scholarly journals TRIM33 Loss in Multiple Myeloma Impairs the DNA Damage Response Resulting in Sensitivity to PARP and ATR Inhibitors

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1570-1570
Author(s):  
Roisin M McAvera ◽  
Jonathan J Morgan ◽  
Ken I Mills ◽  
Lisa J Crawford
Keyword(s):  
Multiple Myeloma ◽  
Dna Damage ◽  
Dna Damage Response ◽  
High Throughput ◽  
Chromosomal Instability ◽  
Conflicts Of Interest ◽  
Parp Cleavage ◽  
Structural Variants ◽  
Damage Response ◽  
The Impact

Abstract Introduction Chromosomal instability is a hallmark of Multiple Myeloma (MM), with most patients displaying cytogenetic abnormalities which can arise due to DNA damage response (DDR) defects. TRIM33 is an E3 ligase and transcriptional co-repressor located on chromosome 1p13.2, a region frequently deleted in MM. Previous studies have shown that TRIM33 plays a role in the DDR and can regulate chromosomal stability, but its precise function remains unknown. In this study we investigated the impact of TRIM33 loss in MM on genomic stability and DDR pathways and whether this could be exploited therapeutically. Methods The CoMMpass dataset (IA15 release) was screened to identify patients with copy number (CN) loss of TRIM33 and this was correlated with overall survival (OS) and structural variants. TRIM33 shRNA knockdown models were established in JJN3 and U266 cells. The effect on DDR signalling was determined by western blotting and immunofluorescence. The Selleckchem DNA Damage/Repair Compound Library was screened on the JJN3 model in a high-throughput manner using the CellTox™ Green cytotoxicity assay. Validation of selected compounds was performed using CellTiter® Glo viability assay or clonogenic assays. Combination indices (CI) were calculated using CompuSyn software. Results Data on CN, OS and structural variants were available for 730 newly diagnosed MM patients and of these, 69 (9.5%) were identified to have a CN loss of TRIM33. These patients have poorer OS compared to those without TRIM33 loss (52.3 months vs 72.6 months; p<0.0001). Moreover, they exhibit a significantly higher median number of structural variants (deletions, duplications, inversions, and translocations; 38 vs 26; p<0.0001), indicative of increased chromosomal instability. Our data in MM cell lines has shown that TRIM33 is rapidly recruited to chromatin within 5 minutes of induced DNA damage. TRIM33 knockdown led to an increase in 53BP1 foci formation and endogenous γH2AX (P<0.001) indicating unrepaired DNA double-strand breaks (DSBs) typical of a DDR defect. In response to these DSBs both ATM and ATR kinases were activated as demonstrated by increased pKAP1 Ser824 and pCHK1 Ser345 respectively (p<0.001). Additionally, we observed a reduction in RAD51 (p<0.05) indicative of a potential defect in the DSB repair pathway homologous recombination (HR). To identify therapeutic vulnerabilities relating to TRIM33 loss, we performed a high-throughput screen to assess sensitivity to 160 unique DNA damaging compounds. TRIM33 knockdown cells exhibited increased sensitivity to 27 compounds across a range of drug classes. Additional studies confirmed that compared to control cells, TRIM33 knockdown sensitized cells to the PARP inhibitor Olaparib and ATR inhibitors BAY-1895344 and VE-821. Further investigation with VE-821 demonstrated that whilst treatment induced PARP cleavage and DSBs in both control and knockdown cells within 48 hours, knockdown cells exhibited significantly more pCHK1 Ser345 inhibition (p<0.01). Furthermore, combining VE-821 with bortezomib yielded synergistic effects in TRIM33 knockdown cells across a range of doses (CI range 0.57-0.9) while no synergy was observed in control cells (CI>1 for all combinations). Conclusion We have identified a subset of MM patients with TRIM33 loss who display high-risk disease characterized by chromosomal abnormalities and defective DDR. Alongside this we have identified PARP and ATR inhibitors as therapeutic vulnerabilities in cell line models of TRIM33 loss. Moreover, we demonstrate that ATR inhibition increases the efficacy of bortezomib in TRIM33 knockdown cells. Further investigation into these compounds could lead to novel therapies for patients with TRIM33 loss. Disclosures No relevant conflicts of interest to declare.

Relationships Between Aberrant Activity of the NF-κB Subunits and Outcome In Chronic Lymphocytic Leukemia: The Dual Role of DNA Damage Sensor Enzymes

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3588-3588
Author(s):  
Evan A Mulligan ◽  
Jill E Hunter ◽  
Arabella EG Baird ◽  
Sarah Elliott ◽  
Geoffrey P Summerfield ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Binding ◽  
Dna Damage Response ◽  
Poor Prognosis ◽  
Conflicts Of Interest ◽  
Excision Repair ◽  
Ex Vivo ◽  
Constitutive Activation ◽  
Damage Response ◽  
The Impact

Abstract Abstract 3588 Poor prognosis patients with chronic lymphocytic leukaemia (CLL) can be identified by cytogenetic abnormalities such as del(17p), and del(11q) and corresponding mutations in TP53 and ATM (ataxia telangiectasia mutated kinase) respectively, which are associated with chemoresistance by virtue of defects in the DNA damage response pathway. We have demonstrated that overexpression of DNA-dependent protein kinase (DNA-PK), which mediates non-homologous end joining, is also associated with poor prognosis CLL1. Recent data show that constitutive activation of the p65 subunit of the transcription factor, NF-κB, confers poor survival in CLL. Since the repertoire of genes activated by NF-κB includes anti-apoptotic and pro-survival genes, NF-κB therefore represents an attractive target for therapeutic intervention. The parthenolide analogue, LC-1, has been shown to be synergisitic with fludarabine in ex vivo studies on CLL cells2, and has validated the concept of targeting NF-κB and prompted clinical trials in CLL patients. Previous data from our laboratory using cell line models demonstrated that DNA damage-induced NF-κB activation requires the base excision repair protein, poly(ADP-ribose) polymerase (PARP), to confer radioresistance3. The DNA damage sensor, ATM, also mediates the response to DNA damage, via phosphorylation of IKK-γ (Inhibitory κ-Kinase/NEMO). In fact, we demonstrated that radio-sensitization by an ATM inhibitor is mediated via NF-κB, rather than by the inhibition of double strand break repair4. Since these observations identify key DNA damage response proteins as regulators of NF-κB activity, we hypothesized that inhibitors of PARP, ATM and DNA-PK, could chemosensitize CLL cells via inhibition of NF-κB. Here, we analyzed NF-κB activity by quantifying NF-κB subunit DNA binding (measured by ELISA) in an unselected CLL cohort. Constitutive activation of the p65 and p50 subunits correlated closely (P= 0.001, n= 57), and predicted shorter time to first treatment (TTFT) and overall survival (OS). Importantly, higher activation occurred in del(17p) (e.g. p50; P= 0.05, n= 49) cases and in those cases that had received treatment. p52 and c-Rel activation correlated with p65 and p50 activation. However, although high p65 and p50 activation predicted shorter OS and TTFT, increased p52 and c-Rel activation were associated with a longer TTFT (up to 40 months) demonstrating the complex crosstalk between the NF-κB subunits in CLL. We were able to correlate p50 and p65 subunit activation with ex vivo resistance to fludarabine and chlorambucil in 12 cases: the most chemoresistant cases had higher p50 and p65 DNA binding activity. Preliminary data indicates that levels of activated NF-κB binding seen in the ELISA correlates with protein expression in nuclear extracts isolated from CLL cells (P= 0.0013, n=26). Here we make the novel observation that the increase in DNA damage-induced NF-κB activation is linked to increased DNA-PK activation. DNA-PK catalytic subunit levels were significantly higher in patients with high p65 activation (P= 0.02, n= 30), regardless of treatment status. Furthermore, a selective inhibitor of DNA-PK, NU7441, increased mitoxantrone-induced cytotoxicity in CLL cells (up to 50-fold) and reduced p65 and p50 DNA binding, indicating a direct link between DNA-PK and NF-κB activation. Ongoing studies are investigating this mechanistic link further, using chromatin immunoprecipitation (ChIP) and using siRNA knockdown. CLL cells were radiosensitized by either the DNA-PK inhibitor, NU7441 or the pan IKK inhibitor, BAY 11–7082. Strikingly, a combination of these two inhibitors had no further effect on radiosensitization, suggesting that DNA-PK and NF-κB act in a common pathway. We have demonstrated that inhibition of ATM sensitizes CLL cells to DNA damage, and future work will assess the impact of ATM and PARP inhibition on NF-κB activity in CLL cells. These data present a novel role for DNA-PK in the regulation of NF-κB and highlight important new therapeutic avenues for the use of DNA-PK inhibitors, which may prove useful in overcoming NF-κB mediated therapeutic resistance in CLL. Willmore E, et al, Clin Cancer Res. 2008. Hewamana S, et al, Clin Cancer Res. 2008. Hunter JE, et al, Proceedings of the AACR. 2009. Veuger SJ et al, Under review, DNA repair. 2010. Disclosures: No relevant conflicts of interest to declare.

Effect of the Pan-PIM Kinase Inhibitor, AZD1208, Alone and in Combination with Bortezomib in Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5726-5726
Author(s):  
Loredana Santo ◽  
Janani Ramachandran ◽  
Diana D Cirstea ◽  
Homare Eda ◽  
Neeharika Nemani ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dna Damage ◽  
Cell Survival ◽  
Cell Lines ◽  
Dna Damage Response ◽  
Kinase Inhibitor ◽  
Parp Cleavage ◽  
Pim Kinases ◽  
Damage Response ◽  
Equity Ownership

Abstract The proto-oncogene proviral integration site for moloney murine leukemia virus (PIM) kinases (PIM-1, PIM-2, and PIM-3) are serine/threonine kinases upregulated in multiple cancer cells including multiple myeloma (MM). Our preliminary data show higher expression of PIM 1/2/3 in patient derived tumor cells (CD138+) compared to the stromal compartment (CD138-), confirming that PIM expression is predominant in MM cell compartment. Although PIM2 expression is significantly higher than PIM1 or PIM3 in patient MM cells and in human MM cell lines, transient knockout of PIM1, PIM2 and PIM3 individually showed a similar cytotoxicity in U266 MM cells 24 hours post transfection, suggesting that all three isoforms play a role in MM cell survival and that an inhibitor of all three isoforms would have a therapeutic advantage in MM. AZD1208 is a potent, highly selective PIM kinase inhibitor that effectively inhibits all three isoforms. Here we investigated the preclinical activity of AZD1208 in MM. Maximal cytotoxicity was observed in 72h culture, with IC50 of 1µM in KMS-12BM. AZD1208 1µM inhibited the phosphorylation of BAD at Ser-112 (a direct PIM kinase substrate) and the phosphorylation of P70S6K T389 (downstream of the mTOR-C1 complex) in KMS12-BM, MM1S and MM.1R MM cells, confirming that PIM kinases regulate mTOR activity. The effect on downstream signaling pathways correlated with the inhibitory effect on KMS-12BM, but not MM1S and MM1R, cell survival, suggesting alternative feedback mechanisms of resistance. AZD1208 (2µM) induced increased PARP cleavage after 24 hr in KMS-12BM cells; however, only high doses of AZD1208 (10µM) induced apoptotic signaling cascade in MM1S and MM1R cells. Since Checkpoint kinase 1 (Chk1) is a critical component of the DNA damage response and is regulated by PIM1 and PIM2 in other hematologic malignancies, we further investigated the effect of AZD1208 on major signaling checkpoints involved in the DNA damage response. Increasing doses of AZD1208 induced phosphorylation of Chk1 (Ser 345), Chk2 (Thr68), histone H2A family member H2A.X (Ser 139), and p53 (Ser15) after 4h of treatment in KMS-12BM cell lines, suggesting that cell death induced by AZD1208 is a consequence of the DNA damage response. We next examined the combination treatment of AZD1208 with bortezomib, which is not known to induce DNA damage. A synergistic cytotoxic effect was observed in MM1S, U266 and KMS-12BM. Importantly, PIM1 and PIM2 expression was increased by bortezomib, suggesting that PIM1 and PIM2 are substrate of proteasome. On going studies aim at understanding the mechanism underlying this synergistic combination and in validating its efficacy in vivo in order to provide the rationale for clinical protocols of PIM kinase inhibitors in MM. Disclosures Hideshima: Acetylon Pharmaceuticals: Consultancy. Huszar:AstraZeneca: Employment, Equity Ownership. Anderson:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Gilead: Consultancy; Sanofi Aventis: Consultancy; BMS: Consultancy; Oncopep/Acetylon: Equity Ownership. Raje:novartis, Amgen, Celgene, Millenium, Onyx: Consultancy; Eli Lilly, Acetylon: Research Funding.

scholarly journals Impact of U2AF1 Mutations on Clinical Characteristics and RNA Splicing in Myelodysplastic Syndromes (MDS)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2979-2979
Author(s):  
Bing Li ◽  
Jinqin Liu ◽  
Tiejun Qin ◽  
Zefeng Xu ◽  
Gang Huang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Gene Set Enrichment Analysis ◽  
Aberrant Splicing ◽  
Hematopoietic Stem ◽  
Damage Response ◽  
Subclonal Mutation ◽  
The Impact

To investigate the clinical impact of U2AF1MT in MDS, we studied a cohort of 511 patients who were performed 112-targeted gene sequencing. We identified U2AF1MT in 86 (17%) subjects, including U2AF1S34F (64%, n=55), U2AF1S34Y (23%, n=20), U2AF1Q157R (6%, n=5), U2AF1Q157P (5%, n=4) and U2AF1R156H (2%, n=2). Compared to many MDS-related gene mutation, U2AF1MT more frequently occurred in younger patients (Median age: 45 vs. 54 yrs; P=0.001). We performed multivariate analysis to characterize the associations between U2AF1MT and other common gene mutations and cytogenetic abnormalities, SF3B1MT (P=0.001, OR=0.214, 95%CI: 0.083-0.55) was inversely associated with U2AF1MT subjects, while isolated +8 (P<0.001, OR=4.671, 95%CI: 2.510-8.689), KRASMT (P=0.008, OR=3.521, 95%CI: 1.388-8.934), and ASXL1MT (P=0.033, OR=2.003, 95%CI: 1.059-3.786) was significantly enriched in U2AF1MT subjects. Using copy number-adjusted VAF, we reconstructed the clonal architecture of U2AF1MT patients to establish whether a U2AF1MT was an ancestral or subclonal mutation. 65 patients with 1 or more mutation, except for U2AF1MT, were analyzed. U2AF1MT was an ancestral mutation in 46 (71%) patients and was a subclonal mutation in 19 (29%) patients. In our cohort, U2AF1MT was associated with worse overall survival (OS, P=0.01). Considering for different type of U2AF1MT, MDS patients with U2AF1Q157/R156MT tended to have a reduced median survival as opposed to patients with S34 (17 vs. 30 months, P=0.318). Then, we performed a comprehensive and systematic analysis to determine the impact of U2AF1MT on pre-mRNA splicing in bone marrow mononuclear cells from 40 patients and 5 healthy controls. We identified 315 misregulated splicing events between patients with U2AF1MT (n=20) and those without any spliceosome mutations (n=17) (Figure 1A). U2AF1MT were associated with a higher proportion of exon skipping (SE, 68%), mutually exclusive exons (MXE, 19%) and alternative 3′ splice site (A3SS, 8%) usage events (Figure 1B). Interestingly, only one gene (MRS2, Magnesium Transporter MRS2) with significantly dysregulated expression was observed from the aberrantly spliced genes. Gene ontology (GO) analysis was performed on the genes showing significant aberrant splicing events and the analysis showed a strong cluster of GOs associated with histone modification, RNA metabolism and cell cycle processing. Gene set enrichment analysis (GSEA) revealed DNA damage response pathway, including DNA Repair and p53 pathway as one of the major gene sets up regulated upon U2AF1MT (Figure 2). Then, the single cell RNA-sequencing was performed in Lin-CD34+CD38-CD90+CD45RA- hematopoietic stem cell (HSC) from one sample with U2AF1MT. After QC filtering, 5 HSC with U2AF1MT and 47 HSC without any spliceosome mutations were used for aberrant splicing events analysis. Totally, 1752 misregulated splicing events were indentified (Figure 3A). Forty three genes with significantly dysregulated expression were observed from the aberrantly spliced genes, including 6 genes (TP53BP1, MDM2, PRC1, RAD51C, IP6K2, TMBIM6) taking part in DNA damage response (Figure 3B). In summary, this comprehensive study provides novel insights into U2AF1MT MDS disease pathophysiology, with newly identified clinical associations, and dysregulated genes and pathways representing potential new therapeutic targets. Disclosures No relevant conflicts of interest to declare.

scholarly journals TCF19 Impacts a Network of Inflammatory and DNA Damage Response Genes in the Pancreatic β-Cell

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 513
Author(s):  
Grace H. Yang ◽  
Danielle A. Fontaine ◽  
Sukanya Lodh ◽  
Joseph T. Blumer ◽  
Avtar Roopra ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Association Studies ◽  
Cell Cycle Gene ◽  
Genome Wide Association Studies ◽  
Β Cell ◽  
Nucleotide Incorporation ◽  
Damage Response ◽  
The Impact

Transcription factor 19 (TCF19) is a gene associated with type 1 diabetes (T1DM) and type 2 diabetes (T2DM) in genome-wide association studies. Prior studies have demonstrated that Tcf19 knockdown impairs β-cell proliferation and increases apoptosis. However, little is known about its role in diabetes pathogenesis or the effects of TCF19 gain-of-function. The aim of this study was to examine the impact of TCF19 overexpression in INS-1 β-cells and human islets on proliferation and gene expression. With TCF19 overexpression, there was an increase in nucleotide incorporation without any change in cell cycle gene expression, alluding to an alternate process of nucleotide incorporation. Analysis of RNA-seq of TCF19 overexpressing cells revealed increased expression of several DNA damage response (DDR) genes, as well as a tightly linked set of genes involved in viral responses, immune system processes, and inflammation. This connectivity between DNA damage and inflammatory gene expression has not been well studied in the β-cell and suggests a novel role for TCF19 in regulating these pathways. Future studies determining how TCF19 may modulate these pathways can provide potential targets for improving β-cell survival.

scholarly journals DNA Damage Response in Multiple Myeloma: The Role of the Tumor Microenvironment

Cancers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 504
Author(s):  
Takayuki Saitoh ◽  
Tsukasa Oda
Keyword(s):  
Multiple Myeloma ◽  
Dna Damage ◽  
Dna Repair ◽  
Tumor Microenvironment ◽  
Dna Damage Response ◽  
Genetic Instability ◽  
Dna Repair Mechanisms ◽  
Damage Response ◽  
Repair Mechanisms

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by genomic instability. MM cells present various forms of genetic instability, including chromosomal instability, microsatellite instability, and base-pair alterations, as well as changes in chromosome number. The tumor microenvironment and an abnormal DNA repair function affect genetic instability in this disease. In addition, states of the tumor microenvironment itself, such as inflammation and hypoxia, influence the DNA damage response, which includes DNA repair mechanisms, cell cycle checkpoints, and apoptotic pathways. Unrepaired DNA damage in tumor cells has been shown to exacerbate genomic instability and aberrant features that enable MM progression and drug resistance. This review provides an overview of the DNA repair pathways, with a special focus on their function in MM, and discusses the role of the tumor microenvironment in governing DNA repair mechanisms.

Viral Infection Sentinel Rig-I Maintains Hematopoietic Stem Cell Function Through Regulating DNA-Damage Response

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1166-1166
Author(s):  
Wu Zhang ◽  
Meng-Lei Ding ◽  
Xian-Yang Li ◽  
He-Zhou Guo ◽  
Hong-Xin Zhang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Dna Lesions ◽  
Type I ◽  
Hematopoietic Stem ◽  
Damage Response ◽  
Underlying Mechanisms ◽  
And Function

Abstract Throughout life hematopoietic stem cells (HSCs) have to cope with various kinds of insults from inflammation to DNA damage constantly to maintain the integrity of stemness. It is possible that certain core factors are commonly implicated in the maintenance of HSC pool and function under discrete physiological and pathological conditions. However, the underlying mechanisms remain largely unexplored. Previous works have demonstrated that retinoic acid inducible gene I (Rig-I) plays an essential role in recognizing viral RNA and activating type I IFN transcription, but whether Rig-I is involved in the core program governing HSCs’ behaviors is unclear. Here, we report that in the steady status Rig-I deficiency significantly increased HSC number by dysregulating the cell-cycling status of HSCs in mice. However, HSCs in Rig-I-/- mice were actually more sensitive to genotoxic treatments such as irradiation as compared to wild type HSCs, causing more Rig-I-/- mice to die of hematopoietic exhaustion. In accordance, HSC transplantation assays showed a significant impact of Rig-I loss on the hematopoietic regeneration capacity. Mechanistically, we found that Rig-I represented a pivotal component of the molecular pathways that mediate DNA-damage response and the repair of DNA lesions. Taken together, these data indicate a crucial role of innate immunity-regulatory factor Rig-I in the maintenance of HSCs. Disclosures: No relevant conflicts of interest to declare.

Induction of the DNA damage response by IAP inhibition triggers natural immunity via upregulation of NKG2D ligands in Hodgkin lymphoma in vitro

2013 ◽  
Vol 394 (10) ◽  
pp. 1325-1331 ◽  
Author(s):  
Maike Sauer ◽  
Katrin S. Reiners ◽  
Hinrich P. Hansen ◽  
Andreas Engert ◽  
Stephan Gasser ◽  
...  
Keyword(s):  
Dna Damage ◽  
Hodgkin Lymphoma ◽  
Dna Damage Response ◽  
Natural Immunity ◽  
Immune Recognition ◽  
Iap Antagonist ◽  
Damage Response ◽  
The Impact ◽  
Apoptosis Proteins

Abstract Evasion of apoptosis is a hallmark of cancer cells. Inhibitor of apoptosis proteins (IAPs) act as endogenous inhibitors of programmed cell death and are overexpressed in several tumors including Hodgkin lymphoma (HL). Preclinical studies indicate antitumor activity of IAP antagonists and clinical studies in hematological malignancies are underway. Here, we investigate the impact of the small molecule IAP antagonist LCL161 on HL cell lines. Although the antagonist caused rapid degradation of cIAP1 leading to TNFα secretion, LCL161 did not promote apoptosis significantly. However, LCL161 induced expression of MICA and MICB, ligands for the activating immune receptor NKG2D, and enhanced the susceptibility of HL cells to NKG2D-dependent lysis by NK cells. MICA/B upregulation was dependent on activation of the DNA damage response upon LCL161 treatment. Taken together, we demonstrate a novel link between IAP inhibition, DNA damage and immune recognition.

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