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

Blood ◽  
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.

ABL Tyrosine Kinase Plays an Important Role in Mechanisms Involved in Genomic Instability in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2087-2087
Author(s):  
Subodh Kumar ◽  
Purushothama Nanjappa ◽  
Srikanth Talluri ◽  
Masood A Shammas ◽  
Nikhil C Munshi
Keyword(s):  
Dna Repair ◽  
Tyrosine Kinase ◽  
Genomic Instability ◽  
Copy Number ◽  
Repair Mechanism ◽  
Genomic Changes ◽  
Abl Kinase ◽  
Abl Tyrosine Kinase ◽  
Rpmi 8226 ◽  
The Impact

Abstract Homologous recombination (HR) is a DNA repair mechanism that uses extensive sequence homology in the participating DNA molecules for an accurate repair. In a normal cellular environment, HR is the most precise DNA repair mechanism and therefore has a unique role in the maintenance of genomic integrity and stability. Normally HR is tightly regulated, however, as it involves incision and recombination of genomic DNA fragments, if dysregulated or dysfunctional, it can also be deleterious. Consistent with this view, we have shown that elevated HR activity mediates genomic instability and development of drug resistance in MM. Here we have now investigated the mechanism that may contribute to dysregulation of HR and genomic instability in MM, as well as evaluated an agent able to decrease acquisition of new genomic changes. It has been shown that Abl kinase regulates recombinase RAD51 by affecting its expression, stability as well as phosphorylation at Y315. Phosphorylation of RAD51 (at Y315) mediates its dissociation from BCR-ABL1 kinase and migration to the nucleus to form nuclear foci, one of the initial steps in HR. We have evaluated nilotinib, a small molecule inhibitor of Abl kinase and observed that it inhibited HR activity in all MM cell lines tested, in a dose-dependent manner. At 5 µM, nilotinib inhibited HR activity in MM1S, RPMI 8226 and U266 MM cells by 64%, 78% and 80%, respectively. Nilotinib led to reduced phosphorylation of RAD51 at Y315, the phosphorylation which affects RAD51 migration. Nilotinib-mediated inhibition of RAD51 and HR activity was also associated with reduced DNA breaks, as indicated by reduced levels of g-H2AX. To determine the impact of nilotinib on genome stability, MM (RPMI 8226) cells were cultured in the presence of nilotinib for three weeks and the impact of this treatment on appearance of new copy number changes was evaluated using SNP arrays. Using day 0 cells as baseline to identify new copy number events at 3 weeks, the acquisition of new genomic changes was inhibited by 50% in the presence of nilotinib. As we have previously reported that induction of HR helps develop dexamethasone resistance in a short period of time, we investigated whether inhibition of HR by nilotinib may improve efficacy of melphalan and dexamethasone in MM. Nilotinib (at 2.5 µM) significantly increased the efficacy of melphalan (10 µM); and dexamethasone (10 nM) in RPMI 8226 cells. The relation between these observed effects and inhibition of HR is being investigated. In conclusion, we have observed that Abl tyrosine kinase plays an important role in genomic instability in myeloma and its inhibition using nilotinib, suppresses the underlying mechanism of genomic instability and reduces acquisition of new genomic changes with potential for clinical application. Disclosures No relevant conflicts of interest to declare.

Factors that influence bidirectional long-tract homozygosis due to double-strand break repair in Candida albicans

Genetics ◽  
2021 ◽  
Author(s):  
Timea Marton ◽  
Murielle Chauvel ◽  
Adeline Feri ◽  
Corinne Maufrais ◽  
Christophe D’enfert ◽  
...  
Keyword(s):  
Dna Repair ◽  
Candida Albicans ◽  
Genome Stability ◽  
Molecular Mechanisms ◽  
Strand Break ◽  
Genomic Rearrangements ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Break Site ◽  
The Impact

Abstract Genomic rearrangements have been associated with the acquisition of adaptive phenotypes, allowing organisms to efficiently generate new favorable genetic combinations. The diploid genome of Candida albicans is highly plastic, displaying numerous genomic rearrangements that are often the by-product of the repair of DNA breaks. For example, DNA double-strand breaks (DSB) repair using homologous-recombination pathways are a major source of loss-of-heterozygosity (LOH), observed ubiquitously in both clinical and laboratory strains of C. albicans. Mechanisms such as break-induced replication (BIR) or mitotic crossover (MCO) can result in long tracts of LOH, spanning hundreds of kilobases until the telomere. Analysis of I-SceI-induced BIR/MCO tracts in C. albicans revealed that the homozygosis tracts can ascend several kilobases towards the centromere, displaying homozygosis from the break site towards the centromere. We sought to investigate the molecular mechanisms that could contribute to this phenotype by characterizing a series of C. albicans DNA repair mutants, including pol32-/-, msh2-/-, mph1-/- and mus81-/-. The impact of deleting these genes on genome stability revealed functional differences between Saccharomyces cerevisiae (a model DNA repair organism) and C. albicans. Additionally, we demonstrated that ascending LOH tracts towards the centromere are associated with intrinsic features of BIR and potentially involve the mismatch repair pathway which acts upon natural heterozygous positions. Overall, this mechanistic approach to study LOH deepens our limited characterization of DNA repair pathways in C. albicans and brings forth the notion that centromere proximal alleles from DNA break sites are not guarded from undergoing LOH.

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

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.

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

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4467-4467
Author(s):  
Chaitanya Yenumula ◽  
Srikanth Talluri ◽  
Tommaso Perini ◽  
Subodh Kumar ◽  
Jialan Shi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Genomic Instability ◽  
Cell Lines ◽  
Genome Stability ◽  
Fold Increase ◽  
Dna Breaks ◽  
Soluble Factors ◽  
Abasic Sites ◽  
The Impact

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.

scholarly journals Elevated APE1 Mediates Dysregulation of Homologous Recombination in Myeloma: Mechanisms and Translational Significance

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2074-2074 ◽  
Author(s):  
Subodh Kumar ◽  
Maria Gkotzamanidou ◽  
Jagannath Pal ◽  
Renquan Lu ◽  
Puru Nanjappa ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genomic Instability ◽  
Small Molecule ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Small Molecule Inhibitor ◽  
Telomere Maintenance ◽  
Dna Breaks ◽  
Molecule Inhibitor ◽  
Rad51 Promoter

Abstract We have previously shown that elevated homologous recombination (HR) activity mediates genomic instability and progression in myeloma. Moreover, elevated HR also plays critical role in tumor growth by contributing to telomere maintenance and other survival mechanisms. We have now investigated molecular mechanisms driving dysregulated HR in MM. We observe that elevated apurinic apyrimidic endonuclease 1 (APE1) significantly contributes to dysregulation of HR, directly through transcriptional control of RAD51 as well as indirectly through its ability to induce DNA breaks. The transgenic suppression using APE1-specifc shRNA inhibits RAD51 expression, HR activity, and genomic instability as measured by SNP array profile in MM cells; whereas its induction leads to increased RAD51 expression, HR activity, genomic instability and oncogenic transformation in normal human cells. We have further investigated how APE1, a base excision repair protein, regulates RAD51, the key component of HR in myeloma and evaluated a novel small molecule inhibitor of APE1 for its impact on HR and associated genomic instability. Using an antibody array we observed that APE1 physically interacts with p73, a known transcriptional regulator of RAD51. To demonstrate that APE1 and P73 interact with RAD51 promoter in MM cells, we conducted chromatin immunoprecipitation (chip) assays and observed both P73 and APE1 binding to adjacent loci on RAD51 promoter. Taken together, these data suggest that elevated APE1 induces RAD51 expression through its interaction with P73. We next evaluated effect of a small molecule inhibitor specifically targeting nuclease function of APE1 in MM cells, and observed that it inhibits RAD51 expression, RAD51 foci, HR activity and reduces DNA breaks as assessed by g-H2AX levels on western blotting. The suppression of APE1 by this small molecule was associated with significant loss of RAD51 promoter activity, as assessed by a RAD51-promoter driven luciferase construct, as well as reduced RAD51 transcript levels. As APE1 is required for DNA repair which plays a critical part in development of drug resistance, we evaluated if APE1 inhibitor can help sensitize MM cells to DNA damaging agents. To investigate this we pretreated RPMI8226 and LR5 MM cells with the small molecule inhibitor of APE1 and then exposed them to various concentrations of melphalan for 48 hrs and cell viability and growth assessed. Pretreatment with APE1 inhibitor not only sensitized RPMI8226 cells to melphalan but also resistant LR5 cell line. These observations suggest that elevated APE1 is a critical target to induce DNA damage or overcome certain type of resistance possibly driven by repair mechanisms. In summary, we conclude that elevated APE1 is a critical intermediate for dysregulated HR and associated genomic instability, and small molecule inhibitor of APE1 has potential to reduce genomic instability, prevent/delay progression and improve clinical outcome in MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals Transcription and mRNA export machineries SAGA and TREX-2 maintain monoubiquitinated H2B balance required for DNA repair

2018 ◽  
Vol 217 (10) ◽  
pp. 3382-3397 ◽  
Author(s):  
Federica M. Evangelista ◽  
Anne Maglott-Roth ◽  
Matthieu Stierle ◽  
Laurent Brino ◽  
Evi Soutoglou ◽  
...  
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
Genomic Instability ◽  
Genome Stability ◽  
Mrna Export ◽  
Genome Integrity ◽  
Nuclear Pore ◽  
Functional Interaction ◽  
Histone H2b ◽  
Coactivator Complex

DNA repair is critical to maintaining genome integrity, and its dysfunction can cause accumulation of unresolved damage that leads to genomic instability. The Spt–Ada–Gcn5 acetyltransferase (SAGA) coactivator complex and the nuclear pore–associated transcription and export complex 2 (TREX-2) couple transcription with mRNA export. In this study, we identify a novel interplay between human TREX-2 and the deubiquitination module (DUBm) of SAGA required for genome stability. We find that the scaffold subunit of TREX-2, GANP, positively regulates DNA repair through homologous recombination (HR). In contrast, DUBm adaptor subunits ENY2 and ATXNL3 are required to limit unscheduled HR. These opposite roles are achieved through monoubiquitinated histone H2B (H2Bub1). Interestingly, the activity of the DUBm of SAGA on H2Bub1 is dependent on the integrity of the TREX-2 complex. Thus, we describe the existence of a functional interaction between human TREX-2 and SAGA DUBm that is key to maintaining the H2B/HB2ub1 balance needed for efficient repair and HR.

scholarly journals In vivo analysis of FANCD2 recruitment at meiotic DNA breaks in Caenorhabditis elegans

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Marcello Germoglio ◽  
Anna Valenti ◽  
Ines Gallo ◽  
Chiara Forenza ◽  
Pamela Santonicola ◽  
...  
Keyword(s):  
Dna Repair ◽  
Caenorhabditis Elegans ◽  
Genome Stability ◽  
Genetic Interaction ◽  
Model Systems ◽  
Dna Breaks ◽  
Strand Breaks ◽  
C Elegans ◽  
In Vivo Analysis

AbstractFanconi Anemia is a rare genetic disease associated with DNA repair defects, congenital abnormalities and infertility. Most of FA pathway is evolutionary conserved, allowing dissection and mechanistic studies in simpler model systems such as Caenorhabditis elegans. In the present study, we employed C. elegans to better understand the role of FA group D2 (FANCD2) protein in vivo, a key player in promoting genome stability. We report that localization of FCD-2/FANCD2 is dynamic during meiotic prophase I and requires its heterodimeric partner FNCI-1/FANCI. Strikingly, we found that FCD-2 recruitment depends on SPO-11-induced double-strand breaks (DSBs) but not RAD-51-mediated strand invasion. Furthermore, exposure to DNA damage-inducing agents boosts FCD-2 recruitment on the chromatin. Finally, analysis of genetic interaction between FCD-2 and BRC-1 (the C. elegans orthologue of mammalian BRCA1) supports a role for these proteins in different DSB repair pathways. Collectively, we showed a direct involvement of FCD-2 at DSBs and speculate on its function in driving meiotic DNA repair.

scholarly journals Homologous Recombination Repair Polymorphisms and the Risk for Osteosarcoma / Polimorfizam Gena Odgovornih Za Reparaciju Dnk Homolognom Rekombinacijom I Rizikza Pojavu Osteosarkoma

2015 ◽  
Vol 34 (2) ◽  
pp. 200-206 ◽  
Author(s):  
Katja Goričar ◽  
Viljem Kovač ◽  
Janez Jazbec ◽  
Janez Lamovec ◽  
Vita Dolžan
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
Genome Stability ◽  
Cancer Susceptibility ◽  
Homologous Recombination Repair ◽  
Nucleotide Polymorphisms ◽  
Dna Repair Mechanisms ◽  
Recombination Repair ◽  
Repair Mechanisms ◽  
Repair Genes

Summary Background: DNA repair mechanisms are essential for maintaining genome stability, and genetic variability in DNA repair genes may contribute to cancer susceptibility. Our aim was to evaluate the influence of polymorphisms in the homologous recombination repair genes XRCC3, RAD51, and NBN on the risk for osteosarcoma. Methods: In total, 79 osteosarcoma cases and 373 controls were genotyped for eight single nucleotide polymorphisms (SNPs) in XRCC3, RAD51, and NBN. Logistic regression was used to determine the association of these SNPs with risk for osteosarcoma. Results: None of the investigated SNPs was associated with risk for osteosarcoma in the whole cohort of patients, however, in patients diagnosed before the age of thirty years XRCC3 rs861539 C>T and NBN rs1805794 G>C were associated with significantly decreased risk for osteosarcoma (P=0.047, OR=0.54, 95% CI=0.30-0.99 and P=0.036, OR=0.42, 95% CI=0.19-0.94, respectively). Moreover, in the carriers of a combination of polymorphic alleles in both SNPs risk for osteosarcoma was decreased even more significantly (Ptrend=0.007). The risk for developing osteosarcoma was the lowest in patients with no wild-type alleles for both SNPs (P=0.039, OR=0.31, 95% CI=0.10-0.94). Conclusions: Our results suggest that polymorphisms in homologous recombination repair genes might contribute to risk for osteosarcoma in patients diagnosed below the age of thirty years.

scholarly journals Targeting Homologous Recombination in Lymphoid Malignancies: Evaluation of Four Small Molecule Inhibitors of RAD51

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2080-2080
Author(s):  
Melinda Day ◽  
Tyler Maclay ◽  
Amber Cyr ◽  
Muneer G Hasham ◽  
Kin-hoe Chow ◽  
...  
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Cell Line ◽  
Genomic Instability ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Burkitt’S Lymphoma ◽  
Lymphoid Malignancies ◽  
Anti Tumor Activity

Genomic instability is recognized as a driver of tumorigenesis and cancer progression. Loss of tumor suppressors or activation of oncogenes can induce DNA damage stress, promoting genomic instability and creating dependencies upon key DNA repair pathways. These dependencies can be targeted therapeutically to induce synthetic lethality. The homologous recombination (HR) repair pathway is an attractive target. HR deficient cancers are hypersensitive to numerous anticancer drugs, and tumors will often induce expression of HR genes to promote drug resistance. RAD51 is a key component of the HR pathway. RAD51 forms nucleoprotein filaments at sites of DNA damage and replication fork stalls, mediating homologous DNA strand exchange to promote recombinational repair of breaks and damaged replication forks. We utilized four small molecule inhibitors of RAD51-mediated HR for evaluation of RAD51 as a potential therapeutic target. Compounds CYT-0851, CYT-0853, CYT-1027, and CYT-1127 were evaluated for anti-cancer activity in vitro and in vivo. To determine the impact of the small molecules on RAD51 and HR, all four were tested for effects on RAD51 focus formation and sister chromatid exchange (SCE) activity. All the compounds showed a reduction in SCE activity, however only CYT-0851 and CYT-0853 produced a measurable reduction in RAD51 foci. We have previously shown that that RAD51 inhibition leads to accumulation of DNA breaks, and ultimately cell death, in cells expressing the DNA mutator protein Activation Induced Cytidine deaminase (AICDA/AID). Cytotoxicity assays were performed in an AID+ (Daudi, Burkitt's Lymphoma) and AID- (WI-38, fibroblast) cell lines. All four compounds were preferentially active in AID+ cells with little to no cytotoxicity observed in the AID-negative WI-38 cell line. CYT-0853 was the most potent in the Daudi cell line with an EC50 of 8nM. All four compounds were orally bioavailable in all preclinical species tested but showed differences in pharmacokinetics. Preclinical cell line derived xenograft models of AID-high Burkitt's lymphoma (Daudi) and B-cell acute lymphoblastic leukemia (CCRF-SB) were used to determine the in vivo anti-tumor activity of the compounds in lymphoid cancer models. CYT-0851 and CYT-0853 both showed significant anti-tumor activity with tumor growth inhibition of greater than 50% in both models. Further analysis showed drug exposure with CYT-0851 was more consistent in the CDX models than CYT-0853. Overall, these data indicate that RAD51 and HR are attractive therapeutic targets for the treatment of lymphoid malignancies and that CYT-0851 is a viable clinical development candidate. Disclosures Day: Cyteir Therapeutics: Employment. Maclay:Cyteir Therapeutics: Employment. Cyr:Cyteir Therapeutics: Employment. Mills:Cyteir Therapeutics: Employment, Equity Ownership.

scholarly journals Small molecule inhibitors of a human recombination-associated ATPase, RAD54

2019 ◽  
Author(s):  
Kirk T. Ehmsen ◽  
Kenny K.H. Ang ◽  
William D. Wright ◽  
Julia L. Davies ◽  
Yassir Younis ◽  
...  
Keyword(s):  
Dna Repair ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Homology Search ◽  
Dna Breaks ◽  
Repair Synthesis ◽  
Molecular Events ◽  
Dna Strand Exchange ◽  
Dna Repair Synthesis

ABSTRACTHomologous recombination (HR) is a principal support pathway for DNA replication and for recovery from DNA breaks and interstrand crosslinks, making it a rational target for inhibition in cancer therapy. The ATPase RAD54 functions in molecular events that promote DNA sequence-preservation during HR-mediated damage repair, including homology search, DNA strand exchange, and transition to DNA repair synthesis within a displacement loop intermediate. We developed a high-throughput biochemical screen to identify small-molecule inhibitors of human RAD54, using a phosphate detection assay to monitor RAD54 ATPase activity in the presence of double-stranded DNA (dsDNA). After filtering potential DNA intercalators and ‘frequent hitters,’ we identified two chemotypes that reproducibly inhibited RAD54 ATPase in vitro. We evaluated these chemotypes for inhibition of RAD54-dsDNA binding and cancer cell survival. A halogenated carbazole/dihydroacridine scaffold inhibited a panel of SWI2/SNF2-related ATPases but not VCP/p97, an unrelated ATPase. Small molecules that interfere with key steps in HR— such as inhibitors of RAD54—may expose DNA repair-dependent vulnerabilities in cancer cells.

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