Interleukin-6 Adversely Impacts Genomic Stability Via Targeting Multiple Pathways in Multiple Myeloma

Abstract In our previous investigation, using whole exome sequencing, we have shown that multiple myeloma (MM) patients display a complex dynamic of clonal evolution and the number of mutations in patient samples correlate with overall and relapse free survival. These results highlight the importance of understanding the mechanisms driving genomic instability in MM. Investigating mechanisms underlying genomic instability, we have shown that dysregulated homologous recombination (HR), nuclease (especially apurinic/apyrimidinic related) and APOBEC deaminase activities contribute to genomic instability in MM. We have also demonstrated that bone marrow microenvironment (BMM) also contributes to genomic instability in MM. So here we have further investigated the soluble factors in BMM that may impact genomic integrity. We treated RPMI8226 MM cells with IL-6, IL-17 and TGFb and evaluated their impact on DNA breaks by monitoring the levels of gH2AX (a DNA break marker). Treatment with all 3 cytokines (IL-6, IL-17 and TGFb) caused DNA breaks, as demonstrated by increase in gH2AX, in MM as well as a solid tumor (esophageal cancer) cell lines. Importantly, among these cytokines, the exposure to IL-6 was associated with the highest induction of gH2AX expression. These observations were confirmed in additional MM cell lines (MM1S, H929, OPM2 and U266) treated with IL-6. Since we have previously demonstrated that elevated HR is a key mechanism of genomic instability in MM, we investigated the role of IL-6 in dysregulation of HR pathway by evaluating its impact on p-RPA32 (a marker of DNA end resection which is a decisive step in the initiation of HR), recombinase (RAD51) expression and HR activity (as assessed by a functional assay). Treatment of MM cells with IL-6 led to increased expression of p-RPA32 and RAD51 (as detected by Western blotting) as well as increased HR activity in MM cells. Increased HR activity was also observed following exposure of MM cells to IL-17, TGFb and IFNb, with the highest induction caused by IL-6. However, the combination of cytokines (IL-6, IL-17 and TGFb) led to a further (> 2-fold) increase in HR activity in these cells, indicating that these soluble factors may interact in inducing mechanisms underlying genomic instability in MM. Based on our data showing important role of apurinic/apyrimidinic (AP) nuclease in regulation of HR and genome stability in MM, we also evaluated the impact of IL-6 on abasic sites, the substrate of AP nuclease activity. An increase in the number of abasic sites (ranging from 1.7- to 2.3-fold increase) was observed with increasing concentration of IL-6 in MM1S cells. To further investigated the impact of IL-6 on number of micronuclei, used as marker of genomic instability. The treatment of MM cell lines with IL-6 for 48 hrs led to a dose-dependent increase (ranging from 2- to > 2.5-fold increase) in the number of micronuclei in MM1S and RPMI cells, indicating increased genomic instability. We have previously shown that inhibition of HR by RAD51 knockdown or ABL kinase inhibitor, nilotinib, significantly reduces genomic instability, as assessed by micronuclei assay as well as direct evaluation of impact on genomewide acquisition of copy number changes over time using SNP arrays. To investigate if HR inhibition can reverse IL-6-induced genomic instability, we treated MM cells with IL-6, nilotinib and combination of both and observed that inhibition of HR by nilotinib can reverse IL-6-induced increase in number of micronuclei (by 48±17%) in MM cells. Similarly, addition of anti-IL-6 antibody also reversed the IL6-induced DNA breaks (as assessed from gH2AX expression) in MM1S cells. Moreover, combination of nilotinib and anti-IL6 antibody resulted in maximum inhibition of IL6-induced DNA breaks in MM1S cells. Taken together, these data suggest that IL-6 significantly contributes to dysregulation of HR and genome stability in MM, and agents targeting HR and/or other mechanisms of genomic instability including anti-IL-6 antibody, have potential to reduce/delay genomic evolution and disease progression. Disclosures Munshi: OncoPep: Other: Board of director.

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Dysregulated APOBEC3G causes DNA damage and promotes genomic instability in multiple myeloma

Blood Cancer Journal ◽

10.1038/s41408-021-00554-9 ◽

2021 ◽

Vol 11 (10) ◽

Author(s):

Srikanth Talluri ◽

Mehmet K. Samur ◽

Leutz Buon ◽

Subodh Kumar ◽

Lakshmi B. Potluri ◽

...

Keyword(s):

Multiple Myeloma ◽

Dna Damage ◽

Genomic Instability ◽

Genome Stability ◽

Myeloma Cell ◽

Dna Breaks ◽

Abasic Sites ◽

Underlying Mechanisms ◽

The Impact

AbstractMultiple myeloma (MM) is a heterogeneous disease characterized by significant genomic instability. Recently, a causal role for the AID/APOBEC deaminases in inducing somatic mutations in myeloma has been reported. We have identified APOBEC/AID as a prominent mutational signature at diagnosis with further increase at relapse in MM. In this study, we identified upregulation of several members of APOBEC3 family (A3A, A3B, A3C, and A3G) with A3G, as one of the most expressed APOBECs. We investigated the role of APOBEC3G in MM and observed that A3G expression and APOBEC deaminase activity is elevated in myeloma cell lines and patient samples. Loss-of and gain-of function studies demonstrated that APOBEC3G significantly contributes to increase in DNA damage (abasic sites and DNA breaks) in MM cells. Evaluation of the impact on genome stability, using SNP arrays and whole genome sequencing, indicated that elevated APOBEC3G contributes to ongoing acquisition of both the copy number and mutational changes in MM cells over time. Elevated APOBEC3G also contributed to increased homologous recombination activity, a mechanism that can utilize increased DNA breaks to mediate genomic rearrangements in cancer cells. These data identify APOBEC3G as a novel gene impacting genomic evolution and underlying mechanisms in MM.

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PDZ Binding Kinase (PBK) - a Novel Gene Driving Genomic Evolution in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-119186 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4474-4474

Author(s):

Subodh Kumar ◽

Leutz Buon ◽

Srikanth Talluri ◽

Jialan Shi ◽

Hervé Avet-Loiseau ◽

...

Keyword(s):

Multiple Myeloma ◽

Genomic Instability ◽

Cell Lines ◽

Excision Repair ◽

Critical Role ◽

Micronucleus Assay ◽

Genomic Evolution ◽

Dna Breaks ◽

Functional Changes ◽

Elevated Expression

Abstract As in all cancers, genomic instability leads to ongoing acquisition of new genetic changes in multiple myeloma (MM). This adaptability underlies the development of drug resistance and progression in MM. This genomic instability is driven by cellular processes, mainly related with DNA repair and perturbed by functional changes in limited number of genes. Since kinases play a critical role in the regulation of biological processes, including DNA damage/repair signaling and are relatively easy to screen for inhibitors, we investigated for novel genes involved in the acquisition of new genomic changes in MM. Using a large genomic database which had both the gene expression and CGH array-based copy number information (gse26863, n=246), we first identified a total of 890 expressed kinases in MM and correlated their expression with genomic instability defined as a change in ≥3 and/or 5 consecutive amplification and/or deletion events. We identified 198 kinases whose elevated expression correlated with increased genomic instability (based on FDR ≤ 0.05). Amongst these kinases, using univariate Cox survival analysis, elevated expression of 15 kinases correlated with poor overall as well as event free survival (P ≤0.05) in two MM datasets (IFM70, n=170; gse24080; n=559). We further confirmed the correlation of these 15 genes in both EFS and OS in additional two MM datasets (MMRF CoMMpass Study, IFM-DFCI 2009) as well as in additional solid tumor datasets from TCGA from patients with lung and pancreatic adenocarcinoma (P values ranging from 0.01 to <0.000002). A pathway analysis identified phosphorylation and regulation of proteasome pathway, mitotic spindle assembly/checkpoint, chromosomal segregation and cell cycle checkpoints as among major pathways regulated by these genes. To investigate the relevance of these genes with genomic instability, we performed a functional siRNA screen to evaluate impact of their suppression on homologous recombination (HR). PDZ Binding Kinase (PBK) was one of the top genes whose knockdown caused the maximal inhibition of HR activity in initial screen. To investigate it further in detail, we suppressed PBK in MM cells using shRNA and confirmed that its suppression significantly reduces HR activity. PBK-knockdown also reduced gH2AX levels (marker of DNA breaks) measured by Western blotting and decreased number of micronuclei (a marker of ongoing genomic rearrangements and instability) as assessed by flow cytometry . A small molecule inhibitor of PBK also confirmed a similar reduction in gH2AX levels as well as micronuclei, indicating inhibition of spontaneous DNA breaks and genomic instability. Using mass spectrometry and co-immunoprecipitation, we identified that PBK interacts with FEN1, a nuclease with roles in base excision repair and HR pathways. We confirmed that PBK induces phosphorylation of FEN1 and that inhibition of PBK, suppressed the phosphorylation of FEN1, RAD51 expression and gH2AX levels and it reversed FEN1-induced HR activity. These results confirm that phosphorylation of FEN1 nuclease by PBK contributes to its ability to impact DNA breaks, HR and genome stability in MM. PBK inhibition also significantly sensitized MM cells to melphalan and inhibited cell viability in a panel of MM cell lines (IC50 in MM cell lines ~20-30 nM vs ~100 nM in normal PBMCs) at the same time also reversed melphalan-induced genomic instability, as assessed by micronucleus assay. These data identify PBK as an important target affecting genomic instability, and its inhibitor as a potential drug, to inhibit genomic evolution and MM cell growth. Disclosures Munshi: OncoPep: Other: Board of director.

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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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Molecular Basis of Genomic Instability and Progression in Multiple Myeloma: Potential Role of Apurinic (Apyrimidinic) Endonuclease.

Blood ◽

10.1182/blood.v106.11.1561.1561 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1561-1561

Author(s):

Masood A. Shammas ◽

Hemanta Koley ◽

Paola Neri ◽

Pierfrancesco Tassone ◽

Ramesh B. Batchu ◽

...

Keyword(s):

Multiple Myeloma ◽

Genomic Instability ◽

Cell Lines ◽

Molecular Basis ◽

Endonuclease Activity ◽

Ap Endonuclease ◽

Myeloma Cells ◽

Genome Wide ◽

New Mutations

Abstract Genetic instability is a prominent feature of most cancers including multiple myeloma (MM) and is responsible for ongoing accrual of mutational changes which may lead to development of drug resistance and metastasis. The molecular basis for the generation of genetic diversity in MM is therefore extremely important to understand carcinogenesis and to identify novel targets for treatment. As genomic rearrangements require excision of DNA, we hypothesized that an elevated endonuclease activity may induce recombination and subsequent genomic instability in cancer cells. We developed a plasmid degradation assay that confirmed significantly elevated endonuclease activity in MM cells compared to normal plasma cells. To identify the pre-dominating endonuclease the degradation assay was carried out in the presence of specific endonuclease inhibitors, which identified apurinic/apyrimidinic endonuclease (Ape1 and Ape2) as the predominant endonucleases in mediating increased endonuclease activity in MM. Gene expression analysis confirmed > 2 fold elevation of Ape1 or Ape2 or both in 5 of 6 MM cell lines and 12 of 15 patient samples. Both immunocytochemistry and western blot analyses confirmed upregulation of Ape1 protein in all MM cell lines and patient samples. Next, we investigated the role of elevated APE endonuclease activity in DNA recombination and subsequent genomic re-arrangements. Using a plasmid-based assay we have previously demonstrated significantly elevated homologous recombination (HR) in MM. To investigate the role of elevated AP endonuclease activity in MM, we cultured myeloma cells in the presence of methoxyamine (MX), which specifically inhibits AP endonuclease activity, and evaluated its effect on HR activity and genome-wide appearance of new mutations. Exposure of intact myeloma cells to MX resulted in > 90% inhibition of HR activity and a significant (71±10.9%; p<0.05) reduction in the appearance of new mutations compared to untreated cells, as assessed by genome-wide loss of heterozygosity (LOH) assay (Affymetrix). We also evaluated the effects of overexpression of Ape1 & 2 in normal fibroblasts which have low endonuclease activity. The transgenic upregulation of AP endonucleases (Ape1 and Ape2) in normal cells led to a significant increase in the lecombination activity, leading to a marked mutational instability as indicated by the appearance of over 20,063 and 20,143 new LOH loci per 100,000 polymorphic regions examined throughout the genome, at population doublings 25 and 50 respectively. Mutational instability was also associated with chromosomal instability confirmed by spectral karyotyping of these cells showing significant numerical and structural chromosomal abnormalities. These changes were associated with indefinite growth of cells and formation of tumors when injected in SCID mice. These data suggest that elevated AP endonuclease may be responsible for mutational and chromosomal instabilities, leading to progression of myeloma.

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Role Of Sirtuin-6 In Maintenance Of Genomic Instability In Multiple Myeloma Cells

Blood ◽

10.1182/blood.v122.21.276.276 ◽

2013 ◽

Vol 122 (21) ◽

pp. 276-276

Author(s):

Michele Cea ◽

Antonia Cagnetta ◽

Mariateresa Fulciniti ◽

Yu-Tzu Tai ◽

Chirag Acharya ◽

...

Keyword(s):

Multiple Myeloma ◽

Dna Damage ◽

Dna Repair ◽

Genomic Instability ◽

Cell Lines ◽

Dsb Repair ◽

Dna Damaging Agents ◽

Genotoxic Agents ◽

Equity Ownership

Abstract Background Deregulation of the DNA damage response (DDR) signaling machinery underlies genomic instability, leading to cancer development and clonal evolution. Multiple Myeloma (MM) remains an incurable disease characterized by a highly unstable genome, with aneuploidy observed in nearly all patients. The mechanism causing this karyotypic instability is largely unknown, but recent observations have correlated these abnormalities with dysfunctional DDR machinery. Mammalian NAD+-dependent deacetylase sirtuin-6 (SIRT6) is emerging as new protein involved in multiple pathways, including maintenance of genome integrity. Methods A panel of 18 MM cell lines, both sensitive and resistant to conventional and novel anti-MM therapies, was used in this study. Blood and BM samples from healthy volunteers and MM patients were obtained after informed consent and mononuclear cells (MNCs) separated by Ficoll-Paque density sedimentation. Patient MM cells were isolated from BM MNCs by CD138-positive selection. Lentiviral delivery was used for expression and knock-down of SIRT6 in MM cell lines. The biologic impact of SIRT6 phenotype was evaluated using cell growth, viability and apoptosis assays. DNA Double-Strand Breaks (DSB) repair occurring via homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways was assessed using a transient direct repeat (DR)-GFP/I-SceI system. Results A comparative gene expression analysis of 414 newly-diagnosed uniformly-treated MM patients showed high levels of SIRT6 mRNA in MM patients versus MGUS or normal donors; moreover, in active MM elevated SIRT6 expression correlated with adverse clinical outcome. Due to its prognostic significance, we further evaluated its role in MM biology. We found higher SIRT6 nuclear expression in MM cell lines and primary cells compared to PBMCs from healthy donors. Targeting SIRT6 by specific shRNA increased MM cell survival by reducing DNA repair efficiency (HR and NHEJ). Whole genome profiling of three different SIRT6 knockout (Sirt6-/-) MM cell lines identified a restricted effect of SIRT6 silencing on transcription of DNA damage genes, which also represented the most down-regulated genes. Consistent with these data, GSEA algorithm revealed that gene set regulating DNA repair were prominently enriched in SIRT6 depleted cells (p<0.0001 and FDR=0.003), confirming the role of SIRT6 in this pathway. We next examined the therapeutic relevance of SIRT6 inhibition in MM by evaluating the effect of SIRT6 depletion on cytotoxicity induced by genotoxic agents. SIRT6 shRNA impaired DNA DSB repair pathways triggered by DNA damaging agents, thereby enhancing overall anti-MM activity of these agents. Finally, in concert with our in vitro data, studies using our human MM xenograft model confirmed that SIRT6 depletion enhanced anti-MM activity of DNA-damaging agents. Conclusion Collectively, our data provide basis for targeting SIRT6 as a novel therapeutic strategy in combination with genotoxic agents to enhance cytotoxicity and improve patient outcome in MM. Disclosures: Tai: Onyx: Consultancy. Hideshima:Acetylon Pharmaceuticals: Consultancy. Chauhan:Vivolux: Consultancy. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

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Flap Structure-Specific Endonuclease 1 (FEN1) May be a Key Mediator of Genome Instability in Myeloma: A Cellular Vulnerability with Potential Therapeutic Significance

Blood ◽

10.1182/blood.v128.22.4440.4440 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4440-4440

Author(s):

Masood A Shammas ◽

Leutz Buon ◽

Subodh Kumar ◽

Mehmet K. Samur ◽

David Alagpulinsa ◽

...

Keyword(s):

Genomic Instability ◽

Cell Lines ◽

Conflicts Of Interest ◽

Genetic Recombination ◽

Nuclease Activity ◽

Gene Signature ◽

Genomic Integrity ◽

Dna Breaks ◽

Poor Overall Survival

Abstract Multiple myeloma is associated with a marked genomic instability which leads to acquisition of mutational changes, some of which underlie disease progression including development of drug resistance and poor clinical outcome. Understanding mechanisms of genomic instability is, therefore, extremely important to develop novel improved therapeutic strategies. Since dysregulated nuclease activity can induce DNA breaks and genetic recombination eventually disrupting genomic integrity, we have evaluated nuclease activity and specific nucleases for their role in genomic instability in MM. We previously identified a nuclease gene signature correlating with genomic instability in a myeloma patient dataset and tested it for correlation with survival in two other datasets. We showed that expression of these genes associated with poor overall as well as event free survival in both datasets, IFM172 (P< 0.00005) and gse24080 (P< 0.0008). We have now further refined this signature to a nine gene signature and tested it for correlation with survival in three different MM patient datasets in which gene expression was evaluated either by microarray (GSE39754, n=170; gse24080; n=559) or RNASeq (n=300). Elevated expression of nine gene signature significantly correlated with poor overall survival in all three datasets (P ≤ 1e-06 for IFM70 and gse24080 and P = 0.002 for RNASeq). To biologically and molecularly validate this signature, we conducted an shRNA screen and evaluated impact of all nine genes in signature as well as four additional nucleases on homologous recombination (HR) activity, using a plasmid based assay in which HR produces a functional luciferase gene. Of thirteen nucleases tested, knockdown of seven was associated with ≥50% inhibition of HR activity; the strongest (~80%) inhibition of HR activity was observed by FEN1 knockdown. To further investigate FEN1, we confirmed the role of this nuclease in HR using a different (DRGFP) assay in which homology-based recombination between two mutated genes, generates a functional GFP gene. Using this assay, we showed that FEN1-knockdown in U2OS cells was also associated with a strong (71%) inhibition of HR activity, confirming the role of this nuclease in dysregulation of HR. Evaluation by Western blotting in three different normal PBMC samples and eleven MM cell lines showed that FEN1 was not detected in normal cells, whereas highly expressed in MM cells. Expression profile using microarray also showed that FEN1 is elevated in a subset of MM patient samples. Knockdown of FEN1 in two MM cell lines, RPMI and H929, led to reduction in overall nuclease activity (by ~40%) as assessed by a fluorescence based nuclease activity assay and a similar (~50%) reduction in the levels of gamma-H2AX, a marker of DNA breaks. These data indicate that FEN1 nuclease activity contributes to increased DNA breaks as well as elevated HR activity in MM cells. To further understand the role of FEN1 in dysregulated HR and genome stability in MM, using mass spectrometry, we have identified the interacting proteins. Role of FEN1 in acquisition of new genomic changes over time in MM cells is cuurently being investigated in our laboratory. In summary, we show that FEN1 is an important component of machinery maintaining genomic integrity and plays a significant role in genome dysregulation in myeloma. The FEN1 dysfunction may provide a cellular vulnerability that can be therapeutically exploited. Disclosures No relevant conflicts of interest to declare.

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Integrated Genomics and Functional Validation Identifies a Global Kinase Gene Signature Impacting Genome Stability in Myeloma

Blood ◽

10.1182/blood-2019-129069 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 363-363

Author(s):

Subodh Kumar ◽

Leutz Buon ◽

Srikanth Talluri ◽

Chengcheng Liao ◽

Jialan Shi ◽

...

Keyword(s):

Dna Repair ◽

Homologous Recombination ◽

Genomic Instability ◽

Small Molecule ◽

Copy Number ◽

Genome Stability ◽

Dna Breaks ◽

Genomic Amplification ◽

Data Set ◽

The Impact

Identification of mechanisms underlying genomic instability is necessary to understand disease progression, including development of drug resistance. Our previous data demonstrates that dysregulation of DNA repair and maintenance/modification activities (including homologous recombination (HR), apurinic/apyrimidinic nuclease and APOBEC) significantly contribute to genomic instability in multiple myeloma (MM). However, how these and other pathways involved in genomic instability are dysregulated, remains to be explored. Since kinases play a critical role in the regulation of the maintenance of genomic integrity, we have performed a genome-wide kinome profiling to identify those involved in genomic instability in cancer. First, we analyzed genomic database for ten human cancers (including MM) from TCGA with both tumor cell gene expression and SNP/CGH array-based copy number information for each patient.We assessed genomic instability in each patient based on the total number of amplification and deletion events. We next interrogated all 550 kinases expressed in humans and identified those whose expression correlated with copy number alteration (based on FDR ≤ 0.05) in all tumor types. We identified six kinases whose elevated expression correlated with increased genomic instability defined by genomic amplification/deletion events in all ten cancers, including MM. To demonstrate functional relevance of these kinases, we conducted a CRISPR-based loss of function screen (using 3 guides per gene) in MM cells and evaluated the impact of each gene-knockout on micronuclei, a marker of ongoing genomic rearrangements and instability. For all six kinases, at least one guide resulted in ≥ 65% inhibition of micronuclei formation. Moreover, for five out of the six kinases, at least two guides showed ≥ 60% inhibition of micronuclei. All together, these data establishes a strong relevance of these kinases with genomic instability in MM. PDZ Binding Kinase (PBK) was among top kinases impacting genome stability in this data set with 2 out of 3 guides causing > 88% and 3rdguide causing 35% inhibition of micronuclei formation. We further report that inhibition of PBK, by knockdown or small molecule, inhibits DNA breaks, RAD51 recombinase expression and homologous recombination in MM cells. We further investigated molecular mechanisms involved in PBK-mediated genomic instability in MM. Expression profiling using RNA sequencing of MM cells treated with a specific PBK inhibitor showed that top ten pathways downregulated by treatment were mostly DNA repair/recombination followed by replication and G2/M checkpoint. Interestingly, we identified a notable overlap between PBK-regulated genes with FOXM1 target genes. FOXM1 is a major transcriptional regulator of genes involved in DNA repair, G2/M regulation and chromosomal stability. We, therefore, investigated PBK/FOXM1 interaction and show that PBK interacts with FOXM1 in MM cells. Moreover, the inhibition of PBK, by knockdown or small molecule, inhibits phosphorylation of FOXM1 as well as downregulates FOXM1-regulated HR and cell cycle genes RAD51, EXO1 and CDC25A. These results suggest that PBK-dependent phosphorylation of FOXM1 activity controls transcriptional networks involved in genomic instability in MM. Ongoing work is investigating role of PBK and other kinases in progression of MGUS/SMM to active MM and their impact on ongoing genomic changes with influence on multiple DNA repair pathways including HR. In conclusion, we describe a kinase panel that may have significant role in maintaining genome stability, and their perturbation may allow to improve genome stability in MM. Disclosures Munshi: Adaptive: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Takeda: Consultancy.

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Nucleotide Excision Repair (NER) Is Frequently Impaired and Affects Outcome in Multiple Myeloma (MM)

Blood ◽

10.1182/blood.v124.21.2055.2055 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2055-2055

Author(s):

Raphael Szalat ◽

Matija Dreze ◽

Mehmet Kemal Samur ◽

Anne S. Calkins ◽

Giovanni Parmigiani ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Dna Repair ◽

Genomic Instability ◽

Nucleotide Excision Repair ◽

Cell Lines ◽

Excision Repair ◽

Sequencing Data ◽

Nucleotide Excision

Abstract Introduction Multiple Myeloma (MM) is a heterogeneous disease characterized by genomic instability and eventual poor outcome. Aberrations in DNA repair-related pathways have been considered to explain the instability. Nucleotide excision repair (NER) is an important pathway involved in the removal of bulky adducts and DNA crosslinks induced by various genotoxins. Little is known about the relationship between NER in MM biology and patient outcomes. Here we assess the role of NER in MM. Methods We evaluated NER efficiency in a panel of MM cell lines (n=18), with a functional assay based on the purified DNA-Damage Binding protein 2 (DDB2) complex (DDB2 proteo-probe, Dreze et al. 2014). NER proficiency was correlated with cytogenetic characteristics, p53 status, sequencing data, gene expression profile, and with melphalan (MLP) sensitivity evaluated by CellTiterGlo (CTG). We then evaluated NER efficiency in patient samples and interrogated the role of NER in MM patients by correlating expression of NER genes with survival (OS) in a cohort of 170 patients (IFM 2005-01) homogeneously treated with alkylating agents. Results NER, measured as the amount of (6-4) photoproducts remaining 2 hours after UV irradiation, showed variability between MM cell lines. Out of 18 cell lines, 7 exhibited various levels of NER deficiencies, defined as less than 90% repair at 2 hours (4 cell lines 90-70% and 3 cell lines <60%). The other 11 cell lines presented more than 90% of repair. P53 loss of function did not associate with NER deficiency. Notably, all t(4;14) cell lines tested (n=5) showed a NER repair rate > 90%. NER deficient cell lines (NER <90%) were sensitive to melphalan. However all melphalan sensitive cells did not exhibit NER deficiency, This suggests that other DNA repair pathways are involved in the repair of melphalan-induced lesions. Furthermore, we performed the assay in patient samples showing variable levels of NER, which may reflect different disease status and prognosis. Whole genome sequencing data from 6 NER deficient cell lines revealed missense mutations in critical NER genes in 2 of these cell lines. MM1S and MM1R cells showed mutations in the Xeroderma Pigmentosum Complementation Group A (XPA) gene (mutation D70H), and MM1R was also mutated in the Cockayne syndrome, ERCC6 gene (mutation L682I). Gene expression profile comparison in 12 of these showed a positive correlation between expression of NER genes and NER efficiency. We next studied expression of 20 NER genes in 170 patients treated with high dose melphalan (IFM 2005-01). The analysis revealed a significant negative correlation between 5 overexpressed NER genes (ERCC3, ERCC4, ERCC6, MMS19 and NTHL1) and overall survival (OS). Conclusion NER efficiency is heterogeneous in MM, in part due to acquired mutations. Impairment of NER is associated with outcome as well as may contribute to genomic instability. Ability to proficiently measure NER in patient samples provides us an opportunity to now evaluate NER as a prognostic marker in myeloma. Disclosures No relevant conflicts of interest to declare.

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Elevated APEX1 Disrupts G2/M Checkpoint, Contributing to Evolution and Survival of Myeloma Cells

Blood ◽

10.1182/blood.v126.23.2997.2997 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2997-2997

Author(s):

Subodh Kumar ◽

Srikanth Talluri ◽

Mariateresa Fulciniti ◽

Masood A Shammas ◽

Nikhil C. Munshi

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Fold Increase ◽

Cell Cycle Checkpoints ◽

Cyclin B ◽

Antibody Array ◽

Myeloma Cells ◽

G2 Checkpoint ◽

The Impact

Abstract Cell cycle checkpoints provide the cell with time to repair chromosomal DNA damage before its replication (G1) and also prior to its segregation (G2), thus ensuring integrity, maintenance and protection of genome. Although proper functioning of both checkpoints is essential, G2/M has a special significance as a potentially lethal double-strand break in DNA escape repair and persist from G2 into mitosis, it may recombine in G1 to produce gene rearrangements. Moreover, G2 is the phase where homologous recombination (HR) can utilize a sister chromatid as a template to provide error-free repair. There is ample evidence that supports the role of defective G2/M checkpoint and dysregulated HR in genomic rearrangements and evolution in cancer. Previously, we have shown that elevated APEX1 contributes to dysregulated HR and genome stability in multiple myeloma (MM), and its upregulation leads to genomic instability and tumorigenesis in animal model. To further understand the role of APEX1 in myeloma, we investigated the impact of elevated APEX1 on cell cycle checkpoint/s and in the cellular response to genotoxic exposure. Our investigation using antibody array and subsequent confirmation with immunoprecipitation experiments demonstrated that APEX1 interacts with cyclin B and PLK in myeloma cells. A key step in progression from G2 to mitosis is the activation of cyclin B-CDK1 complex, which subsequently activates PLK to ensure G2/M progression. Based on observed interaction of APEX1 with cyclin B/PLK, we hypothesized that elevated APEX1 disrupts G2 checkpoint by mediating progression into mitosis. To test this, we inhibited APEX1 in myeloma cells by a small molecule as well as by shRNA targeting this gene, and investigated the impact on cell cycle checkpoints using a unique phospho-antibody array which allows investigation of 238 relevant proteins and their phosphorylation status. APEX1 inhibition by small molecule led to downregulation (> 2-fold) of many proteins/phosphorylations involved in the activation of cyclin B-CDK1 complex and other mediators of G2/M progression (including CDC25A, CDC25A, CDK1, ABL1), and upregulation of proteins/phosphorylations involved in G2/M arrest, including CDK1-phospho-Tyr15, 14-3-3 zeta-phospho-Ser58, p53-phospho-Ser15, and MYT and WEE which are involved in negative regulation of cyclin B-CDK1 complex. To further investigate the role of APEX1 in G2/M progression, myeloma cell lines (ARP, RPMI 8226, MM1S, LR5, H929) were treated with APEX1 inhibitor and subjected to cell cycle analysis using flow cytometery. Compared to control cells, all five APEX1-inhibitor treated cell lines showed a strong G2/M block, ranging from 5- to 10-fold increase in the fraction of cells in G2 phase in a dose dependent manner. The G2/M cell cycle arrest of APEX1-treated myeloma cells was further supported by reduced cell viability of treated myeloma cells (RPMI, H929, MM1S, ARP and U266); IC50 of inhibitor in myeloma cell lines ranged from 1.2 to 4 µM. Co-treatment with APEX1 inhbitor also sensitized myeloma cells to Melphalan. Consistent with these data, shRNA-mediated knockdown (KD) of APEX1 in RPMI cells was associated with 4-fold increase in the fraction of cells in G2, relative to control cells. APEX1-KD was also associated with reduction in cell viability (by 40%) and sensitization to melphalan. Our results therefore suggest that elevated APEX1 disrupts G2 checkpoint and sets a stage for genomic rearrangements by allowing persistance of DNA damage from G2 into mitosis. Dysfunctional G2 checkpoint, combined with APEX1-mediated dysregulation of HR, could be attributed to APEX1 associated genomic instability and oncogenic transformation. Therefore, inhibitors of APEX1, alone or in combination with other agents, have potential to make myeloma cells static. Disclosures No relevant conflicts of interest to declare.

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A High Throughput Functional Screen Identifies a Novel Apex Inhibitor: Augments Cytotoxicity While Significantly Decreasing Genomic Evolution in Myeloma

Blood ◽

10.1182/blood-2020-139945 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 10-11

Author(s):

Srikanth Talluri ◽

Jialan Shi ◽

Mychell Neptune-Buon ◽

Subodh Kumar ◽

Lakshmi B. Potluri ◽

...

Keyword(s):

Genomic Instability ◽

Cell Lines ◽

Genome Stability ◽

Genomic Evolution ◽

Dna Breaks ◽

Private Company ◽

Functional Screen ◽

Dna End Resection ◽

End Resection ◽

Dose Dependent

Multiple myeloma (MM) is molecularly heterogenous disease with significant genomic instability. It carries number of mutations at diagnosis (median > 7000) and acquires additional changes overtime. With this background, we have evaluated the molecular intermediates of genomic instability in MM. Based on our large transcriptomic data we have identified apurinic/apyrimidinic deoxyribonuclease (APEX) as an important target whose elevated activity contributes to dysregulation of homologous recombination (HR) and genome stability in MM. Our investigation also demonstrates that transgenic overexpression of APEX nucleases induces genomic instability, leading to oncogenic transformation and tumorigenesis in a murine and Zebrafish models. Importantly, we have now observed that both transgenic as well as chemical inhibition of APEX1, reduces DNA breaks, HR activity and genomic instability as measured by micronucleus assay, and induces G2/M arrest in myeloma as well as esophageal cancer cells. To identify novel and effective inhibitors of APEX1, we optimized a high throughput APEX1 activity assay and screened a custom library of 100,000 small molecules. We identified API-93 as an effective APEX inhibitor in both the primary and secondary screens, and have now investigated it, alone as well as in combination, with existing myeloma drugs, for impact on different parameters of growth and genome maintenance. Although API-93 had minimal single agent cytotoxic effect on MM cells, it synergistically increased cytotoxicity of chemotherapeutic agent cyclophosphamide in both MM cell lines (MM1S and RPMI) tested, and also increased the efficacy of melphalan in several MM cell lines (RPMI, MM1R, KK1 and LR5) tested. A strong synergistic effect of API-93 was also observed in combination with lenalidomide in 5 MM cell lines tested (RPMI, MM1S, MM1R, KK1, H929; combination indexes < 1) as well as velcade in MM cells. For evaluation of impact on genomic changes, myeloma cells were treated with API-93, live cells purified and evaluated for impact on DNA breaks (by measuring levels of γ-H2AX), DNA end resection (a decisive step in the initiation of HR, by monitoring levels of p-RPA32), and genome stability by investigating micronuclei (the marker of genomic instability). Treatment with API-93 reduced spontaneous as well as melphalan-induced DNA breaks, DNA end resection as well as genomic instability (as assessed by significant reduction in micronuclei) in MM cells, in a dose-dependent manner. To further confirm the impact on genome stability, the MM cells were cultured in the presence or absence of API-93, and the acquisition of new copy number changes over 3 weeks were measured using SNP arrays, in the cultured relative to Day 0 cells (representing baseline genome). Relative to control cells, the cells treated with API-93 resulted in dose-dependent decrease in the acquisition of new copy number events, from 50% to > 90%. In conclusion, these data demonstrate APEX gene as being associated with MM cell survival and with genome instability. The novel APEX1 inhibitor, identified in a functional screen, provides an important tool to augment cytotoxicity of the current therapeutics while significantly decreasing genomic evolution in MM. Disclosures Munshi: Adaptive: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Legend: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy.

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