scholarly journals Targeting DNA Repair to Overcome Drug Resistance in Hodgkin Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 26-26
Author(s):  
Julie Devin ◽  
Quentin Denis ◽  
Caroline Bret ◽  
Jerome Moreaux
Keyword(s):  
Drug Resistance ◽  
Dna Repair ◽  
High Risk ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Brdu Incorporation ◽  
Synthetic Lethal ◽  
Genotoxic Agents ◽  
Chk1 Inhibitor ◽  
Dna Repair Inhibitors

Classical Hodgkin lymphoma (cHL) is characterized by rare tumor-initiating Hodgkin and Reed-Sternberg cells (HRS) surrounded by a microenvironment with a reactional immune cells infiltrate. cHL accounts for 15% to 25% of all lymphomas. This neoplasm is curable in the majority of cases with chemotherapy including ABVD (Doxorubicin, Bleomycin, Vinblastine, Dacarbazine) or BEACOPP (Bleomycin, etoposide, Doxorubicin, cyclophosphamide, Vincristine, procarbazine and Prednisone) and/or radiation. However, 15-20% of high-risk patients ultimately relapse and high dose chemotherapy in combination with autologous stem-cell transplantation is successful in only half of the patients with relapsed/refractory cHL (Eichenaueret al., Eur. Soc. Med. Oncol,2018). HRS cells are characterized by genetic instability, abnormal DNA damage response and repair that may play a role in drug resistance in high-risk cHL (Reichelet al., Blood,2015, Monroyet al. Mol. Carcinog,2011). In this work, we hypothesized that inhibiting DNA repair mechanisms using small molecules might represent a promising strategy to overcome drug resistance to genotoxic agents in cHL, such as cyclophosphamide and topoisomerase II inhibitors (O'Connor,Mol. Cell, 2015; Shaheenet al., Blood 2011). We characterized the drug-response of 4 cHL cell lines to DNA repair inhibitors including PJ34 (PARP inhibitor), NU7441 (DNAPK inhibitor), KU55933 (ATM inhibitor), PF477736 (CHK1 inhibitor), AZD6738 (ATR inhibitor), AZD1775 (Wee1 inhibitor), MP-470 (Rad51 inhibitor) and genotoxic agents used in standard chemotherapy (cyclophosphamide, doxorubicin and etoposide). We showed that DNA repair inhibitors targeting ATR, CHK1 or Wee1 significantly induced apoptosis of cHL cell lines (AnnexinV staining, p<0.05), significant inhibition of proliferation (BrdU incorporation). ATR inhibitor induces a significant accumulation of cHL cells in G0/G1 phase (p<0.05) whereas CHK1 and Wee1 inhibitors induced a significant accumulation in G2/M phase (p<0.01). Moreover, ATR, CHK1 and Wee1 inhibitors induced double strand breaks monitored by 53BP1 foci formation, γH2A.X staining (p<0.01) and chromosomic instability characterized by a higher frequency of micronucleation (p<0.05) and nucleoplasmic bridges formation (p<0.01). Since DNA repair pathways play a role in drug resistance, we sought to identify new synthetic lethal and synergistic combinations associating IC20 DNA repair targeted treatments with conventional genotoxic agents in cHL. Applying a standard threshold of 2 SDs below the IC50 of the genotoxic agent alone (Srivaset al., Mol. Cell, 2016), a total of two synthetic lethal combinations in cHL cells have been identified: CHK1 inhibitor (PF477736) combined with a Wee1 inhibitor (AZD1775) (IC50 reduced from 5.00 µM to 1.75 µM for L-428 and from 4.81 µM to 1.86 µM for KMH2) and cyclophosphamide combined with an ATR inhibitor (AZD6738) (IC50 reduced from 3.26 µM to 1.12 µM for L-428 and from 1.27µM to 0.10µM for KMH2). In addition, we identified 3 synergistic/additive combinations (combination index < 1): doxorubicin combined with a DNAPK inhibitor (NU7441) (IC50 reduced from 44 nM to 22 nM for L-428 and from 27 nM to 8 nM for KMH2), cyclophosphamide combined with CHK1 inhibitor (PF477736) (IC50 reduced from 2.9 µM to 2.4 µM for L-428 and from 1.2µM to 0.9µM for KMH2) and etoposide combined with an ATR inhibitor (AZD6738) (IC50 reduced from 420 nM to 195 nM for KMH2). The treatment of Hodgkin's lymphoma is successful for a majority of patients. However, patients with advanced-stage or high-risk disease are only cured in ∼70% of cases. Alternative treatment options for the subsets of patients for whom first- or second-line therapies fail are needed. These results open new perspectives to improve the treatment of relasped/refractory cHL patients and provide new strategies to overcome drug resistance. Disclosures Moreaux: Diag2Tec:Consultancy.

Microsatellite Instability (MSI) In High Risk Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukaemia (AML) Cells Promotes Frameshift Mutations In DNA Repair Genes: Indications for PARP Inhibitor Therapy.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1194-1194
Author(s):  
Terry J Gaymes ◽  
Azim Mohamedali ◽  
Austin G Kulasekararaj ◽  
Sydney Shall ◽  
Ghulam J. Mufti
Keyword(s):  
Dna Repair ◽  
High Risk ◽  
Cell Lines ◽  
Chromosomal Abnormalities ◽  
Gene Transcript ◽  
Dna Repair Genes ◽  
Monosomy 7 ◽  
Frameshift Mutations ◽  
Group 3 ◽  
Repair Genes

Abstract Abstract 1194 Despite major advances in the biology and pathogenesis of myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) identification of the most effective and safest form of treatment continue to present a formidable challenge particularly in older patients. Older patients (>70 years) that constitute the majority of MDS/AML patients are often resistant to chemotherapy, achieve short lived remission and are not candidates for stem cell transplantation. Therefore the emphasis is to prolong survival or improve the quality of life. Currently, a number of therapeutic strategies are being evaluated and these include treatment with DNA methyltransferase or histone deacetylase inhibitors. Seminal work in breast cancer have shown that inhibitors of poly ADP ribose polymerase (PARP) activity can selectively target tumour cells through exploitation of inherent DNA repair defects. MDS/AML are characterized by genomic instability (GI) and single nucleotide polymorphism arrays (SNPA) karyotyping show that loss of heterozygosity (LOH) and uniparental disomy (UPD) are common in MDS/AML and it has been suggested that the underlying cause of this GI is a defect in double strand DNA repair. We have demonstrated that non homologous end joining, a major pathway for the repair of double strand DNA breaks is overactive and associated with extensive joining errors in primary AML cells. Hence, potentially MDS/AML patients are candidates for PI therapy. We have also shown more recently, that 15% of MDS/AML primary patient cells and cell lines are sensitive to PARP inhibitors (PI) through exploitation of homologous recombination DNA repair defects. To further elucidate the mechanisms that underlie PI sensitivity in MDS/AML we tested for microsatellite instability (MSI) in MDS/AML cell lines and high risk MDS patients and the presence of frameshift mutations in specific DNA repair genes that confer PI sensitivity. MSI is a change in length of a microsatellite allele caused by insertion or deletion of nucleotides that are misincorporated during DNA replication and not removed by the mismatch repair pathway. Using fluorescent PCR analysis, PI sensitive cell lines, P39, KG-1 and Molm-13 showed MSI-high (instability at ≥ 2 loci) at 5 mononucleotide microsatellites, in contrast to 12 PI insensitive cell lines that showed no MSI at these loci. We also show using fluorescent PCR and DNA sequencing that these MSI positive cell lines demonstrate MSI (monoallelic 1–2 base pair [bp] deletion) in the coding region microsatellites of DNA repair genes, Ataxia telancgiectasia mutated gene (ATM), CTiP, and MRE11. Monoallelic 1–2 bp base pair deletions at these loci produced frameshift mutations that induced aberrant gene splicing transcripts in ATM and MRE11 and a markedly truncated CTiP gene transcript. No MSI was detected in DNA repair genes CHK1, RAD50, PTEN, BLM and ATR in these cell lines and no mutations were observed at any DNA repair gene microsatellite in the 13 PI insensitive cell lines. We then determined MSI in high risk MDS patients with or without monosomy 7 (-7/del7q). 13 of 63 (21%) high risk MDS patients showed MSI (9 MSI-low and 4 MSI-high). Of the 13 MSI positive patients, 7 (4 MSI-high, 3 MSI-low) had monosomy 7 and other complex chromosomal abnormalities (Group 1, 54%), 2 (MSI-low) patients had isolated monosomy 7 (Group 2, 15%) whilst 4 patients (MSI-low) had normal cytogenetics (Group 3, 30%). Constitutional DNA from these patients did not show MSI at these loci. Significantly, however, Group 3 with MSI and normal cytogenetics all had widespread UPD and cryptic chromosome changes determined by SNPA. Strikingly, thus all 13 patients with MSI possessed chromosomal abnormalities, both gross and cryptic. Furthermore, 12 patients (19%) found to be cytogenetically normal and lacking UPD and genomic aberrations by SNPA did not show MSI. We have also identified that 3 patients with MSI-high (Group 1) and 1 patient with MSI-low (Group 3) had a monoallelic 1 bp deletion in the CTiP exon coding microsatellite. 1 bp deletion within the coding exon of CTiP resulted in an abbreviated CTiP gene transcript. In conclusion, we have made the important correlation between MSI and subsequent frameshift mutations in specific DNA repair genes with the gross and cryptic chromosomal changes observed in MDS/AML. Identification of a cohort of MDS/AML patients with MSI would herald a significant advancement for the selection of candidates for PI therapy. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
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.

scholarly journals DNA Junction Ligands Trigger DNA Damage and Are Synthetic Lethal with DNA Repair Inhibitors in Cancer Cells

2019 ◽  
Vol 142 (1) ◽  
pp. 424-435 ◽  
Author(s):  
Katerina Duskova ◽  
Pauline Lejault ◽  
Élie Benchimol ◽  
Régis Guillot ◽  
Sébastien Britton ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Cancer Cells ◽  
Synthetic Lethal ◽  
Dna Repair Inhibitors

scholarly journals 5-Fluorotroxacitabine Displays Potent Anti-Leukemic Effects and Circumvents Resistance to Ara-C

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3939-3939 ◽  
Author(s):  
Aniket Bankar ◽  
Thirushi Piyumika Siriwardena ◽  
Biljana Rizoska ◽  
Christina Rydergård ◽  
Helen Kylefjord ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Lines ◽  
K562 Cells ◽  
Nucleoside Analogues ◽  
Brdu Incorporation ◽  
Wild Type ◽  
Employment Equity ◽  
Over Expression ◽  
Equity Ownership ◽  
Further Development

Abstract The cytotoxic nucleoside cytarabine (Ara-C) is a cornerstone of AML induction and consolidation therapies, but drug resistance contributes to disease relapse. Among clinically relevant mechanisms of Ara-C resistance is the over-expression of cytidine deaminase (CDA). CDA degrades Ara-C through deamination into inactive metabolites and its increased expression in AML cells results in drug resistance. Therefore, nucleoside analogues that are not degraded by CDA may be new therapeutic agents for this disease. Through our efforts to develop novel nucleoside analogues that overcome mechanisms of resistance to Ara-C, we identified 5-fluorotroxacitabine (5FTRX), a chain-terminating cytidine-based L-nucleoside. First, we tested whether 5FTRX was a substrate of CDA by evaluating its cytotoxicity in HEK-293 cells over-expressing CDA. Compared to wild type cells, Ara-C was 6-fold less active in cells over-expressing CDA (IC50 wild type HEK293: 3.7µM (95% CI: 3.2-4.2uM) vs IC50 CDA over-expression: 25.6uM (95% CI 21.6-30.3uM), consistent with degradation of Ara-C by CDA. In contrast, cells over-expressing CDA were more sensitive to 5FTRX, compared to wild type cells (IC50 wild type HEK293: 5.5uM (95% CI 4.7-6.3uM) vs IC50 CDA over-expression: 0.4uM (95% CI 0.4-0.5uM), potentially due to depletion of endogenous nucleotide pools by increased CDA. Thus, 5FTRX is not a substrate of CDA. We then treated 6 AML cell lines for 72 hours with increasing concentrations of 5FTRX and then measured cell growth and viability using the MTS assay. 5FTRX reduced the growth of 5 of 6 tested AML cell lines, with mean IC50 values (n= > 3) of 92nM (TEX), 130nM (KG1a), 150nM (MV4-11), 410nM (NB4), and 250nM (OCI-AML2). In contrast, K562 cells that have mutant p53 were resistant with an IC50 >50,000nM. The K562 cells were also resistant to Ara-C. 5FTRX reduced the clonogenic growth of primary AML samples (n=2) with a >90% reduction in growth at 50nM, demonstrating that 5FTRX targets leukemia initiating cells. To test whether 5FTRX induced DNA damage in AML cells, we measured changes in phosphorylated H2AX (pH2AX) after 5FTRX treatment. In the tested cell lines (OCI-AML2, TEX, NB4 and MV4-11 cells), 5FTRX increased pH2AX at concentrations associated with loss of viability. Finally, we evaluated the efficacy and toxicity of 5FTRX in mouse models of AML. 5FTRX displayed robust and dose-dependent inhibition of MV4-11 and OCI-AML2 tumors in mouse xenograft models, with complete tumor regressions and long-lasting tumor growth delays (>20 days at 100mg/kg in MV4-11 and >30 days in OCI-AML2) after a single cycle of 5 days of once-daily drug treatment, with no changes in body weight or behavior. The efficacy of 5FTRX was superior to Ara-C dosed for daily for 10 days at its MTD, 60 mg/kg (35%TGI, no regressions). MV4-11 tumors treated with 5FTRX displayed induction of pH2AX, reduction in proliferation (BrdU incorporation) and induction of necrosis, consistent with the mode of action and dramatic tumor regressions. The anti-leukemic effects of 5FTRX were further evaluated in mice engrafted intrafemorally with primary AML cells. 5FTRX (100 mg/kg i.p, x 5 days) reduced primary AML engraftment >95% compared to controls without toxicity (Fig 1). In summary, 5FTRX was identified as a potent inhibitor of AML cell proliferation in vitro and in vivo. In contrast to Ara-C, 5FTRX was not a substrate for CDA. Further development of analogues of 5FTRX is ongoing using protide prodrugs of the 5FTRX monophosphate to further increase potency and evade additional resistance mechanisms. Taken together, our findings support the further development of 5FTRX-based therapies for the treatment of AML, including AML patients with reduced sensitivity to Ara-C through high CDA expression. #AB and TPS contributed equally to this work. #ADS and MA contributed equally to this work. Disclosures Rizoska: Medivir AB: Employment, Equity Ownership. Rydergård:Medivir AB: Employment, Equity Ownership. Kylefjord:Medivir AB: Employment, Equity Ownership. Rraklli:Medivir AB: Employment. Eneroth:Medivir AB: Employment, Equity Ownership. Pinho:Medivir AB: Employment, Equity Ownership. Norin:Medivir AB: Employment, Equity Ownership. Bylund:Medivir AB: Employment, Equity Ownership. Moses:Medivir AB: Employment, Equity Ownership. Bethell:Medivir AB: Employment, Equity Ownership. Targett-Adams:Medivir AB: Employment, Equity Ownership. Schimmer:Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka Pharmaceuticals: Consultancy; Medivir AB: Research Funding. Albertella:Medivir AB: Employment, Equity Ownership.

DNA Ligase III Alpha and (Poly-ADP) Ribose Polymerase (PARP1) Are Therapeutic Targets in Imatinib-Resistant (IR) Chronic Myeloid Leukemia (CML).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 853-853
Author(s):  
Lisa A. Tobin ◽  
Aaron P. Rapoport ◽  
Ivana Gojo ◽  
Maria R. Baer ◽  
Alan E. Tomkinson ◽  
...  
Keyword(s):  
Dna Repair ◽  
Tyrosine Kinase ◽  
Genomic Instability ◽  
Cell Lines ◽  
Combined Treatment ◽  
Therapeutic Targets ◽  
Dna Ligase ◽  
Dsb Repair ◽  
Dna Repair Inhibitors ◽  
Colony Survival

Abstract Abstract 853 Therapy with the tyrosine kinase inhibitor imatinib, targeting the constitutively active BCR-ABL kinase has been remarkably successful in Philadelphia chromosome-positive (Ph+) CML, but resistance to tyrosine kinase inhibitors is a growing clinical problem, prompting the search for new therapeutic targets. BCR-ABL expression leads to increased reactive oxygen species (ROS), repair errors and genomic instability. We have previously shown that an error-prone alternative non-homologous end-joining (ALT NHEJ) pathway involving PARP1 and DNA ligase IIIa/XRCC1 is upregulated in Ph+ CML, providing a mechanism for the repair errors and genomic instability. To determine whether ALT NHEJ components may be novel therapeutic targets in IR CML, we characterized two IR cell lines (P210Mo7eIR, Baf3P210IR) for DSB repair abnormalities. Both IR cell lines demonstrate significantly higher levels of DSBs and NHEJ abnormalities (P<0.05) compared with their imatinib-sensitive (IS) counterparts. Notably, whereas steady state levels of the ALT NHEJ components DNA ligase IIIa and PARP1 are increased in IS P210Mo7e and Baf3P210 cells, compared with parental Mo7e and Baf3, the levels of these proteins are increased even further in the IR cells. Presence of increased DNA ligase IIIa and PARP1 levels in the IR cell lines suggests that these enzymes may be targets for therapy using the DNA ligase inhibitors that we have previously identified and PARP1 inhibitors, which have been used successfully in the treatment of cancers with DSB repair defects. Initial tests for cytotoxicity in BCR-ABL-positive cell lines and parental controls showed that the DNA ligase inhibitor L67, which specifically inhibits DNA ligase I and IIIα, is cytotoxic in BCR-ABL-positive cells and parental controls at concentrations of >10 μM, and that cytotoxicity is not influenced by BCR-ABL1 expression. Therefore, we examined the effect of a subtoxic concentration of L67 (0.3 μM) in the presence or absence of the PARP1 inhibitor Nu1025 (Calbiochem) at 50 μM in IR versus IS and parental cells. Combined treatment with L67 and Nu1025 significantly (p<0.001) reduces survival of IR cells compared with IS and parental controls, which were not significantly affected. To determine whether cells from CML patients that are resistant to imatinib are also sensitive to the combination of DNA ligase and PARP inhibitors, we next tested primary bone marrow mononuclear cells (BM MNC) from 6 CML patients with IR disease, compared with normal BM MNC. Cells from 3 of the 6 patients demonstrated a significant decrease in colony survival in response to the combination of DNA repair inhibitors, similar to the sensitivity demonstrated by the two IR cell lines studied. Interestingly, the patient demonstrating the highest sensitivity to the combination of DNA repair inhibitors had significantly increased levels of both DNA ligase IIIa and PARP1, whereas patients demonstrating less sensitivity had increased levels of either DNA ligase IIIa or PARP1, compared with normal BM MNC. Importantly, sensitivity to the DNA repair inhibitors is not correlated with mutations in BCR-ABL because the BCR-ABL mutation T315I that is found in Baf3P210IR cells when overexpressed in Baf3 cells has no effect on colony survival following drug treatment. Together, our results suggest that the process of acquiring IR may select for cells with high levels of PARP1 and DNA ligase IIIa and/or may upregulate ALT NHEJ pathways. Thus, patients with high levels of these proteins are likely to benefit from therapy using inhibitors of ALT NHEJ. Disclosures: No relevant conflicts of interest to declare.

Rational Drug Design: Proteasome Inhibitor Mediated Down-Regulation of the FA/BRCA Pathway Is Synergistic with PARP Inhibition in Myeloma Cell Lines

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2922-2922
Author(s):  
Liang Nong ◽  
Linda Mathews ◽  
Mark B Meads ◽  
William Dalton ◽  
Kenneth H. Shain
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Dna Repair ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Myeloma Cell ◽  
Sequential Treatment ◽  
Parp Inhibition ◽  
Repair Pathway ◽  
Myeloma Cells

Abstract Abstract 2922 The development of new and biologically-based therapeutic regimens is critical for the successful control, if not cure, of multiple myeloma. Incorporation of the novel agents, including the proteasome inhibitor bortezomib, harbored large strides in disease modification. However, even with the success of bortezomib containing regimens, drug resistance and disease relapse remain inevitable. As such, it is critical that we use preclinical models to not only develop drugs, but also to consider strategies for co-development of novel drug combinations capitalizing on complementary biological activities. Our investigations in drug resistance recently revealed that increased homologous recombination (HR) potential, via over-expression of the FA/BRCA DNA repair pathway (FA/BRCA pathway), contributed to acquired melphalan-resistance in myeloma cell lines.(Yarde et al 2009) Drug resistance was causally linked to a novel transcriptional regulation of the FA/BRCA by NF-κB. Further examination demonstrated that bortezomib attenuated this component of the HR repair pathway and reversed melphalan resistance. To this end, we anticipated that bortezomib treatment may sensitize cells to inhibitors of complementary DNA repair pathways in a manner similar to the synthetic lethality elicited in by PARP1/2 inhibition in BRCA1 or FANCD1/ BRCA2 mutant cancers.(Farmer 2005, Bryant 2005) Consistent with this rationale, treatment of myeloma cells with bortezomib and the PARP inhibitor AZD2281/olaparib demonstrated synergism in specific myeloma cell lines. Pre-treatment of RPMI8226 myeloma cells with bortezomib for 6 hours greatly enhanced myeloma cell sensitivity to PARP inhibition with AZD2281/olaparib. The inhibitory concentration(IC)-50 was decreased by 17.7-fold (n=3; IC50 AZD2281 alone: 62.7 microM (39.0–84.0) and pretreated with bortezomib 3.54 microM (2.4–4.6)). Combination Index (CI) demonstrated a mean of 0.41 in 8226 and 0.43 in U266 myeloma cells, consistent with a synergistic relationship. Further analysis confirmed that synergism correlated with decreased expression of FANCD2 mRNA and protein by 6 hours. In contrast to sequential treatment, concomitant treatment with these agents did not elicit the synergistic phenotype. Interestingly, sequential treatment of NCIH929 myeloma cells did not demonstrate the same synergistic response (CI :0.89, slight synergism). Consistent with this, treatment of NCIH929 cells with bortezomib did not negatively regulate FANCD2 mRNA or protein expression, suggesting that FA/BRCA pathway can be differentially regulated in myeloma cells. To more specifically determine if FANCD2 was a key factor regulated by bortezomib, we targeted FANCD2 with siRNA. Pretreatment of myeloma cells with FANCD2 siRNA also sensitized cells to AZD2281/olaparib relative to siRNA control (IC50: 19.0 microM vs 35.0 microM n=4; p<0.05). These results show that bortezomib (or other proteosome inhibitors) and AZD2281/olaparib (or other PARP inhibitors) may represent an exciting new combination therapy for myeloma. We are currently examining the applicability of these studies to other proteosome inhibitors and the clinical relevance with ex vivo studies with myeloma patient samples. We believe that data presented here are innovative as they introduce a novel biological rationale, the abrogation complementary pathways in DNA damage repair, for the preclinical development of novel targeted drug combinations in myeloma. Further, we anticipate that although this study has focused on multiple myeloma, the results of the proposed research will be applicable to a wide range of hematologic and solid tumors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals WRN Helicase is a Synthetic Lethal Target in Microsatellite Unstable Cancers

2018 ◽  
Author(s):  
Edmond Chan ◽  
Tsukasa Shibue ◽  
James McFarland ◽  
Benjamin Gaeta ◽  
Justine McPartlan ◽  
...  
Keyword(s):  
Dna Repair ◽  
Cell Lines ◽  
Large Scale ◽  
Synthetic Lethality ◽  
Cancer Therapeutics ◽  
Dna Helicase ◽  
Dna Breaks ◽  
Synthetic Lethal ◽  
Dna Mismatch ◽  
Double Strand Dna Breaks

Synthetic lethality, an interaction whereby the co-occurrence of two or more genetic events lead to cell death but one event alone does not, can be exploited to develop novel cancer therapeutics. DNA repair processes represent attractive synthetic lethal targets since many cancers exhibit an impaired DNA repair pathway, which can lead these cells to become dependent on specific repair proteins. The success of poly (ADP-ribose) polymerase 1 (PARP-1) inhibitors in homologous recombination-deficient cancers highlights the potential of this approach in clinical oncology. Hypothesizing that other DNA repair defects would give rise to alternative synthetic lethal relationships, we asked if there are specific dependencies in cancers with microsatellite instability (MSI), which results from impaired DNA mismatch repair (MMR). Here we analyzed data from large-scale CRISPR/Cas9 knockout and RNA interference (RNAi) silencing screens and found that the RecQ DNA helicase was selectively essential in MSI cell lines, yet dispensable in microsatellite stable (MSS) cell lines. WRN depletion induced double-strand DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models specifically required the helicase activity, but not the exonuclease activity of WRN. These findings expose WRN as a synthetic lethal vulnerability and promising drug target in MSI cancers.

scholarly journals Genome-Scale CRISPR-Cas9 Synthetic Lethal Screening of AML Cell Line Identified Functional Modulators of Etoposide Resistance Predictive of Clinical Outcome in AML Patients

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2685-2685
Author(s):  
Roya Rafiee ◽  
Amin Sobh ◽  
Abdelrahman H. Elsayed ◽  
Abderrahmane Tagmount ◽  
Chris D Vulpe ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Clinical Outcome ◽  
Candidate Genes ◽  
Cell Lines ◽  
Chemotherapeutic Agents ◽  
Functional Screening ◽  
Synthetic Lethal ◽  
Effective Manner ◽  
Two Samples ◽  
Genome Scale

Despite the use of intensive chemotherapeutic regimens containing ara-C, daunorubicin and etoposide (ADE), in combination with stem cell transplantation for high-risk group patients, approximately significant patients experience relapse. Resistant and relapsed disease remains the most prevalent forms of clinical failure in treating this disease. The current scientific challenge is to identify candidate genes that are truly essential to drug resistance and thus may be pharmacologically targeted in a clinically effective manner. The development of the CRISPR/cas9 genome editing tool has dramatically improved the capabilities for functional screening in multiple systems including AML where genome-wide drop-out screens in AML cancer cell lines identified AML-essential genes. None of the studies have unfortunately integrated the CRISPR/cas9 screening tool with patient outcome data. In this study, we have successfully performed Genome scale Crispr knock out GeCKO -CRISPR/cas9 screening using Brunello library (targeting 19,114 genes) in K562 cell lines followed by treatments with etoposide, dauno and cytarabine. We performed a large-scale transduction into ~30 x 106 cells at a low multiplicity of infection (0.3). After puromycin selection the mutant cell library was exposed in triplicate to IC30 concentration of chemotherapeutic agents for up to 18 days with collection of samples at day 4 (ara-C, dauno, etoposide) days 12 and 18 (dauno and etoposide). A vehicle-only exposure was similarly carried out for 18 days. Direct sequencing of PCR amplified sgRNA guides from the pooled cells by short read sequencing (Illumina) was used to quantify the representation of each knockout clone in the pooled population (schema shown in Fig 1). The relative enrichment/depletion of each knockout clone was determined by ratio of the abundance of each clone between two samples. The FASTX-Toolkit, was used to extract the unique sgRNA sequences which were assembled into a Burrows-Wheeler index using the Bowtie build-index function and number of uniquely aligned reads for each sgRNA were calculated. The MaGeCK algorithms was used to analyze the read count data and perform statistical comparisons. The results for the etoposide screening are shown in Fig 2, overall 17 genes showed significant association with responsiveness to etoposide at all the three different time points (4, 12 and 18 days). Further investigation of these candidate genes in AML patients treated on AML02 trial demonstrated consistent and significant association between expression levels of several genes identified in CRISPR screening and clinical outcome. Fig 3 shows a representative result for 2 of the genes identified in etoposide screening, ABCC1and RAD54L2 both were associated with etoposide resistance in CRISPR screening and concordantly high expression was associated with greater induction 1 MRD, inferior event free survival (EFS, Fig 3) and overall survival (OS) in the AML02 cohort. ABCC1 is a drug efflux transporter that has been implicated in resistance to etoposide and daunorubicin as well as has been associated with poor prognosis in AML consistent with our results. RAD54L2, is a helicase involved in DNA damage repair response pathway. Other genes of interest identified in etoposide screening with significant association with clinical endpoints as MRD, EFS and OS included TKT, involved in pentose phosphate pathway and implicated in regulation of metabolic switch in cancer. A nucleotide excision repair gene, RAD23B, implicated in breast cancer progression as well as in CML and ALL. Similarly, for daunorubicin and cytarabine CRISPR/cas9 synthetic lethal screening identified target genes of clinical relevance that will be discussed in the presentation. Our approach shows that using CRISPR/cas9 targeted synthetic lethal screening as a reliable approach to not only identify and establish genes predictive of drug resistance and poor outcome but also potential targets for novel drug development that could be used in combination with currently approved agents. Disclosures No relevant conflicts of interest to declare.

scholarly journals Independent Mechanisms Lead to Genomic Instability in Hodgkin Lymphoma: Microsatellite or Chromosomal Instability

Cancers ◽  
2018 ◽  
Vol 10 (7) ◽  
pp. 233 ◽  
Author(s):  
Corina Cuceu ◽  
Bruno Colicchio ◽  
Eric Jeandidier ◽  
Steffen Junker ◽  
François Plassa ◽  
...  
Keyword(s):  
Dna Repair ◽  
Hodgkin Lymphoma ◽  
Genomic Instability ◽  
Cell Lines ◽  
Chromosomal Instability ◽  
Chromosomal Rearrangements ◽  
Peripheral Blood Lymphocytes ◽  
Small Cells ◽  
Molecular Approaches ◽  
Circulating Lymphocytes

Background: Microsatellite and chromosomal instability have been investigated in Hodgkin lymphoma (HL). Materials and Methods: We studied seven HL cell lines (five Nodular Sclerosis (NS) and two Mixed Cellularity (MC)) and patient peripheral blood lymphocytes (100 NS-HL and 23 MC-HL). Microsatellite instability (MSI) was assessed by PCR. Chromosomal instability and telomere dysfunction were investigated by FISH. DNA repair mechanisms were studied by transcriptomic and molecular approaches. Results: In the cell lines, we observed high MSI in L428 (4/5), KMH2, and HDLM2 (3/5), low MSI in L540, L591, and SUP-HD1, and none in L1236. NS-HL cell lines showed telomere shortening, associated with alterations of nuclear shape. Small cells were characterized by telomere loss and deletion, leading to chromosomal fusion, large nucleoplasmic bridges, and breakage/fusion/bridge (B/F/B) cycles, leading to chromosomal instability. The MC-HL cell lines showed substantial heterogeneity of telomere length. Intrachromosmal double strand breaks induced dicentric chromosome formation, high levels of micronucleus formation, and small nucleoplasmic bridges. B/F/B cycles induced complex chromosomal rearrangements. We observed a similar pattern in circulating lymphocytes of NS-HL and MC-HL patients. Transcriptome analysis confirmed the differences in the DNA repair pathways between the NS and MC cell lines. In addition, the NS-HL cell lines were radiosensitive and the MC-cell lines resistant to apoptosis after radiation exposure. Conclusions: In mononuclear NS-HL cells, loss of telomere integrity may present the first step in the ongoing process of chromosomal instability. Here, we identified, MSI as an additional mechanism for genomic instability in HL.

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