Synergistic Effect of PARP Inhibitor and BRD4 Inhibitor in Multiple Models of Ovarian Cancer

Background: Ovarian cancer has the highest fatality rate among patients with gynaecological tumours. Current therapies including poly-ADP ribose polymerase (PARP) inhibitors have limitations due to the frequent recurrence of ovarian cancer after treatment and resistance to therapy.Methods: In this study, we used multiple models with different genetic backgrounds to investigate the potential synergism effect and mechanism between the bromodomain-containing protein 4 (BRD4) inhibitor AZD5153 and the PARP inhibitor Olaparib. The models were two-dimensional (2D) and 3D cell lines, patient-derived organoids (PDO) and patient-derived xenografts (PDX). Results: Cotreatment with Olaparib and AZD5153 exhibited marked synergistic effects, and significantly attenuated cell viability, whereas it increased DNA replication fork instability, chromosomal breakage and apoptosis compared to treatment with either drug alone. Mechanistically, the tumor upregulates PTEN after Olaparib treatment to make its DNA and chromosome more stable and therefore induces Olaparib resistance. AZD5153 can downregulate PTEN to reverse Olaparib resistance and thus increase joint lethal effect with Olaparib.Conclusion: This study reveals that AZD5153 can downregulate PTEN to reverse Olaparib resistance and thus increase joint lethal effect on DNA replication fork instability, chromosomal breakage, and apoptosis with Olaparib.

Case Series: Bone Marrow Failure in Teen Siblings with Unique RPS19 Variant

Aplastic anemia occurs when there is a lack of hematopoiesis in the bone marrow leading to peripheral pancytopenia. It is a rare entity and is often idiopathic. However, upon presentation, inherited bone marrow failure syndromes and acquired etiologies must be considered. Investigating causality is particularly important when multiple family members are affected, especially in a short amount of time. It is also essential to identify novel causative genetic variants of bone marrow failure in order to direct treatment in these patients. In our case series, we describe two siblings who presented two weeks apart with severe pancytopenia. The first patient is a 13 year-old non-binary female (prefers pronouns they/them/theirs) who presented from clinic after getting routine labs with white blood cell (WBC) 1.83 x10(3)/mcL, absolute neutrophil count (ANC) 0.16 x10(3)/mcL, hemoglobin (Hgb) 4.8 gm/dL, platelet 13 x10(3)/mcL. Bone marrow biopsy revealed marked hypocellularity (0-10%) with hypoplasia. The second patient is their 16 year-old brother who presented two weeks later with new-onset petechial rash and was found to have WBC 4 x10(3)/mcL, ANC 2.19 x10(3)/mcL, Hgb 6.3 gm/dL, platelet 16 x10(3)/mcL. His bone marrow biopsy demonstrated variable cellularity (10-70%), but after months of transfusion-dependence he met criteria for severe aplastic anemia. Laboratory evaluation for acquired etiologies such as infection was negative. Both patients had a shared medical history of depression briefly treated with fluoxetine, but otherwise no potential medical triggers or environmental exposures were identified. Telomere length analysis was normal and chromosomal breakage analysis was negative. Upon genetic evaluation, both patients were found to have a heterozygous variant of unknown significance of RPS19 (c.-163>T), which substitutes a moderately conserved nucleotide in the noncoding exon 1 in 5' untranslated region of RPS19. Although this variant has not been classified as pathogenic, three other variants in the 5' untranslated region of RPS19 have been reported in patients with Diamond-Blackfan Anemia (DBA). In contrast to classic DBA, these patients did not present in infancy or early childhood. Likewise, they lacked congenital anomalies or other classic phenotypic characteristics of disease. Lab studies showed normal erythrocyte adenosine deaminase levels, though it should be noted that these levels could not be obtained prior to transfusion in both patients. Given lack of other identified etiologies, however, an inherited bone marrow failure syndrome, possibly due to this variant, was presumed. With suspected but potentially unidentifiable genetic predisposition, matched sibling donor transplant was deferred and both patients underwent a matched unrelated donor bone marrow transplant with reduced-intensity conditioning. With these cases, we aim to share a unique presentation of aplastic anemia that reveals a potentially novel pathogenic variant as well as to provide our approach to medical management in pediatric aplastic anemia in the setting of uncertainty. Identification of other patients with bone marrow failure and this genetic variant will be important to determine its pathogenicity. Disclosures No relevant conflicts of interest to declare.

Genomic and transcriptomic analyses reveal a tandem amplification unit of 11 genes and mutations in mismatch repair genes in methotrexate-resistant HT-29 cells

DHFR gene amplification is commonly present in methotrexate (MTX)-resistant colon cancer cells and acute lymphoblastic leukemia. In this study, we proposed an integrative framework to characterize the amplified region by using a combination of single-molecule real-time sequencing, next-generation optical mapping, and chromosome conformation capture (Hi-C). We identified an amplification unit spanning 11 genes, from the DHFR gene to the ATP6AP1L gene position, with high adjusted interaction frequencies on chromosome 5 (~2.2 Mbp) and a twenty-fold tandemly amplified region, and novel inversions at the start and end positions of the amplified region as well as frameshift insertions in most of the MSH and MLH genes were detected. These mutations might stimulate chromosomal breakage and cause the dysregulation of mismatch repair. Characterizing the tandem gene-amplified unit may be critical for identifying the mechanisms that trigger genomic rearrangements. These findings may provide new insight into the mechanisms underlying the amplification process and the evolution of drug resistance.

ATM inhibition enhances Auranofin-induced oxidative stress and cell death in lung cell lines

Ataxia-Telangiectasia (A-T), a pleiotropic chromosomal breakage syndrome, is caused by the loss of the kinase Ataxia-telangiectasia mutated (ATM). ATM is not only involved in the response to DNA damage, but also in sensing and counteracting oxidative stress. Since a disturbed redox balance has been implicated in the pathophysiology of A-T lung disease, we aimed to further explore the interplay between ATM and oxidative stress in lung cells. Using a kinetic trapping approach, we could demonstrate an interaction between the trapping mutant TRX1-CS and ATM upon oxidative stress. We could further show that combined inhibition of thioredoxin reductase (TrxR) and ATM kinase activity, using Auranofin and KU55933 respectively, induced an increase in cellular reactive oxygen species (ROS) levels and protein oxidation in lung cells. Furthermore, ATM inhibition sensitized lung cells to Auranofin-induced cell death that could be rescued by ROS scavengers. As a consequence, targeted reduction of ATM by TRX1 could serve as a regulator of oxidative ATM activation and contribute to the maintenance of the cellular redox homeostasis. These results highlight the importance of the redox-active function of ATM in preventing ROS accumulation and cell death in lung cells.

Sites of chromosomal instability in the context of nuclear architecture and function

Chromosomal fragile sites are described as areas within the tightly packed mitotic chromatin that appear as breaks or gaps mostly tracing back to a loosened structure and not a real nicked break within the DNA molecule. Most facts about fragile sites result from studies in mitotic cells, mainly during metaphase and mainly in lymphocytes. Here, we synthesize facts about the genomic regions that are prone to form gaps and breaks on metaphase chromosomes in the context of interphase. We conclude that nuclear architecture shapes the activity profile of the cell, i.e. replication timing and transcriptional activity, thereby influencing genomic integrity during interphase with the potential to cause fragility in mitosis. We further propose fragile sites as examples of regions specifically positioned in the interphase nucleus with putative anchoring points at the nuclear lamina to enable a tightly regulated replication–transcription profile and diverse signalling functions in the cell. Consequently, fragility starts before the actual display as chromosomal breakage in metaphase to balance the initial contradiction of cellular overgrowth or malfunctioning and maintaining diversity in molecular evolution.

Spectrum of Hematological Malignancies in 130 Patients with Germline Predisposition Syndromes - Mayo Clinic Germline Predisposition Study

Introduction Germline predisposition syndromes (GPS) are inherited disorders associated with germinal aberrations that increase the risk of malignancies. While aberrations in certain genes increase the risk for all types of malignancies (Tp53, ATM, CDKN2A, CHEK2), there is a growing list of genes associated specifically with hematological malignancies (GATA2, RUNX1, DDX41, ETV6, ANKRD26). At our institution, we have established a hematology GPS clinic to diagnose and manage GPS and with this report, detail our experience with 130 patients. Methods GPS were investigated in pediatric and adult patients with one or more first degree relatives with hematological/visceral malignancies or in those with antecedent thrombocytopenia (ANKRD26, RUNX1, ETV6), or with specific syndromic features (short telomere syndromes/STS, GATA2 haploinsufficiency, Fanconi anemia/FA, Shwachman-Diamond syndrome/SDS). Depending on the phenotype, specific functional assays such as flow-FISH for telomere length assessment and chromosomal breakage assays were ordered. After informed consent and genetic counselling, germline testing was carried out on peripheral blood mononuclear cell, skin fibroblast, or hair follicle-derived DNA. A custom-designed marrow failure NGS panel (200 genes) was used in most cases and interrogation of variants, in silico studies, and functional assays were carried out as previously described (Mangaonkar et al MC Proc 2019). Copy number variations were identified by aCGH. At the time of progression/worsening cytopenias, bone marrow/lymph node biopsies and NGS (next generation sequencing) were carried out where indicated. Results 130 patients with germline predisposition have been identified to date. The spectrum of disorders seen include STS 29 (22%), FA 17 (13%), GATA2 16 (12%), Diamond Blackfan anemia/DBA 13 (10%), RUNX1-FPD 12 (9%), ATM deletions/mutations 11 (8%), ANKRD26 6 (5%), SDS 5 (4%), DDX41 4 (3%), MPL 3 (2%), CHEK2, MECOM, Tp53 mutations 2 (2%) each, and CBL, CEPBA, ELANE, NF1, CDKN2A, CSF3R, ETV6, and GATA1 mutations, 1 (1%) each. Evidence for clonal evolution (CCUS) and hematological malignancies were seen in 51 (39%) patients, involving all the aforementioned genes/syndromes with the exception of DBA, CBL, ETV6, MPL, CSF3R, and GATA1. Seven (64%) of 11 patients with germline ATM deletions/mutations developed lymphoid malignancies; homozygous ATM (Follicular NHL-1, Burkitt lymphoma-1, T-ALL-1, T-LPD-1) and heterozygous ATM (T-PLL-1, DLBCL-1, CLL-1). Clonal evolution occurred in 11 (69%) of 16 GATA2 haploinsufficient patients (CCUS-2, MDS-3, CMML-1, AML-5) and in 7 (58%) of 12 RUNX1-FPD patients (CCUS-1, MDS-1, MDS/MPN-3, AML-2). Five of 29 (17%) STS patients had clonal progression (CCUS-2, MDS-2, AML-1), and 5 (29%) of 17 FA patients progressed to MDS-2 or AML-3. JMML was seen in one patient with a germline NF1 mutation, while 1 (20%) of 5 SDS patients progressed to AML. NGS data at progression was available in 24 (55%) of 44 myeloid/CCUS progressions, with somatic truncating ASXL1 mutations being most frequent (29%), followed by RAS pathway mutations (15%). AML/MDS progressions in STS, FA, and SDS were universally associated with complex/monosomal karyotypes, translating to refractory disease. Seventeen (39%) of 44 patients with myeloid predisposition underwent allogenic HCT (GATA2-7, FA-3, RUNX1-FPD-3, STS-2, NF1-1, Tp53-1), with 10 (59%) being alive at last follow up (Table 1). Conclusion We demonstrate the spectrum of germline aberrations associated with predisposition to hematological malignancies and outline the phenotypic heterogeneity of clonal transformation. The advent of NGS allows identification of clonal progression earlier than morphological changes, with mutations in ASXL1 and RAS pathway genes being commonly implicated. This study supports the universal development of dedicated germline predisposition clinics. Disclosures Pruthi: CSL Behring: Honoraria; Genentech Inc.: Honoraria; Bayer Healthcare: Honoraria; HEMA Biologics: Honoraria; Instrumentation Laboratory: Honoraria; Merck: Honoraria.

p50 mono-ubiquitination and interaction with BARD1 regulates cell cycle progression and maintains genome stability

p50, the mature product of NFKB1, is constitutively produced from its precursor, p105. Here, we identify BARD1 as a p50-interacting factor. p50 directly associates with the BARD1 BRCT domains via a C-terminal phospho-serine motif. This interaction is induced by ATR and results in mono-ubiquitination of p50 by the BARD1/BRCA1 complex. During the cell cycle, p50 is mono-ubiquitinated in S phase and loss of this post-translational modification increases S phase progression and chromosomal breakage. Genome-wide studies reveal a substantial decrease in p50 chromatin enrichment in S phase and Cycln E is identified as a factor regulated by p50 during the G1 to S transition. Functionally, interaction with BARD1 promotes p50 protein stability and consistent with this, in human cancer specimens, low nuclear BARD1 protein strongly correlates with low nuclear p50. These data indicate that p50 mono-ubiquitination by BARD1/BRCA1 during the cell cycle regulates S phase progression to maintain genome integrity.

Interferon-stimulated gene 15 accelerates replication fork progression inducing chromosomal breakage

DNA replication is highly regulated by the ubiquitin system, which plays key roles upon stress. The ubiquitin-like modifier ISG15 (interferon-stimulated gene 15) is induced by interferons, bacterial and viral infection, and DNA damage, but it is also constitutively expressed in many types of cancer, although its role in tumorigenesis is still largely elusive. Here, we show that ISG15 localizes at the replication forks, in complex with PCNA and the nascent DNA, where it regulates DNA synthesis. Indeed, high levels of ISG15, intrinsic or induced by interferon-β, accelerate DNA replication fork progression, resulting in extensive DNA damage and chromosomal aberrations. This effect is largely independent of ISG15 conjugation and relies on ISG15 functional interaction with the DNA helicase RECQ1, which promotes restart of stalled replication forks. Additionally, elevated ISG15 levels sensitize cells to cancer chemotherapeutic treatments. We propose that ISG15 up-regulation exposes cells to replication stress, impacting genome stability and response to genotoxic drugs.