratumumab resistance (Nijhof et al, Blood (2016) 128:959). As a result, there is significant interest in elucidating the regulation and role of CD38 in MM. Recently, All Trans Retinoic Acid (ATRA), a known small molecule inducer of CD38 in myeloid cells, as well as the FDA-approved histone deacetylase inhibitor panobinostat, were both demonstrated to induce CD38 in MM plasma cells leading to increased lysis by daratumumab. Examining ENCODE data, we found the presence of a CpG island at the first exon of CD38. We hypothesized that removing methylation sites from this CpG island may de-repress CD38 transcription and lead to increased CD38 protein at the cell surface in MM plasma cells. Therefore, here we studied the role of DNA methyl-transferase inhibitors (DNMTis), currently FDA-approved for treatment of myelodysplastic syndrome, as agents to potentiate daratumumab therapy. Methods: We treated MM cell lines (RPMI-8226, MM.1S, XG-1, KMS12-PE) with two different DNMTis, 5-Azacytidine and decitabine, and assessed CD38 cell surface expression by flow cytometry. Similarly, we treated MM patient bone marrow aspirates ex vivo and assessed induction of CD38 expression in the CD138 positive population by flow cytometry. We analyzed CD38 mRNA levels and total CD38 protein levels by qRT-PCR and western blotting respectively. ATRA was used as a positive control in all experiments. We further tested the functional effect of DNMTi treatment on MM cell lines using an Antibody Dependent Cell Cytotoxicity (ADCC) assay. Briefly, live treated cells were incubated overnight with daratumumab and NK92-CD16 transgenic cells at and E:T ratio of 20:1, and lysis was measured using CytoTox-Glo (Promega). Results: Flow analysis revealed that DNMTi treatment induces a 1.2-2 fold increase in CD38 surface protein expression in a dose-dependent manner across MM cell lines. DNMTi treatment consistently yielded similar or higher increases in CD38 expression than that seen in ATRA- or panobinostat-treated cells. Despite significantly lower single-agent cytotoxicity, we found that decitabine led to similar surface CD38 induction as 5-Azacytidine. By RT-qPCR, 5-Azacytidine increased CD38 mRNA expression ~3 fold versus DMSO control, compared to ~2 fold mRNA increase with ATRA. In functional ADCC assays, DNMTi treatment also led to greater lysis than ATRA. Furthermore, the combination of both DNMTi and ATRA was additive, leading to the greatest lysis by NK cells. In contrast, in ex vivo-treated patient samples, ATRA induced greater CD38 expression than 5-Azacytidine on malignant plasma cells. However, this result is expected since MM plasma cells from patients typically do not proliferate in standard ex vivo culture, and active DNA replication is a requirement for successful DNMT inhibition based on known mechanism of action. In patients, however, we anticipate that continual plasma cell proliferation will lead to effective increases in CD38 after DNMTi treatment, as found in MM cell lines here. Summary and Conclusions: Our results here demonstrate that CD38 expression in MM cells is regulated by DNA methylation and targeting DNMTs with small molecule inhibitors leads to increased vulnerability to Daratumumab treatment. We propose that combination treatment with DNMTi and Daratumumab can lead to higher efficacy of daratumumab in daratumumab-naïve MM, as well as reversal of daratumumab-resistance. These combinations should be tested in clinical trials. Disclosures Wiita: Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

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Role of Base Excision Repair Pathway in the Processing of Complex DNA Damage Generated by Oxidative Stress and Anticancer Drugs

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.617884 ◽

2021 ◽

Vol 8 ◽

Author(s):

Yeldar Baiken ◽

Damira Kanayeva ◽

Sabira Taipakova ◽

Regina Groisman ◽

Alexander A. Ishchenko ◽

...

Keyword(s):

Dna Damage ◽

Base Excision Repair ◽

Genome Instability ◽

Excision Repair ◽

Chromosomal Rearrangements ◽

Strand Break ◽

Dna Lesions ◽

Complex Nature ◽

Base Excision

Chemical alterations in DNA induced by genotoxic factors can have a complex nature such as bulky DNA adducts, interstrand DNA cross-links (ICLs), and clustered DNA lesions (including double-strand breaks, DSB). Complex DNA damage (CDD) has a complex character/structure as compared to singular lesions like randomly distributed abasic sites, deaminated, alkylated, and oxidized DNA bases. CDD is thought to be critical since they are more challenging to repair than singular lesions. Although CDD naturally constitutes a relatively minor fraction of the overall DNA damage induced by free radicals, DNA cross-linking agents, and ionizing radiation, if left unrepaired, these lesions cause a number of serious consequences, such as gross chromosomal rearrangements and genome instability. If not tightly controlled, the repair of ICLs and clustered bi-stranded oxidized bases via DNA excision repair will either inhibit initial steps of repair or produce persistent chromosomal breaks and consequently be lethal for the cells. Biochemical and genetic evidences indicate that the removal of CDD requires concurrent involvement of a number of distinct DNA repair pathways including poly(ADP-ribose) polymerase (PARP)-mediated DNA strand break repair, base excision repair (BER), nucleotide incision repair (NIR), global genome and transcription coupled nucleotide excision repair (GG-NER and TC-NER, respectively), mismatch repair (MMR), homologous recombination (HR), non-homologous end joining (NHEJ), and translesion DNA synthesis (TLS) pathways. In this review, we describe the role of DNA glycosylase-mediated BER pathway in the removal of complex DNA lesions.

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Evidence for Ongoing DNA Damage in Multiple Myeloma as Revealed by Constitutive Phosphorylation of H2AX.

Blood ◽

10.1182/blood.v114.22.2817.2817 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2817-2817

Author(s):

Denise K. Walters ◽

Renee C. Tschumper ◽

Xiaosheng Wu ◽

Kimberly J. Henderson ◽

Angela Dispenzieri ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Dna Damage ◽

Cell Lines ◽

Plasma Cells ◽

Clonal Evolution ◽

S Phase ◽

Intratumor Heterogeneity ◽

H2ax Phosphorylation ◽

Γh2ax Foci

Abstract Abstract 2817 Poster Board II-793 Abnormal plasma cells (PC) present in patients with multiple myeloma (MM) and its precursor condition, monoclonal gammopathy of undetermined significance (MGUS), characteristically possess multiple chromosomal abnormalities. Moreover, both stages of disease exhibit considerable intratumor heterogeneity, which often becomes even more complex during disease progression. The precise mechanism(s) underlying this process remains unknown. However, we hypothesize that DNA double-strand breaks (DSBs) and compromised repair of these deleterious lesions may underlie intratumor heterogeneity and clonal evolution in the monoclonal gammopathies. In this regard, H2AX, a member of the H2A family of histones, plays a particularly important role in the DSB response and prevention of cancer. Immediately following DSB formation, one or more of the PI3K-like kinases become activated and rapidly phosphorylate H2AX on a conserved serine residue. Phosphorylated H2AX (γH2AX) is then rapidly recruited to the DSB site and is readily detectable as DNA damage foci by immunohistochemistry. The precise function of γH2AX has yet to be determined, however, it is hypothesized that γH2AX may recruit DNA repair proteins to the DSB site and may aid in keeping severed DNA ends in place in order to avoid erroneous end joining. Despite the functional uncertainty of γH2AX, the presence of γH2AX nuclear foci serves as an excellent indicator of DSBs. Therefore, the goal of our study was to assess MM cells for evidence of DSBs. We began our studies using a panel of 8 human MM cell lines. Of note, the number of foci was found to vary among the MM cell lines and to vary from cell to cell with the number of γH2AX foci per cell ranging from 0 to 28. The presence of γH2AX in these cells was also confirmed via flow cytometry and western blotting. We also wished to determine if primary MM and MGUS PCs displayed evidence of DSBs. Among primary patient samples, freshly isolated PCs from 13/18 MM patients and 1/3 MGUS patients exhibited evidence of γH2AX foci. Taken together with the MM cell line data, the number of γH2AX foci was found to increase across the disease spectrum of MGUS to MM patient sample to MM cell line. Endogenous γH2AX foci have previously been detected in a variety of tumor cell lines. Although these foci have been hypothesized to derive from multiple factors, the extent of phosphorylation has been shown to be associated with the number of chromosomal aberrations as well as the phase of the cell cycle. In general, S and G2/M phase cells tend to demonstrate higher levels of H2AX phosphorylation, which is most likely due to doubling of histone content during the cell cycle and the fact that chromatin condensation during DNA replication can also trigger H2AX phosphorylation. Thus, it remained possible that the γH2AX displayed by the cell lines simply reflected cells in the S phase of the cell cycle. To address this possibility, we labeled cells with BrdU and then measured levels of γH2AX in cells in the G1, S and G2/M phases of the cell cycle. However, we observed nearly equal levels of γH2AX in G1 and S phase cells suggesting some level of γH2AX foci was independent of DNA replication. These results were also consistent with our observation that there is no correlation between the plasma cell labeling index and the number of γH2AX foci in CD138+ plasma cells isolated from 18 MM patients. Thus, endogenous γH2AX in MM cells does not appear to be primarily attributed to cycling cells and may be indeed reflective of DSBs. Finally, to further demonstrate that the γH2AX foci genuinely reflected sites of DSBs, we performed double staining for γH2AX foci and 53BP1, a protein that is known to be recruited to DSB sites following DNA damage. Results revealed clear colocalization of γH2AX and 53BP1 in both MM cell lines and MM patient samples. Given that DSBs can lead to genomic instability and tumor progression, our observations that primary MGUS and MM PCs display evidence of DSBs at isolation are intriguing and suggest a mechanism whereby clonal evolution occurs in the monoclonal gammopathies. The presence of a higher frequency of γH2AX foci in MM cell lines is consistent with their derivation from MM patients with aggressive disease. Collectively, these studies suggest MGUS/MM PCs may display an impaired ability to repair DNA damage and studies designed to examine this possibility are underway. Disclosures: Dispenzieri: Celgene: Research Funding.

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Identification of EpHA3 As a New Potential Molecular Target in Multiple Myeloma.

Blood ◽

10.1182/blood.v120.21.2926.2926 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2926-2926

Author(s):

Antonella Caivano ◽

Francesco La Rocca ◽

Alessandra Favole ◽

Sonia Carturan ◽

Enrico Bracco ◽

...

Keyword(s):

Multiple Myeloma ◽

Monoclonal Antibody ◽

Cell Lines ◽

Plasma Cells ◽

Molecular Target ◽

Over Expression ◽

In Vitro Angiogenesis ◽

Novel Therapeutic

Abstract Abstract 2926 Introduction Angiogenesis plays a central role in the progression of both solid and hematological tumors. In particular, in multiple myeloma (MM) the critical role of bone marrow (BM) microenvironment and angiogenesis has been well documented. The past decade has witnessed a dramatic improvement in the therapeutic options in MM. However, the disease remains incurable, underscoring the need for continued efforts towards understanding MM biology and exploitation of novel therapeutic approaches. In this setting, monoclonal antibodies against myeloma-specific cell surface antigens represent a promising therapeutic approach, which is however hampered by a lack of appropriate target structures expressed across all pathogenic myeloma cells. The Eph receptors, a large family of receptor tyrosine kinases (RTKs) activated by ephrins binding, have been implicated in many processes involved in malignancy, including alteration of the tumor microenvironment and in angiogenesis, in both of which EpHA3 likely plays an active role. Aberrant expression of EpHA3 is seen in many types of hematolologic malignancies (some leukemic cell lines, T-cell lymphoma, acute lymphoblastic leukemia, myeloproliferative neoplasms) although it is not expressed ubiquitously. Finally, the over-expression of Eph is believed to be sufficient to confer tumorigenic potential although probably further mechanisms can occur to abnormally activate the receptor. Basing on the role of EpHA3 in haematological malignancies, a first-in-class engineered IgG1 antibody targeting the EpHA (KB004) was developed and it is now under phase I clinical trials in USA and Australia for the treatment of EpHA3 overexpressing hematological myeloid malignancies refractory to conventional treatment. We investigated the EpHA3 role and its preferential membrane–bound by GPI linker ligand EFNA5, in MM patients in order to define EpHA3 as new molecular target for a novel therapeutic approach with a specific anti EpHA3 monoclonal antibody. The EpHA3 expression has been studied through a comparative proteomic analysis between BM endothelial cells (ECs) of patients with MM (MMECs) or with monoclonal gammopathy of undetermined significance (MGECs), of control subjects (normal ECs) and in MM cell lines. Methods After written informed consent, BM aspirates have been collected from 20 MM and 4 MGUS patients. Normal ECs were derived from 3 BM aspirates of subjects with anemia due to iron or vitamin B12 deficiency. We analyzed the expression levels of EpHA3 in normal ECs, MGECs and MMECs and MM cell lines evaluating the mRNA and protein levels by RT-qPCR and by WB coupled to ImmunoFluorescence analysis. The biological effects of EpHA3 targeting in MMECs have been studied silencing the EpHA3 mRNA in MMECs and testing them at 72h after silencing in series of functinal assays including viability assay by trypan blue exclusion staining and by in vitro angiogenesis assay followed by measurement of mesh areas and vessel length. Moreover, we studied EFNA5 mRNA expression levels in Normal ECs, MGECs and MMECs and in MM cell lines by PCR. Results Our data showed that EpHA3 mRNA levels are progressively increased from ECs to MGECs reaching the highest values in MMECs. Subsequent analysis by WB and immunofluorescence confirmed EpHA3 protein upregulation among the different EC types. The MMECs in which EpHA3 has been silenced revealed a protein level reduction of approximately 60% when compared to the control. We could not detect major viability defects. Furthermore, in vitro angiogenesis inhibition was marginal when compared to the not silenced counterpart. To know whether EpHA3 may impact not only MM angiogenesis but also plasma cells, three MM cell lines were studied for the EpHA3 expression. We found the plasma cell lines gave constant over expression of EpHA3. Finally, the preliminary data regarding EFNA5 mRNA expression level showed it is expressed in either MMECs and MM plasma cell lines. The evaluation of KB004 effect on MMECs in term of apoptosis induction and in vitro tube formation inhibition, as well as the analysis of EpHA3 levels in primary MM plasma cells are in progress. Conclusions From this study we expect to characterize the role of the EpHA3in MM patients and to provide experimental evidences supporting the possibility of using EpHA3 as a new molecular target for MM by proving the in vitro efficacy of a monoclonal antibody to target the angiogenesis of MM. Disclosures: No relevant conflicts of interest to declare.

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Compromised Nuclear Sirtuins Activity Sensitizes BRCA-Proficient multiple Myeloma Cells to DNA Damage Agents

Blood ◽

10.1182/blood.v120.21.723.723 ◽

2012 ◽

Vol 120 (21) ◽

pp. 723-723

Author(s):

Michele Cea ◽

Antonia Cagnetta ◽

Aditya Munshi ◽

Yu-Tzu Tai ◽

Teru Hideshima ◽

...

Keyword(s):

Multiple Myeloma ◽

Dna Damage ◽

Dna Repair ◽

Cell Lines ◽

Plasma Cells ◽

Dsb Repair ◽

Dna Repair Mechanisms ◽

Dna Damaging Agents ◽

Normal Plasma ◽

Repair Mechanisms

Abstract Abstract 723 Background: Multiple myeloma (MM) is a clonal malignancy of plasma cells with hallmark genetic instability resulting in large-scale changes at diagnosis, as well as further evolution contributing to disease progression. Inhibition of DNA repair mechanisms leads to significant reduction in acquisition of new genetic changes and associated progression of MM. Mammalian sirtuins are class III NAD+-dependent deacetylases emerging as innovative proteins involved in multiple pathways, including genome maintenance. Methods: A panel of 18 MM cell lines, both sensitive and resistant to conventional and novel anti-MM therapies, was used in the study. The antitumor effect of a pan-sirtuins inhibitor, Nicotinamide (Nam), alone and combined with DNA-damaging agents, was investigated by CTG assay and Annexin-V/propidium iodide staining. Mechanistic studies were performed with thymidine incorporation, Western-blotting, lentivirus-mediated shRNAs and immunofluorescence assay. Analysis of DNA DSB repair was done using chromosomally integrated reporter constructs, followed by cytometer analysis. Results: We analyzed an Affymetrix GeneChip (GSE6477) array of patient MM cells (n=162) compared with normal plasma cells, and found that transcript levels of two nuclear sirtuins (SIRT6 and SIRT7) were significantly higher in monoclonal gammopathy of undetermined significance (MGUS), smoldering MM, active MM, and relapsed MM compared with normal plasma cells. Importantly, protein analysis confirmed increased nuclear levels of these deacetylases in MM cell lines, including those resistant to DNA-damaging agents (MM.1R, LR-5, Dox40), as well as in patient CD138+ MM cells compared to PBMCs from healthy donors. Next we evaluated the functional role of these Sirtuins in MM cells by using loss of function approaches with RNAi. SIRT6 and SIRT7 silencing by knockdown reduced MM cell proliferation compared with control scrambled cells, with only a modest induction of cytotoxicity. We also examined the effects of Nam on DNA-damage response signaling triggered by conventional anti-MM agents melphalan and doxorubicin. Nam treatment did not appreciably affect MM cell viability; however, pretreatment with Nam impaired DNA double-strand breaks (DSBs) repair as well as DNA repair mechanisms triggered by conventional DNA damaging agents, evidenced by γH2AX and RPA phosphorylation, respectively. Consistent with these findings, Nam-pretreated MM cells formed fewer RAD51 foci in response to Doxorubicin and Melphalan, thereby conferring sensitivity to these agents. Importantly, this sensitizing effect was also observed in MM cells resistant to doxorubicin (RPMI-Doxo40) or melphalan (LR5), indicating that Nam increases chemosensitivity in both drug-sensitive and –resistant MM cells. Similarly, lentivirus-mediated shRNA knockdown of SIRT-6 and −7 sensitized MM cells to melphalan and doxorubicin. Finally, both chemical and genetic approaches improved the efficiency of DNA DSB repair mechanisms (Homologous and non-Homologous end-joining Recombination) in MM cell lines containing chromosomally integrated green fluorescent protein-based reporter constructs. Ongoing in vivo experiments are assessing how the chemical susceptibility of SIRT6 and/or 7-deficient cells can be exploited therapeutically. Conclusion: Our study demonstrates a link between nuclear sirtuins and DNA instability in MM cells, providing the basis for incorporation of inhibitors of these SIRTs into innovative anti-MM therapeutic approaches. Disclosures: Munshi: Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.

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