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

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

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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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XRCC5 Plays an Important Role in Homologous Recombination, Genome Stability and Survival of Myeloma Cells

Blood ◽

10.1182/blood.v126.23.1218.1218 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1218-1218 ◽

Cited By ~ 1

Author(s):

Edward Laane ◽

Purushothama Nanjappa ◽

Subodh Kumar ◽

Florence Magrangeas ◽

Stephane Minvielle ◽

...

Keyword(s):

Cell Cycle ◽

Board Of Directors ◽

Genomic Instability ◽

Cell Lines ◽

Copy Number ◽

Genome Stability ◽

Myeloma Cell ◽

Advisory Committees ◽

Myeloma Cells ◽

Elevated Expression

Abstract Understanding mechanisms underlying genomic instability is critical in delineating pathogenesis and development of new treatments for prevention and treatment of cancer. We have previously shown that dysregulated homologous recombination (HR) significantly contributes to genomic instability and progression in multiple myeloma (MM). To identify the regulators of HR and genome stability in MM, we conducted a functional shRNA screen and identified XRCC5 (Ku80) as a novel regulator of HR in MM cells. XRCC5 has been known to work as part of DNA ligase IV-XRCC4 complex in the repair of DNA breaks by non-homologous end joining (NHEJ) and the completion of V(D)J recombination events. Evaluation by Western blotting showed that all myeloma cell lines tested (RPMI, MM1S, OPM2, MM1R, U266, ARP, H929) had elevated expression of XRCC5, ranging from 3- to 10-fold elevation relative to average expression in two normal PBMC samples. Expression profiling showed a wide range of XRCC5 expression in myeloma patients, with a subset of patients with very high expression. To investigate the role of XRCC5 in ongoing acquisition of genomic changes, we investigated the association of XRCC5 with genomic instability using two different patient datasets (gse26863, n=246 and IFM 170 pt dataset) in which both the gene expression and genomic copy number information for each patient was available. Copy events were defined as changes observed in ≥ 3 and/or 5 consecutive SNPs. Higher XRCC5 expression significantly correlated with increase in the number of copy number change events in both the 170 dataset (p ≤ 0.005 for amplifications and p = 0.0001 for deletions) as well as in gse26863 dataset (p ≤ 0.004 for amplifications and p ≤ 0.00003 for deletions). To understand mechanisms by which XRCC5 regulates HR in myeloma cells, we investigatedprotein-protein interactions using a custom protein array coated with antibodies against major DNA repair and cell cycle proteins. Array was sequentially incubated with MM cell lysate and HRP-conjugated anti-XRCC5 antibody, and interacting partners were then identified by their address on the array. Investigation in two different cell lines (RPMI and U266) showed that XRCC5 in myeloma interacts with XRCC4 (an NHEJ protein), a panel of major HR regulators (RAD51, RAD52, BRCA2, BRCA1, BARD1, P73, P53, C-ABL) and with components of cell cycle including CDC42, CDK1 (which controls entry from G2 to mitosis), CDK4, CDK6, CHK, CDC36, CDC34, and cyclins E and H. Consistent with these data, knockdown (KD) of XRCC5 was associated with reduced HR as well as reduced proliferation rate followed by a complete cell death over a period of two to three weeks in different experiments, in all 3 myeloma cell lines tested. Moreover, the investigation in U266 cells showed that XRCC5-KD is associated with 3-fold increase in the fraction of cells in G2 phase of cell cycle. Importantly, the elevated expression of XRCC5 was associated with shorter event free (p < 0.013) as well as poor overall survival (p < 0.008) in 170 patient dataset. We evaluted the expression and clinical correlation of XRCC5 in RNA-seq data from 311 newly-diagnosed MM patients and observed that the elevated expression of XRCC5 also correlated with event free survival (p = 0.03). In summary, we report that XRCC5, besides its known role in NHEJ, has important roles in HR, cell cycle and may be involved in the crosstalk among these DNA repair pathways. Elevated XRCC5 expression is associated with dysregulation of HR with consequent impact on survival of myeloma patients. Elevated XRCC5 is, therefore, a promising new target to inhibit/reduce genomic evolution as well as MM cell growth. Disclosures Avet-Loiseau: celgene: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees.

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Human SIRT6 Promotes DNA End Resection Through CtIP Deacetylation

Science ◽

10.1126/science.1192049 ◽

2010 ◽

Vol 329 (5997) ◽

pp. 1348-1353 ◽

Cited By ~ 261

Author(s):

Abderrahmane Kaidi ◽

Brian T. Weinert ◽

Chunaram Choudhary ◽

Stephen P. Jackson

Keyword(s):

Homologous Recombination ◽

Genome Stability ◽

Protein A ◽

Strand Break ◽

Dsb Repair ◽

Interacting Protein ◽

Dna Double Strand Break ◽

Dna End Resection ◽

Lysine Deacetylases ◽

End Resection

SIRT6 belongs to the sirtuin family of protein lysine deacetylases, which regulate aging and genome stability. We found that human SIRT6 has a role in promoting DNA end resection, a crucial step in DNA double-strand break (DSB) repair by homologous recombination. SIRT6 depletion impaired the accumulation of replication protein A and single-stranded DNA at DNA damage sites, reduced rates of homologous recombination, and sensitized cells to DSB-inducing agents. We identified the DSB resection protein CtIP [C-terminal binding protein (CtBP) interacting protein] as a SIRT6 interaction partner and showed that SIRT6-dependent CtIP deacetylation promotes resection. A nonacetylatable CtIP mutant alleviated the effect of SIRT6 depletion on resection, thus identifying CtIP as a key substrate by which SIRT6 facilitates DSB processing and homologous recombination. These findings further clarify how SIRT6 promotes genome stability.

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Abraxas suppresses DNA end resection and limits break-induced replication by controlling SLX4/MUS81 chromatin loading in response to TOP1 inhibitor-induced DNA damage

Nature Communications ◽

10.1038/s41467-021-24665-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Xiao Wu ◽

Bin Wang

Keyword(s):

Dna Synthesis ◽

Genome Stability ◽

Double Strand Breaks ◽

Strand Breaks ◽

Topoisomerase 1 ◽

Dna End Resection ◽

Break Induced Replication ◽

End Resection ◽

Mutagenic Mechanism

AbstractAlthough homologous recombination (HR) is indicated as a high-fidelity repair mechanism, break-induced replication (BIR), a subtype of HR, is a mutagenic mechanism that leads to chromosome rearrangements. It remains poorly understood how cells suppress mutagenic BIR. Trapping of Topoisomerase 1 by camptothecin (CPT) in a cleavage complex on the DNA can be transformed into single-ended double-strand breaks (seDSBs) upon DNA replication or colliding with transcriptional machinery. Here, we demonstrate a role of Abraxas in limiting seDSBs undergoing BIR-dependent mitotic DNA synthesis. Through counteracting K63-linked ubiquitin modification, Abraxas restricts SLX4/Mus81 recruitment to CPT damage sites for cleavage and subsequent resection processed by MRE11 endonuclease, CtIP, and DNA2/BLM. Uncontrolled SLX4/MUS81 loading and excessive end resection due to Abraxas-deficiency leads to increased mitotic DNA synthesis via RAD52- and POLD3- dependent, RAD51-independent BIR and extensive chromosome aberrations. Our work implicates Abraxas/BRCA1-A complex as a critical regulator that restrains BIR for protection of genome stability.

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Dysregulated Aid/Apobec Family Proteins Promote Genomic Instability in Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.803.803 ◽

2016 ◽

Vol 128 (22) ◽

pp. 803-803

Author(s):

Srikanth Talluri ◽

Mehmet Kemal Samur ◽

Leutz Buon ◽

Stekla A Megan ◽

Purushothama Nanjappa ◽

...

Keyword(s):

Genomic Instability ◽

Cell Lines ◽

Copy Number ◽

Cytidine Deaminase ◽

Myeloma Cell ◽

Mrna Editing ◽

Genomic Changes ◽

Elevated Expression ◽

Apobec Family

Abstract The AID/APOBEC family of cytidine deaminase proteins includes AID (activity induced deaminase), and 10 related APOBEC enzymes (A1, A2, A3A, A3B, A3C, A3D, A3F, A3G, A3H and A4). AID has been well-studied for its role in somatic hyper mutation and class switch recombination of immunoglobulin genes whereas APOBECs (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) have been shown to have roles in mRNA editing and in antiviral immunity. Dysregulated activity of APOBECs causes C >T transitions or C>G, C>A transversions in DNA. We have recently shown APOBEC signature mutation pattern in multiple myeloma (MM) genomes (Bolli et al Nat. Comm. 2014), and interestingly, the APOBEC mutation signature correlates with sub clonal diversity in myeloma. A role for the AID/APOBECs in generation of somatic mutations has also been proposed in a variety of other cancers based on identification of APOBEC signature mutations In order to understand which APOBECs are dysregulated in myeloma, we performed RNA sequencing analysis of primary myeloma cells from 409 newly-diagnosed MM patients and myeloma cell lines. Our analysis showed elevated expression of several APOBEC family members; mainly A3A, A3B, A3C, and A3G. We then optimized a plasmid-based functional assay and found high cytidine deaminase activity in extracts from a number of myeloma cell lines and patient derived CD138+ cells compared to CD138+ cells from healthy donors, suggesting that APOBECs are dysregulated in myeloma. We then investigated the impact of elevated APOBEC expression/function on overall genome maintenance and acquisition of genomic changes (such as amplifications, deletions) overtime. We used shRNA-mediated knockdown of specific APOBEC proteins in myeloma cell lines and investigated the acquisition of genomic changes in control and knockdown cells during their growth in culture, using SNP (Single Nucleotide Polymorphism) arrays and WGS (whole genome sequencing) platforms. Our results with both approaches showed significant reduction in the accumulation of copy number changes (both amplifications and deletions) and overall mutation load after APOBEC knockdown. Evaluation with both the SNP and WGS showed that when control and APOBEC knockdown cells were cultured for three weeks, the acquisition of new copy number and mutational changes throughout genome were reduced by ~50%. We next investigated the relationship between APOBEC expression/activity in MM and other DNA repair pathways. Using an in vitro HR activity assay, we measured HR activity in extracts from control and APOBEC knockdown cells. Depletion of APOBEC proteins resulted in 50-80% reduction in in vitro HR activity of the extracts. We also evaluated correlation between HR activity and gene expression using RNA-seq data from myeloma cells derived from 100 patients at diagnosis and identified the genes whose expression correlated with HR activity. Elevated expression of APOBECs 3D, 3G and 3F significantly correlated with high HR activity (R=0.3; P≤0.02), suggesting their relevance to HR. Analyzing genomic copy number information for each patient we have also observed significant correlation between higher expression of A3G and increased genomic instability in this dataset (P=0.0045). In summary, our study shows that dysregulated APOBECs induce mutations and genomic instability, and inhibiting APOBEC activity could reduce the rate of accumulation of ongoing genomic changes. This data sheds light on biology of the disease as well as clonal evolution. Disclosures Munshi: Amgen: Consultancy; Oncopep: Patents & Royalties; Celgene: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Merck: Consultancy; Pfizer: Consultancy.

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Fanci-FANCD2 Promotes Genome Stability and DNA Repair By Down-Regulating BLM Helicase Activity

Blood ◽

10.1182/blood-2021-152218 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1113-1113

Author(s):

Fengshan Liang ◽

Arvindhan Nagarajan ◽

Manoj M Pillai ◽

Patrick Sung ◽

Gary M. Kupfer

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Replication Fork ◽

Genome Stability ◽

Head And Neck Tumor ◽

Amino Acid Deletion ◽

Blm Helicase ◽

Dna End Resection ◽

End Resection ◽

Mutant Cells

Abstract Background: Fanconi anemia (FA) is a genetic disease characterized by bone marrow failure, developmental defects, and higher risk of cancer. Mutations in FA genes have been detected commonly in a large swath of cancers. In the FA DNA repair pathway, DNA damage induces the mono-ubiquitination of the FANCI-FANCD2 (ID2) heterodimer and this regulation licenses the execution of downstream DNA damage signaling and repair steps. In response to replication stress, FANCD2 also prevents replication fork collapse during S phase. Bloom syndrome (BS) is also a genomic instability disease, characterized by growth abnormalities and cancer predisposition. The single BS protein, BLM helicase, participates in DNA repair by promoting DNA end resection and double Holliday junction dissolution. It has been shown that BLM is involved in restart of stalled replication fork. FA and BS have functional interactions. In tumor DNA sequencing of the Yale Precision Tumor board, we identified a somatic 6 amino acid deletion in FANCD2 in a head and neck tumor, while a germline point mutation was found on the other allele. We have identified a FANCD2-L822A mutant with defective BLM binding, which was used to further investigate the role of FANCD2-BLM interaction in genome stability and DNA repair. Methods: Highly purified proteins were used to investigate how ID2 affects helicase and DNA end resection activity of the BLM complex. HeLa, FANCD2-deficient, and FANCD2 corrected fibroblast cell lines were used to examine pRPA2 and RAD51 foci formation. We also used DNA fiber assay to detect end resection and isolation of proteins on nascent DNA (iPOND) assay to examine the RAD51 recruitment on replication fork. Results: A somatic 6 amino acid deletion (p819-824) in FANCD2 was identified in a head and neck tumor. FA-D2 mutant cells expressing the mutant cDNA demonstrated defects in FANCD2 mono-ubiquitination and DNA damage hypersensitivity. A FANCD2-L822A mutant with defective BLM binding was identified (Figure A, B). We found that Bloom helicase and its DNA end resection activity within BLM-DNA2-RPA were negatively regulated by the heterodimer ID2 (Figure C, D). Both DNA and BLM binding of the ID2 are required for the inhibitory function. The premature DNA end resection and HU sensitivity in FANCD2 deficient and mutant cells are rescued by BLM knockdown. By iPOND assay, we discovered that FANCD2 antagonizes BLM to promote RAD51 recruitment on HU-stalled replication fork. Conclusions: Our study suggests that the DNA end resection activity of BLM-DNA2 is tightly regulated by FANCD2 to ensure that the nuclease DNA2 normally resects the DNA intermediate needed for efficient DNA repair and RAD51 recruitment to protect replication forks. Our findings highlight that ID2-BLM interaction functions in DNA damage repair to maintain genome stability. Figure 1 Figure 1. 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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Integrated genomics and comprehensive validation reveal novel drivers of genomic evolution in esophageal adenocarcinoma

10.1101/2020.04.23.058230 ◽

2020 ◽

Author(s):

Subodh Kumar ◽

Leutz Buon ◽

Srikanth Talluri ◽

Marco Roncador ◽

Chengcheng Liao ◽

...

Keyword(s):

Tumor Growth ◽

Genomic Instability ◽

Esophageal Adenocarcinoma ◽

Gene Signature ◽

Genomic Evolution ◽

Functional Studies ◽

Treatment And Prevention ◽

Elevated Expression ◽

Integrated Genomics

AbstractIdentification of genes driving genomic evolution can provide novel targets for cancer treatment and prevention. Here we show identification of a genomic instability gene signature, using an integrated genomics approach. Elevated expression of this signature correlated with poor survival in esophageal adenocarcinoma (EAC) as well as three other human cancers. Knockout and overexpression screens confirmed the relevance of this signature to genomic instability. Indepth evaluation of TTK (a kinase), TPX2 (spindle assembly factor) and RAD54B (recombination protein) further confirmed their role in genomic instability and tumor growth. Mutational signatures identified by whole genome sequencing and functional studies demonstrated that DNA damage and homologous recombination were common mechanisms of genomic instability induced by these genes. Consistently, a TTK inhibitor impaired EAC cell growth in vivo, and increased chemotherapy-induced cytotoxicity while inhibiting genomic instability in surviving cells. Thus inhibitors of TTK and other genes identified in this study have potential to inhibit/delay genomic evolution and tumor growth. Such inhibitors also have potential to increase chemotherapy-induced cytotoxicity while reducing its harmful genomic impact in EAC and possibly other cancers.

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Competing interactions modulate the activity of Sgs1 during DNA end resection

10.1101/515791 ◽

2019 ◽

Author(s):

Kristina Kasaciunaite ◽

Fergus Fettes ◽

Maryna Levikova ◽

Peter Daldrop ◽

Petr Cejka ◽

...

Keyword(s):

Single Molecule ◽

Genome Stability ◽

Strand Break ◽

Cellular Functions ◽

Single Stranded Dna ◽

Single Molecule Level ◽

Protein Partners ◽

Dna Double Strand Break ◽

Dna End Resection ◽

End Resection

AbstractDNA double-strand break repair by homologous recombination employs long-range resection of the 5’ DNA ends at the break points. In Saccharomyces cerevisiae, this process can be performed by the RecQ helicase Sgs1 and the helicase-nuclease Dna2. Though functional interplay has been shown, it remains unclear whether and how the proteins cooperate on the molecular level. Here, we resolved the dynamics of DNA unwinding by Sgs1 at the single molecule level and investigated its regulation by Dna2, the single-stranded DNA binding protein RPA and the Top3-Rmi1 complex. We found that Dna2 modulates the velocity of Sgs1, indicating that during end resection the proteins form a physical complex and couple their activities. Sgs1 unwinds DNA and feeds single-stranded DNA to Dna2 for degradation. RPA is found to regulate the processivity and the affinity of Sgs1 to the DNA fork, while Top3-Rmi1 modulated the velocity of Sgs1. We think that the differential regulation of the Sgs1 activity by its protein partners is important to allow diverse cellular functions of Sgs1 during the maintenance of genome stability.

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High-Copy-Number Expression of Sub2p, a Member of the RNA Helicase Superfamily, Suppresses hpr1-Mediated Genomic Instability

Molecular and Cellular Biology ◽

10.1128/mcb.21.16.5459-5470.2001 ◽

2001 ◽

Vol 21 (16) ◽

pp. 5459-5470 ◽

Cited By ~ 34

Author(s):

Hua-Ying Fan ◽

Robert J. Merker ◽

Hannah L. Klein

Keyword(s):

Rna Polymerase Ii ◽

Genomic Instability ◽

Copy Number ◽

Genome Stability ◽

Genome Instability ◽

Protein A ◽

Mrna Splicing ◽

Splicing Factor ◽

High Copy Number ◽

Permissive Temperature

ABSTRACT We report on a novel role for a pre-mRNA splicing component in genome stability. The Hpr1 protein, a component of an RNA polymerase II complex and required for transcription elongation, is also required for genome stability. Deletion of HPR1 results in a 1,000-fold increase in genome instability, detected as direct-repeat instability. This instability can be suppressed by the high-copy-numberSUB2 gene, which is the Saccharomyces cerevisiae homologue of the human splicing factor hUAP56. Although SUB2 is essential, conditional alleles grown at the permissive temperature complement the essential function of SUB2 yet reveal nonessential phenotypes. These studies have uncovered a role for SUB2 in preventing genome instability. The genomic instability observed in sub2 mutants can be suppressed by high-copy-numberHPR1. A deletion mutant of CDC73, a component of a PolII complex, is also unstable for direct repeats. This too is suppressed by high-copy-number SUB2. Thus, defects in both the transcriptional machinery and the pre-mRNA splicing machinery can be sources of genome instability. The ability of a pre-mRNA splicing factor to suppress the hyperrecombination phenotype of a defective PolII complex raises the possibility of integrating transcription, RNA processing, and genome stability or a second role for SUB2.

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