PARP Inhibitors and Myeloid Neoplasms: A Double-Edged Sword

Despite recent discoveries and therapeutic advances in aggressive myeloid neoplasms, there remains a pressing need for improved therapies. For instance, in acute myeloid leukemia (AML), while most patients achieve a complete remission with conventional chemotherapy or the combination of a hypomethylating agent and venetoclax, de novo or acquired drug resistance often presents an insurmountable challenge, especially in older patients. Poly(ADP-ribose) polymerase (PARP) enzymes, PARP1 and PARP2, are involved in detecting DNA damage and repairing it through multiple pathways, including base excision repair, single-strand break repair, and double-strand break repair. In the context of AML, PARP inhibitors (PARPi) could potentially exploit the frequently dysfunctional DNA repair pathways that, similar to deficiencies in homologous recombination in BRCA-mutant disease, set the stage for cell killing. PARPi appear to be especially effective in AML with certain gene rearrangements and molecular characteristics (RUNX1-RUNX1T1 and PML-RARA fusions, FLT3- and IDH1-mutated). In addition, PARPi can enhance the efficacy of other agents, particularly alkylating agents, TOP1 poisons, and hypomethylating agents, that induce lesions ordinarily repaired via PARP1-dependent mechanisms. Conversely, emerging reports suggest that long-term treatment with PARPi for solid tumors is associated with an increased incidence of myelodysplastic syndrome (MDS) and AML. Here, we (i) review the pre-clinical and clinical data on the role of PARPi, specifically olaparib, talazoparib, and veliparib, in aggressive myeloid neoplasms and (ii) discuss the reported risk of MDS/AML with PARPi, especially as the indications for PARPi use expand to include patients with potentially curable cancer.

XRCC1 protects transcription from toxic PARP1 activity during DNA base excision repair

Genetic defects in the repair of DNA single-strand breaks (SSBs) can result in neurological disease triggered by toxic activity of the single-strand-break sensor protein PARP1. However, the mechanism(s) by which this toxic PARP1 activity triggers cellular dysfunction are unclear. Here we show that human cells lacking XRCC1 fail to rapidly recover transcription following DNA base damage, a phenotype also observed in patient-derived fibroblasts with XRCC1 mutations and Xrcc1−/− mouse neurons. This defect is caused by excessive/aberrant PARP1 activity during DNA base excision repair, resulting from the loss of PARP1 regulation by XRCC1. We show that aberrant PARP1 activity suppresses transcriptional recovery during base excision repair by promoting excessive recruitment and activity of the ubiquitin protease USP3, which as a result reduces the level of monoubiquitinated histones important for normal transcriptional regulation. Importantly, inhibition and/or deletion of PARP1 or USP3 restores transcriptional recovery in XRCC1−/− cells, highlighting PARP1 and USP3 as possible therapeutic targets in neurological disease.

Assessing Stealth and Sensed Base Editing in Human Hematopoietic Stem/Progenitor Cells

Genome editing represents a promising tool to manipulate human hematopoietic stem and progenitor cells (HSPCs) opening the possibility to correct hematopoietic diseases avoiding the risk of insertional mutagenesis and uncontrolled expression of the transgene, issues that emerged with retroviral and lentiviral gene therapy. Engineered nucleases such as CRISPR/Cas9 have enable targeted genetic manipulation in human HSPCs for therapeutic purposes. Still, nuclease-induced DNA double-strand breaks (DSBs) trigger p53-dependent DNA damage response affecting HSPC properties and may lead to unintended chromosomal rearrangements. Base editing (BE) holds the promise for precise editing by the introduction of specific single-nucleotide variants (SNVs) while bypassing the requirement for DSBs. In particular, base editors are composed by: i) a deamination domain that directly modifies nucleotides comprised within a defined editing window in one of the two genomic strands, and ii) a nickase Cas9 that introduces a single-strand break (SSB) on the other strand to promote more efficient base editing. Depending on the type of modification introduced editors are classified in Cytosine (C-) BE (C-G transition to T-A) and Adenine (A-) BE (A-T transition to G-C). However, a comprehensive characterization of efficiency, tolerability and genotoxicity of CBE and ABE in human HSPCs is lacking and is required to instruct the rationale towards safe and effective clinical translation. Here, we developed an optimized mRNA-based protocol for BE in human HSPCs and compared CBE4max, ABE8.20-m and Cas9 nuclease by targeting the same locus (B2M) using the same sgRNA. Common outcome for all editors is disruption of targeted gene expression, which is measured by flow cytometry and Next Generation Sequencing. ABE8.20-m showed higher efficiency than CBE4max and Cas9 nuclease at the target locus (up to 90, 40 and 50%), which was consistent across HSPC subpopulations comprising the most primitive compartment endowed with long term repopulation potential and cell sources (such as cord blood- and mobilized peripheral blood-derived HSPCs). Importantly, Cas9, but not CBE4max and ABE8.20m, treated HSPCs showed lower in-vitro clonogenic capacity than mock electroporated cells. Transcriptional analyses uncovered that CBE4max, but not ABE8.20-m, triggered p53 pathway activation, albeit at lower extent as compared to Cas9 and presumably consequent to a fraction of single-strand nicks turning into DSB upon DNA replication. Additionally, BE, and particularly CBE4max, upregulated the expression of interferon-stimulated genes, which was not ascribed to mRNA delivery. Remarkably, despite edited HSPCs showed long-term multilineage capacity in xenotransplanted mice, CBE4max edited cells tended to decrease over time in the graft pointing to some detrimental response to the treatment of the long-term engrafting HSC subset. Overall, our results prompt further investigation on BE sensing in human HSPCs. On-going studies are aimed to investigate clonal dynamics and genome integrity of base-edited HSPCs with the final goal of building confidence for their perspective clinical translation. Disclosures Naldini: Genenta Science: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

New Hybrid Compounds Combining Fragments of Usnic Acid and Thioether Are Inhibitors of Human Enzymes TDP1, TDP2 and PARP1

Tyrosyl-DNA phosphodiesterase 1 (TDP1) catalyzes the cleavage of the phosphodiester bond between the tyrosine residue of topoisomerase 1 (TOP1) and the 3′ phosphate of DNA in the single-strand break generated by TOP1. TDP1 promotes the cleavage of the stable DNA–TOP1 complexes with the TOP1 inhibitor topotecan, which is a clinically used anticancer drug. This article reports the synthesis and study of usnic acid thioether and sulfoxide derivatives that efficiently suppress TDP1 activity, with IC50 values in the 1.4–25.2 μM range. The structure of the heterocyclic substituent introduced into the dibenzofuran core affects the TDP1 inhibitory efficiency of the compounds. A five-membered heterocyclic fragment was shown to be most pharmacophoric among the others. Sulfoxide derivatives were less cytotoxic than their thioester analogs. We observed an uncompetitive type of inhibition for the four most effective inhibitors of TDP1. The anticancer effect of TOP1 inhibitors can be enhanced by the simultaneous inhibition of PARP1, TDP1, and TDP2. Some of the compounds inhibited not only TDP1 but also TDP2 and/or PARP1, but at significantly higher concentration ranges than TDP1. Leader compound 10a showed promising synergy on HeLa cells in conjunction with the TOP1 inhibitor topotecan.

Induction of Micronuclei, Cellular Stress Response, and DNA Strand Breakage in Two Common Fish Species From Rivers and Reservoirs in Ilorin, Nigeria.

Indiscriminate discharge of home, agricultural, and industrial wastes into water bodies, most rivers and reservoirs around the world are becoming polluted. The ecotoxicological potential of this in fish species gathered from important reservoirs and rivers in Ilorin, Nigeria, was explored in this study. Unilorin reservoir, Asa reservoir, Apodu reservoir, Asa river (Unity), and Asa river (Unity) water samples were collected and physicochemical characteristics were investigated at five distinct sites: Unilorin reservoir, Asa reservoir, Apodu reservoir, Asa river (Unity), and Asa river (Unity) (Harmony). In Tilapia zillii and Clarias gariepinus, we measured serum biochemical (AST, ALT, ALP, serum ALB), histopathological (gills, lungs), and serum antioxidant enzyme responses (SOD, CAT, GPx, GR, GST) as a biomarker for oxidative stress, while micronucleus and comet assays were used to detect DNA damage. Except for DO, which was very low in the two rivers, the physicochemical parameters and heavy metals evaluated in the five separate water bodies were within the allowed levels of the NSDWQ and WHO standard for drinking water. In comparison to the Unilorin, Apodu, and Asa reservoirs, a slight increase in Pb was observed across the five sampling sites, which could contribute to increased biochemical and haematological profiles, histopathological lesions in the gill and lungs, inductions of MN, NA, and DNA single strand break in T. zillii and C. gariepinus collected from Asa rivers. This could be due to the indiscriminate dumping of effluents from adjacent industries, agricultural wastes, and household wastes into rivers.

Reproductive Toxicity Induced By Benzo[a]pyrene Exposure: First Exploration Highlighting The Multi-stage Molecular Mechanism In Female Scallop Chlamys Farreri

Reproductive toxicity induced by Benzo[a]pyrene (B[a]P) exposure have received great ecotoxicological concerns. However, huge gaps on molecular mechanism still exist in bivalves. In this study, reproduction-related indicators during reproductive periods (proliferative, growth, mature, and spawn stage) were investigated in female scallops Chlamys farreri, which under gradient concentration of B[a]P at 0, 0.04, 0.4 and 4 μg/L. To elucidate the potential molecular mechanisms of reproductive toxicology, a multi-stage ovarian transcriptome analysis under 4 μg/L B[a]P exposure was also conducted. The results indicated that life-cycle exposure to 0.4 and 4 μg/L B[a]P had significantly decreased GSI and sex steroid levels. Even 0.04 μg/L B[a]P could play the wicked role on DNA integrity at mature and spawn stages. Ovarian histological sections showed the inhibitions on oocyte maturation and ovulation of B[a]P with dose-dependent effects. Through the functional enrichment analysis of DEGs from transcriptome data, 18 genes involved in endocrine disruption effects, DNA damage and repair, and oogenesis damage were selected and further determined by qRT-PCR. The down-regulate of steroidogenic and estrogen signaling pathways genes indicated the endocrine disruption mechanisms by B[a]P, which emphasized the functions of receptor independent and dependent pathways under B[a]P exposure. The variation of DNA single strand break and repair gene expressions implied there might exist the similar toxic mechanism with that in vertebrates. Gene expression data involved in cell cycle, apoptosis and cell adhesion exhibited the possibly toxic mechanisms of oogenesis caused by B[a]P. Taken together, this study is a pioneer to take advantage of genome-wide transcriptomic analysis and its corresponding reproductive indicators to explore the toxic mechanism under B[a]P exposure in bivalves. Meanwhile, some selected genes were firstly identified in bivalves, and the expression data might be useful in establishing new hypotheses and discovering new biomarkers for marine biomonitoring.

Cellular stress response, induction of micronuclei and DNA strand breaks in two common fish species of Rivers and Reservoirs in Ilorin, North Central, Nigeria.

Most rivers and reservoirs in the world are prone to pollution because of indiscriminate disposal of domestic, agricultural and industrial wastes into the water bodies. In this study, we investigated the ecotoxicological potential this could pose in fish species collected from major reservoirs and rivers in Ilorin, north central, Nigeria. Water samples were collected and the physicochemical parameters were examined from five different sites; Unilorin reservoir, Asa reservoir, Apodu reservoir, Asa river (Unity) and Asa river (Harmony). We determined serum biochemical (AST, ALT, ALP, serum ALB), histopathological (gill, lungs), serum antioxidant enzyme responses (SOD, CAT, GPx, GR, GST) which serves as a biomarker for evaluating oxidative stress while micronucleus and comet assays were used to detect level of DNA damage in Tilapia zillii and Clarias gariepinus. The physicochemical parameters and heavy metal analysed (Pb, Mn, Cu, Ni, N, P, Fe, Cl, and Ca) in the five different water bodies were below the permissible limits of WHO and USEPA except the DO, which was very low in the two rivers, indicating hypoxia. Our results showed significant increase in biochemical and hematological profiles, histopathological lesions in the gill and lungs, inductions of MN, NA and DNA single strand break in Tilapia zillii and Clarias gariepinus collected from Asa rivers compared to the Unilorin, Apodun and Asa reservoirs. This may be attributed to indiscriminate discharge of effluents from nearby industries, agricultural and domestic wastes into the rivers.

Breast Cancer Predisposition Genes and Synthetic Lethality

BRCA1 and BRCA2 are tumor suppressor genes with pivotal roles in the development of breast and ovarian cancers. These genes are essential for DNA double-strand break repair via homologous recombination (HR), which is a virtually error-free DNA repair mechanism. Following BRCA1 or BRCA2 mutations, HR is compromised, forcing cells to adopt alternative error-prone repair pathways that often result in tumorigenesis. Synthetic lethality refers to cell death caused by simultaneous perturbations of two genes while change of any one of them alone is nonlethal. Therefore, synthetic lethality can be instrumental in identifying new therapeutic targets for BRCA1/2 mutations. PARP is an established synthetic lethal partner of the BRCA genes. Its role is imperative in the single-strand break DNA repair system. Recently, Olaparib (a PARP inhibitor) was approved for treatment of BRCA1/2 breast and ovarian cancer as the first successful synthetic lethality-based therapy, showing considerable success in the development of effective targeted cancer therapeutics. Nevertheless, the possibility of drug resistance to targeted cancer therapy based on synthetic lethality necessitates the development of additional therapeutic options. This literature review addresses cancer predisposition genes, including BRCA1, BRCA2, and PALB2, synthetic lethality in the context of DNA repair machinery, as well as available treatment options.