Latin American Challenges and Recommendations for Poly Adenosine Diphosphate Ribose Polymerase Inhibitor Treatment in Metastatic Castration Resistant Prostate Cancer: An Expert Overview.

In Latin America, prostate cancer is the third most common cancer overall and the most common in men, with the highest mortality rate of all cancers. In 2022, there were approximately 22,985 new prostate cancer cases and 61,056 deaths from prostate cancer in the region. Patients with metastatic disease that is resistant to cure by castration now have multiple therapeutic options, including poly-ADP ribose polymerase inhibitors. These treatment advances present new challenges, such as developing monitoring protocols for early detection of disease progression to castration resistance. The Americas Health Foundation organized a 3-day meeting with 8 regional oncologists and pathologists to create a paper on metastatic castration-resistant prostate cancer diagnosis and therapy, including the new poly-ADP ribose polymerase inhibitors. The panel examined metastatic castration-resistant prostate cancer in Latin America and recommended ways to improve patient care using published literature and their expertise. Gene mutations play an important role in prostate cancer development. Precision medicine innovations highlight the importance of genotyping DNA variants and tumor biomarkers for targeted treatment. Access to appropriate genetic testing is difficult, medications are available but expensive, and there is a lack of infrastructure and regulatory frameworks that prevent patients from benefiting from innovative therapies. The panel recommends developing a population database and biobank and creating tumor tissue collection, processing, and storage facilities. Multi-stakeholder collaboration is needed to integrate the information gathered, train staff, select target populations, improve patient accessibility, and reduce the cost burden of drugs, genetic counselors, and cancer geneticists in Latin America. Collaboration is essential among healthcare professionals, policymakers, patient advocacy groups, pharmaceutical companies, and international organizations to address these challenges and needs in Latin America.

Comparative effectiveness of first-line systemic treatments for metastatic castration-resistant prostate cancer: a systematic review and network meta-analysis.

OBJECTIVES: No head-to-head trials had been performed to estimate the relative effectiveness of poly ADP-ribose polymerase inhibitor (PARPi) and androgen receptor signaling inhibitor (ARSi) in the first-line treatment for metastatic castration-resistant prostate cancer (mCRPC). We aimed to perform a systematic review and network meta-analysis to evaluate the comparative effectiveness of various systemic treatment agents for patients with mCRPC. METHODS: A comprehensive literature search was conducted for abstracts and full-text articles from the database's inception through April 27, 2023. The study concentrated on assessing radiographic progression-free survival (rPFS) for both overall and homologous recombination repair mutation (HRRm) population, with overall survival (OS) as the secondary measure. Under the Bayesian framework, the overall effect was pooled using the fixed-effects model in base case analysis. Scenario analysis using restricted mean survival time (RMST) methods was performed to test the robustness of the results. RESULTS: Nine studies with 6,830 patients and 8 unique treatment options were included. Network meta-analysis demonstrated that talazoparib in combination with enzalutamide (TALA + ENZA; overall population, hazard ratio [HR], 0.20; 95% credible interval [CrI]: 0.16-0.26; RMST, 3.51; 95% confidence interval [CI] 2.46-4.60; HRRm population, HR, 0.15; 95% CrI: 0.09-0.23; RMST, 4.14; 95% CI 2.84-5.39) was superior to other treatments in the first-line setting in terms of rPFS. The results of Bayesian framework and RMST models showed consistent efficacy ranks. When extrapolated to overall survival benefit, within the Bayesian framework, olaparib plus abiraterone acetate and prednisone (OLAP + AAP) achieved the highest OS benefit for the overall population, which was not statistically significant when compared to TALA + ENZA. However, TALA + ENZA achieved the highest OS benefit at 3 years by applying RMST. CONCLUSIONS: We suggest that talazoparib in combination with enzalutamide is probably a preferred treatment agent for the overall population and HRRm patients with mCRPC. Given the limitations of network framework and the modeling assumptions undertaken to finalize the analyses, results should be cautiously interpreted.

Network meta-analysis of pembrolizumab as monotherapy and in combination with chemotherapy for first-line treatment of recurrent or metastatic head and neck squamous cell carcinoma / Metanálise em rede comparativa de pembrolizumabe como monoterapia e em combinação com quimioterapia para tratamento de primeira linha de carcinoma de células escamosas da cabeça e pescoço recorrente ou metastático

Pembrolizumab monotherapy or in combination with chemotherapy is approved as first-line treatment in recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) based on improved overall survival (OS) versus EXTREME regimen in the KEYNOTE-048 trial. The clinical outcomes of pembrolizumab were compared with other recommended first-line treatments in R/M HNSCC in this study through a Bayesian network meta-analysis. A systematic literature review was conducted in July 2022, from which six trials that matched the KEYNOTE-048 patient eligibility criteria were included in the network. The OS and progression-free survival (PFS) outcomes were compared in the approved pembrolizumab indication (i.e., total population for pembrolizumab in combination with chemotherapy and combined positive score [CPS] ≥ 1 population for pembrolizumab monotherapy). A significant OS improvement was observed for pembrolizumab in combination with chemotherapy and pembrolizumab monotherapy versus EXTREME regimen (hazard ratio, 95% credible interval: 0.72, 0.60-0.86; 0.73, 0.60-0.88), platinum+5- FU (0.58, 0.43-0.76; 0.58, 0.44-0.78), and platinum+paclitaxel (0.53, 0.35-0.79; 0.53, 0.35-0.81), respectively. A non-significant numeric trend in OS improvement was observed versus the TPEx regimen. PFS was comparable with most first-line treatments and was improved versus platinum+5-FU (0.48, 0.36-0.64; 0.59, 0.45-0.79). Additional analyses in higher CPS subgroups also showed consistent results. Overall, our study results showed an improvement in OS outcomes versus alternative first-line treatments, consistent with the findings of the KEYNOTE-048 trial. These data support using pembrolizumab as a suitable firstline treatment option in R/M HNSCC.

Pembrolizumabe em monoterapia ou em combinação com quimioterapia é aprovado como tratamento de primeira linha em carcinoma de células escamosas recorrente/metastático de cabeça e pescoço (CECCP R/M) com base na melhora da sobrevida global (OS), em comparação com o esquema EXTREME no estudo KEYNOTE-048. Esse estudo comparou os resultados clínicos de pembrolizumabe com outros tratamentos recomendados de primeira linha em CECCP R/M por meio de uma metanálise de rede bayesiana. Uma revisão sistemática da literatura foi conduzida em julho de 2022, a partir da qual seis ensaios clínicos que atendiam aos critérios de elegibilidade de pacientes do KEYNOTE-048 foram incluídos na rede. Os desfechos de OS e sobrevida livre de progressão (PFS) foram comparados na indicação de pembrolizumabe (população total para pembrolizumabe em combinação com quimioterapia e população com escore positivo combinado [CPS] ≥ 1 em monoterapia com pembrolizumabe). Foi observada melhora significativa na OS para pembrolizumabe em combinação com quimioterapia e monoterapia com pembrolizumabe versus o esquema EXTREME (razão de risco, intervalo de confiança de 95%: 0,72, 0,60-0,86; 0,73, 0,60-0,88), platina+5-FU (0,58, 0,43-0,76; 0,58, 0,44-0,78) e platina+paclitaxel (0,53, 0,35-0,79; 0,53, 0,35-0,81), respectivamente. Uma tendência numérica não significativa de melhoria na OS foi observada em relação ao esquema TPEx. A PFS foi comparável com a maioria dos tratamentos de primeira linha e melhor em relação à platina+5-FU (0,48, 0,36-0,64; 0,59, 0,45-0,79). Análises adicionais em subgrupos com CPS mais elevado também mostraram resultados consistentes. No geral, os resultados de nosso estudo mostraram melhora nos desfechos de OS em comparação aos tratamentos de primeira linha alternativos, consistentes com os achados do estudo KEYNOTE-048. Esses dados apoiam o uso de pembrolizumabe como opção de tratamento em primeira linha em pacientes com CECCP R/M.

Design of Two New Sulfur Derivatives of Perezone: In Silico Study Simulation Targeting PARP-1 and In Vitro Study Validation Using Cancer Cell Lines.

Poly-ADP-Ribose Polymerase (PARP-1) is an overexpressed enzyme in several carcinomas; consequently, the design of PARP-1 inhibitors has acquired special attention. Hence, in the present study, three compounds (8-10) were produced through a Michael addition protocol, using phenylmethanethiol, 5-fluoro-2-mercaptobenzyl alcohol, and 4-mercaptophenylacetic acid, respectively, as nucleophiles and perezone as the substrate, expecting them to be convenient candidates that inhibit PARP-1. It is convenient to note that in the first stage of the whole study, the molecular dynamics (MD) simulations and the quantum chemistry studies of four secondary metabolites, i.e., perezone (1), perezone angelate (2), hydroxyperezone (3), and hydroxyperezone monoangelate (4), were performed, to investigate their interactions in the active site of PARP-1. Complementarily, a docking study of a set of eleven sulfur derivatives of perezone (5-15) was projected to explore novel compounds, with remarkable affinity to PARP-1. The molecules 8-10 provided the most adequate results; therefore, they were evaluated in vitro to determine their activity towards PARP-1, with 9 having the best IC50 (0.317 µM) value. Additionally, theoretical calculations were carried out using the density functional theory (DFT) with the hybrid method B3LYP with a set of base functions 6-311++G(d,p), and the reactivity properties were compared between the natural derivatives of perezone and the three synthesized compounds, and the obtained results exhibited that 9 has the best properties to bind with PARP-1. Finally, it is important to mention that 9 displays significant inhibitory activity against MDA-MB-231 and MCF-7 cells, i.e., 145.01 and 83.17 µM, respectively.

Análise de custo de sequência de tratamento de câncer de ovário avançado, HRD/ BRCAwt, sob a perspectiva do sistema de saúde suplementar brasileiro / Cost Analysis of advanced ovarian cancer treatment sequence, HRD/BRCAwt from the Brazilian supplementary healthcare system perspective

Objetivo: Estimar o custo da sequência de tratamento considerando as terapias com niraparibe e bevacizumabe, respectivamente, como terapias de manutenção de 1L e 2L para pacientes com câncer de ovário (CO) epitelial com deficiência de recombinação homóloga (HRD) e BRCA selvagem (BRCAwt) em um horizonte temporal de cinco anos, sob a perspectiva do sistema de saúde suplementar brasileiro. Métodos: Foi desenvolvido um modelo de sobrevida particionado com três transições de estados de saúde, considerando os seguintes regimes em 1L e 2L, respectivamente: carboplatina + paclitaxel seguido de terapia de manutenção com niraparibe; carboplatina + gencitabina + bevacizumabe seguido pela continuação de bevacizumabe. As posologias em bula e as curvas de sobrevida livre de progressão dos respectivos estudos pivotais em cada uma das linhas terapêuticas foram utilizadas na análise, e o custo de tratamento foi calculado a partir da lista oficial de preços de medicamentos da CMED de abril de 2023. Resultados: O custo em 1L e 2L foi de BRL 868.830 e BRL 403.407, totalizando BRL 1.272.237 em um horizonte temporal de cinco anos, com 2,28 e 0,52 anos de vida livre de progressão, respectivamente, na 1L e 2L, com o total de 2,8 anos. Conclusões: O resultado da análise de custo de sequência de tratamento de câncer de ovário HRD/BRCAwt apresentou um custo total estimado de BRL 1.272.237, com 2,8 anos de vida livre de progressão. Essa análise contribui no entendimento dos custos e da eficácia esperada com o uso da terapia de manutenção de niraparibe em 1L e bevacizumabe em 2L em um horizonte temporal de cinco anos.

Objective: To estimate the cost of the treatment sequence, considering the maintenance therapies niraparib and bevacizumab, respectively, as maintenance therapies in 1L and 2L for patients with epithelial ovarian cancer with homologous recombination deficiency (HRD) and BRCA wild-type (BRCAwt) over a 5-year time horizon from the perspective of the Brazilian supplementary health system. Methods: A partitioned survival model was developed with three health state transitions, considering the following regimens in the 1L and 2L, respectively: carboplatin + paclitaxel followed by maintenance therapy with niraparib; carboplatin + gemcitabine + bevacizumab followed by the continuation of bevacizumab. The product's label and progression-free survival curves from the respective pivotal studies in each of the therapeutic lines were used in the analysis and the cost of treatment was calculated using as a reference the official CMED drug price list from April 2023. Results: The cost in 1L and 2L was BRL 868,830 and BRL 403,407, totaling BRL 1,272,237 over a 5-year period, with 2.28 and 0.52 years of progression-free survival, respectively in 1L and 2L, with a total of 2.8 years. Conclusions: The result of the analysis of the cost of the treatment sequence of ovarian cancer HRD/BRCAwt presented an estimated total cost of 1,272,237 with 2.8 year of progression-free survival. This analysis contributes to understand the expected cost and effectiveness with the use of maintenance therapy niraparib in 1L and bevacizumab in 2L over a 5-year time horizon.

Inhibitors of Rho kinases (ROCK) induce multiple mitotic defects and synthetic lethality in BRCA2-deficient cells.

The trapping of Poly-ADP-ribose polymerase (PARP) on DNA caused by PARP inhibitors (PARPi) triggers acute DNA replication stress and synthetic lethality (SL) in BRCA2-deficient cells. Hence, DNA damage is accepted as a prerequisite for SL in BRCA2-deficient cells. In contrast, here we show that inhibiting ROCK in BRCA2-deficient cells triggers SL independently from acute replication stress. Such SL is preceded by polyploidy and binucleation resulting from cytokinesis failure. Such initial mitosis abnormalities are followed by other M phase defects, including anaphase bridges and abnormal mitotic figures associated with multipolar spindles, supernumerary centrosomes and multinucleation. SL was also triggered by inhibiting Citron Rho-interacting kinase, another enzyme that, similarly to ROCK, regulates cytokinesis. Together, these observations demonstrate that cytokinesis failure triggers mitotic abnormalities and SL in BRCA2-deficient cells. Furthermore, the prevention of mitotic entry by depletion of Early mitotic inhibitor 1 (EMI1) augmented the survival of BRCA2-deficient cells treated with ROCK inhibitors, thus reinforcing the association between M phase and cell death in BRCA2-deficient cells. This novel SL differs from the one triggered by PARPi and uncovers mitosis as an Achilles heel of BRCA2-deficient cells.

Unveiling the vulnerabilities of synthetic lethality in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most invasive molecular subtype of breast cancer (BC), accounting for about nearly 15% of all BC cases reported annually. The absence of the three major BC hormone receptors, Estrogen (ER), Progesterone (PR), and Human Epidermal Growth Factor 2 (HER2) receptor, accounts for the characteristic "Triple negative" phraseology. The absence of these marked receptors makes this cancer insensitive to classical endocrine therapeutic approaches. Hence, the available treatment options remain solemnly limited to only conventional realms of chemotherapy and radiation therapy. Moreover, these therapeutic regimes are often accompanied by numerous treatment side-effects that account for early distant metastasis, relapse, and shorter overall survival in TNBC patients. The rigorous ongoing research in the field of clinical oncology has identified certain gene-based selective tumor-targeting susceptibilities, which are known to account for the molecular fallacies and mutation-based genetic alterations that develop the progression of TNBC. One such promising approach is synthetic lethality, which identifies novel drug targets of cancer, from undruggable oncogenes or tumor-suppressor genes, which cannot be otherwise clasped by the conventional approaches of mutational analysis. Herein, a holistic scientific review is presented, to undermine the mechanisms of synthetic lethal (SL) interactions in TNBC, the epigenetic crosstalks encountered, the role of Poly (ADP-ribose) polymerase inhibitors (PARPi) in inducing SL interactions, and the limitations faced by the lethal interactors. Thus, the future predicament of synthetic lethal interactions in the advancement of modern translational TNBC research is assessed with specific emphasis on patient-specific personalized medicine.

RAD50 Deficient in a Breast Cancer Model Predicts Sensitivity to PARP Inhibitors.

BACKGROUND: Breast and ovarian tumors with pathogenic variants in BRCA1 or BRCA2 genes are more sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi) treatment than wildtype tumors. Pathogenic variants in non-BRCA1/2 homologous recombination repair genes (HRR) also concede sensitivity to PARPi treatment. RAD50 participates in the Mre11-RAD50-Nbn (MRN) complex of the HRR pathway and plays an important role in DNA repair. OBJECTIVE: The objective of this study is to evaluate whether RAD50 protein deficiency modulates the PARPi response in breast cancer cell lines. METHODS: T47D breast cancer cell line was modified using small interfering RNA and CRISPR/Cas9 technology, to knockout the RAD50 gene. PARPi response (niraparib, olaparib and rucaparib alone or in combination with carboplatin), in T47D and T47D-edited clones, was evaluated by cell viability, cell cycle, apoptosis and protein expression analyses. RESULTS: Treatment with niraparib and carboplatin exerted a synergistic effect on T47D-RAD50 deficient cells and an antagonistic effect on T47D cells parental. Cell cycle analysis demonstrated an increase in the G2/M population in cells treated with niraparib or rucaparib alone or in combination with carboplatin. T47D-RAD50 deficient cells treated with rucaparib and carboplatin exhibited twofold levels in late apoptosis, also showing differences in PARP activation. All T47D RAD50 deficient clones treated with niraparib or rucaparib combined with carboplatin, or rucaparib alone showed increased levels of H2AX phosphorylation. CONCLUSIONS: T47D RAD50 deficient cells treated with PARP inhibitors alone or in combination with carboplatin showed cell cycle arrest in the G2/M phase, leading to death by apoptosis. Thus, RAD50 deficiency may be a good biomarker for predicting PARPi response.

Análise de impacto orçamentário dos inibidores da PARP no tratamento do carcinoma de ovário avançado a partir de evidências de mundo real sob a perspectiva de um hospital federal referência em oncologia / Budget impact analysis of PARP inhibitors in the treatment of advanced ovarian cancer from on real-world evidence the perspective of a federal hospital reference in oncology

Objetivo: Avaliar o impacto orçamentário do tratamento com iPARP como primeira linha de manutenção, comparado ao tratamento-padrão a partir de evidências de mundo real sob a perspectiva de um hospital público referência em oncologia no Rio de Janeiro. Métodos: Foi aplicada uma análise de impacto orçamentário para estimar a introdução das tecnologias iPARP, olaparibe e niraparibe, em comparação com o cenário referência, utilizando dados de eficácia e evidências de mundo real, e considerando os custos globais de tratamento da doença em cinco anos. Este estudo foi aprovado pelo Comitê de Ética em Pesquisa, CAAE: 95157018.9.0000.5274. Resultados: A análise demonstrou que o cenário referência apresentou um impacto orçamentário no valor de R$ 3.578.768,04 em cinco anos. No cenário alternativo, o custo incremental do olaparibe chegou a ser 23,8% maior, comparado ao niraparibe, atingindo um custo de R$ 23.736.459,20 versus R$ 18.076.951,81, respectivamente. Os parâmetros que apresentaram maior impacto nas análises para a tecnologia olaparibe foram a difusão da tecnologia e o preço do medicamento. Contudo, para o niraparibe, os parâmetros de maior impacto foram a duração do tratamento, a difusão da tecnologia e a dose utilizada, demonstrando maior suscetibilidade de variação. Conclusão: Os iPARP no tratamento de pacientes com carcinoma de ovário avançado, apesar de apresentarem custo incremental de aproximadamente R$ 23 milhões em cinco anos, apontam para uma potencial redução de custos associados à progressão da doença.

Objective: Assess the budgetary impact of treatment with iPARP as a first line of maintenance, compared to standard treatment based on real-world evidence from the perspective of a public hospital reference in oncology at Rio de Janeiro. Methods: A budget impact analysis was applied to estimate the introduction of iPARP, olaparib and niraparib technologies, compared to the reference scenario, using efficacy data and real-world evidence, and considering the global costs of treating the disease in five years. This study was approved by the Research Ethics Committee, CAAE: 95157018.9.0000.5274. Results: The analysis showed that the reference scenario presented a budgetary impact of R$ 3,578,768.04 in five years. In the alternative scenario, the incremental cost of olaparib reached 23.8% higher compared to niraparib, reaching a cost of R$ 23,736,459.20 versus R$ 18,076,951.81, respectively. The parameters that had the greatest impact on the analyzes for the olaparib technology were technology diffusion and drug price. However, for niraparib, the parameters with the greatest impact were the duration of treatment, the diffusion of the technology and the dose used, demonstrating greater susceptibility to variation. Conclusion: iPARP in the treatment of patients with advanced ovarian carcinoma, despite having an incremental cost of approximately R$ 23 million in five years, point to a potential reduction in costs associated with disease progression.

Genotoxicity and Anticancer Effects of the Aminothiophene Derivatives SB-44, SB- 83, and SB-200 in Cancer Cells.

INTRODUCTION: Thiophene derivatives have been widely studied as promising options for the treatment of solid tumors. Previous studies have shown that thiophene derivatives have antileishmanial activity and cytotoxic activity against breast, colon, and ovarian cancer cells. METHODS: In our study, we evaluated the anticancer activities of three aminothiophene derivatives: SB-44, SB-83, and SB-200, in prostate and cervical adenocarcinoma cells. Several in vitro methods were performed, including cytotoxicity, clonogenic migration, mutagenic, and cleaved Poly (ADP-ribose) polymerase (PARP) assays and annexin V staining. RESULTS: Significant cytotoxicity was observed in cell lines with IC50 values less than 35 µM (15.38-34.04 µM). All aminothiophene derivatives significantly reduced clone formation but had no effect on cell motility. SB-83 and SB-44 induced a significant increase in the percentage of cells in the sub-G1 phase, while SB-200 derivatives significantly decreased the percentage of S/G2/M as well as induced apoptosis, with an increase of cleaved PARP. SBs compounds also showed significant mutagenic potential. Beyond that, in silico analyses revealed that all three thiophene derivatives fulfilled the criteria for oral druggability, which underscores the potential of using them in anticancer therapies. CONCLUSION: Our findings show that the thiophene nucleus may be used to treat solid tumors, including prostate cancer and cervical adenocarcinoma.

Deoxycytidine kinase (dCK) inhibition is synthetic lethal with BRCA2 deficiency.

BRCA2 is a well-established cancer driver in several human malignancies. While the remarkable success of PARP inhibitors proved the clinical potential of targeting BRCA deficiencies, the emergence of resistance mechanisms underscores the importance of seeking novel Synthetic Lethal (SL) targets for future drug development efforts. In this work, we performed a BRCA2-centric SL screen with a collection of plant-derived compounds from South America. We identified the steroidal alkaloid Solanocapsine as a selective SL inducer, and we were able to substantially increase its potency by deriving multiple analogs. The use of two complementary chemoproteomic approaches led to the identification of the nucleotide salvage pathway enzyme deoxycytidine kinase (dCK) as Solanocapsine's target responsible for its BRCA2-linked SL induction. Additional confirmatory evidence was obtained by using the highly specific dCK inhibitor (DI-87), which induces SL in multiple BRCA2-deficient and KO contexts. Interestingly, dCK-induced SL is mechanistically different from the one induced by PARP inhibitors. dCK inhibition generates substantially lower levels of DNA damage, and cytotoxic phenotypes are associated exclusively with mitosis, thus suggesting that the fine-tuning of nucleotide supply in mitosis is critical for the survival of BRCA2-deficient cells. Moreover, by using a xenograft model of contralateral tumors, we show that dCK impairment suffices to trigger SL in-vivo. Taken together, our findings unveil dCK as a promising new target for BRCA2-deficient cancers, thus setting the ground for future therapeutic alternatives to PARP inhibitors.

Trypanosoma cruzi PARP is enriched in the nucleolus and is present in a thread connecting nuclei during mitosis.

Poly (ADP-ribose) polymerase (PARP) is responsible for the synthesis of ADP-ribose polymers, which are involved in a wide range of cellular processes such as preservation of genome integrity, DNA damage signaling and repair, molecular switches between distinct cell death pathways, and cell cycle progression. Previously, we demonstrated that the only PARP present in T. cruzi migrates to the nucleus upon genotoxic stimulus. In this work, we identify the N-terminal domain as being sufficient for TcPARP nuclear localization and describe for the first time that TcPARP is enriched in the parasite's nucleolus. We also describe that TcPARP is present in a thread-like structure that connects two dividing nuclei and co-localizes with nucleolar material and microtubules. Furthermore, ADP-ribose polymers could also be detected in this thread during mitosis. These findings represent a first approach to new potential TcPARP functions inside the nucleus and will help understand its role well beyond the largely described DNA damage response protein in trypanosomatids.

BRCA1-associated RING domain-1 (BARD1) loss and GBP1 expression enhance sensitivity to DNA damage in Ewing sarcoma.

Ewing sarcoma is a fusion oncoprotein-driven primary bone tumor. A subset of patients (~10%) with Ewing sarcoma are known to harbor germline variants in a growing number of genes involved in DNA damage repair. We recently reported our discovery of a germline mutation in the DNA damage repair protein BARD1 (BRCA1-associated RING domain-1) in a patient with Ewing sarcoma. BARD1 is recruited to the site of DNA double stranded breaks via the poly(ADP-ribose) polymerase (PARP) protein and plays a critical role in DNA damage response pathways including homologous recombination. We thus questioned the impact of BARD1 loss on Ewing cell sensitivity to DNA damage and the Ewing sarcoma transcriptome. We demonstrate that PSaRC318 cells, a novel patient-derived cell line harboring a pathogenic BARD1 variant, are sensitive to PARP inhibition and by testing the effect of BARD1 depletion in additional Ewing sarcoma cell lines, we confirm that BARD1 loss enhances cell sensitivity to PARP inhibition plus radiation. Additionally, RNA-seq analysis revealed that loss of BARD1 results in the upregulation of GBP1 (guanylate-binding protein 1), a protein whose expression is associated with variable response to therapy depending on the adult carcinoma subtype examined. Here, we demonstrate that GBP1 contributes to the enhanced sensitivity of BARD1 deficient Ewing cells to DNA damage. Together, our findings demonstrate the impact of loss-of function mutations in DNA damage repair genes, such as BARD1, on Ewing sarcoma treatment response.

Effect of Beclin-1 gene silencing on autophagy and apoptosis of the prostatic hyperplasia epithelial cells.

OBJECTIVES: This study aims to explore the effect of silencing Beclin-1 gene on autophagy and apoptosis of Benign Prostatic Hyperplasia (BPH) (BPH-1) cells under the condition of Androgen Deprivation (AD) and Autophagy Inhibition (AI). METHODS: Control group (BPH-1 group), empty carrier group (sh-RNA-BPH-1 group) and Beclin-1 silenced group (sh-Beclin1-BPH-1 group) were set. The Beclin-1 gene silencing efficiency was detected by RT-PCR and Western blot. Autophagic flux was monitored by GFP-LC3 cleavage assay and cell apoptosis was analyzed by flow cytometry. The protein expression levels of LC3, Caspase-3, PARP-1, Bcl-2, and Bax were detected by Western blot. RESULTS: The transfection of sh-Beclin-1 obviously down-regulated the expression of Beclin-1 at both mRNA and protein levels. Under the conditions of AD and AI, silencing of Beclin-1 restrained the autophagy of BPH-1 cells, as evidenced by a decreased number of autophagosomes and down-regulation of LC3-II protein (p < 0.001). The results of flow cytometry showed that the apoptotic rate of sh-Beclin-1 group was elevated significantly compared to the other two groups (p < 0.01). Western blot results showed that silencing of Beclin-1 promoted 89 kd fragmentation of PARP-1 (p < 0.001) and Caspase-3 activation (p < 0.01). Moreover, silencing of Beclin-1 resulted in declined Bcl-2 and augmented Bax protein expression in BPH-1 cells (p < 0.01), which ultimately led to a decreased Bcl-2/Bax ratio. CONCLUSIONS: The results indicated that the silencing of Beclin-1 gene hampered autophagy while activating apoptosis in BPH-1 cells. Thus, Beclin-1 may participate in an antagonistic relationship between autophagy and apoptosis in BPH.

Effects of the PARP Inhibitor Olaparib on the Response of Human Peripheral Blood Leukocytes to Bacterial Challenge or Oxidative Stress.

Prior studies demonstrate the activation of poly-(ADP-ribose) polymerase 1 (PARP1) in various pathophysiological conditions, including sepsis. We have assessed the effect of olaparib, a clinically used PARP1 inhibitor, on the responses of human peripheral blood leukocytes (PBMCs) obtained from healthy volunteers in response to challenging with live bacteria, bacterial lipopolysaccharide (LPS), or oxidative stress (hydrogen peroxide, H2O2). The viability of PBMCs exposed to olaparib or to the earlier generation PARP inhibitor PJ-34 (0.1-1000 µM) was monitored using Annexin V and 7-aminoactinomycin D. To evaluate the effects of olaparib on the expression of PARP1 and its effects on protein PARylation, PBMCs were stimulated with Staphylococcus aureus with or without olaparib (1-10 µM). Changes in cellular levels of nicotinamide adenine dinucleotide (NAD+) and adenosine triphosphate (ATP), as well as changes in mitochondrial membrane potential (MMP), were measured in PBMCs exposed to H2O2. Bacterial killing was evaluated in PBMCs and polymorphonuclear leukocytes (PMNs) incubated with S. aureus. Cytokine production was measured in supernatants using a cytometric bead array. Reactive oxygen species (ROS), nitric oxide (NO) production, and phagocytic activity of monocytes and neutrophils were measured in whole blood. For ROS and NO production, samples were incubated with heat-killed S. aureus; phagocytic activity was assessed using killed Escherichia coli conjugated to FITC. Olaparib (0.1-100 µM) did not adversely affect lymphocyte viability. Olaparib also did not interfere with PARP1 expression but inhibits S. aureus-induced protein PARylation. In cells challenged with H2O2, olaparib prevented NAD+ and ATP depletion and attenuated mitochondrial membrane depolarization. LPS-induced production of TNF-α, MIP-1α, and IL-10 by PBMCs was also reduced by olaparib. Monocytes and neutrophils displayed significant increases in the production of ROS and NO after stimulation with S. aureus and phagocytic (E. coli) and microbicidal activity, and these responses were not suppressed by olaparib. We conclude that, at clinically relevant concentrations, olaparib exerts cytoprotective effects and modulates inflammatory cytokine production without exerting adverse effects on the cells' ability to phagocytose or eradicate pathogens. The current data support the concept of repurposing olaparib as a potential experimental therapy for septic shock.

Synergistic antitumor effect of a penicillin derivative combined with thapsigargin in melanoma cells.

PURPOSE: To investigate the effect of TAP7f, a penicillin derivative previously characterized as a potent antitumor agent that promotes ER stress and apoptosis, in combination with thapsigargin, an ER stress inducer, on melanoma cells. METHODS: The synergistic antiproliferative effect of TAP7f in combination with thapsigargin was studied in vitro in murine B16-F0 melanoma cells, and in human A375 and SB2 melanoma cells. In vivo assays were performed with C57BL/6J mice challenged with B16-F0 cells. Immunofluorescence and Western blot assays were carried out to characterize the induction of ER stress and apoptosis. Necrotic tumor areas and the potential toxicity of the combined therapy were examined by histological analysis of tissue sections after hematoxylin-eosin staining. RESULTS: In vitro, the combination of TAP7f with thapsigargin synergistically inhibited the proliferation of murine B16-F0, and human A375 and SB2 melanoma cells. When non-inhibitory doses of each drug were simultaneously administered to C57BL/6J mice challenged with B16-F0 cells, a 50% reduction in tumor volumes was obtained in the combined group. An apoptotic response characterized by higher expression levels of Baxenhanced PARP-1 cleavage and the presence of active caspase 3 was observed in tumors from the combined treatment. In addition, higher expression levels of GADD153/CHOP and ATF4 were found in tumors of mice treated with both drugs with respect to each drug used alone, indicating the induction of an ER stress response. No signs of tissue toxicity were observed in histological sections of different organs extracted from mice receiving the combination. CONCLUSION: The synergistic and effective antitumor action of TAP7f in combination with thapsigargin could be considered as a potential therapeutic strategy for melanoma treatment.

Calotropis Procera Induced Caspase-Dependent Apoptosis and Impaired Akt/mTOR Signaling in 4T1 Breast Cancer Cells.

INTRODUCTION: Calotropis procera (Aiton) Dryand (Apocynaceae) is an herb that has been commonly used in folk medicine to treat various diseases for more than 1500 years. AIMS: Our goal was to investigate the anti-metastatic effects of phenolics extracted from C. procera (CphE) against 4T1 breast cancer cells and in BALB/c mice. METHODS: 4T1 cells were treated with CphE and quercetin (positive control) at concentrations that inhibited cell viability by 50% (IC50). Levels of reactive oxygen species (ROS), wound healing, and protein expressions were determined following standard protocols. For the in vivo pilot study, the syngeneic BALB/c mouse model was used. 4T1 cells were injected into mammary fat pads. Tumors were allowed to grow for 9 days before gavage treatment with CphE (150 mg GAE/kg/day) or PBS (controls) for one week. Excised tumors, liver, and lungs were analyzed for gene and protein expression and histology. RESULTS: In vitro results showed that CphE suppressed cell viability through apoptosis induction, via caspase-3 cleavage and total PARP reduction. CphE also scavenged ROS and suppressed Akt, mTOR, ERK1/2, CREB, and Src activation contributing to cell motility inhibition. CphE reduced IR, PTEN, TSC2, p70S6, and RPS6, protein levels, which are proteins involved in the PI3K/Akt/mTOR pathway, suggesting this pathway as CphE primary target. In vivo results showed downregulation of ERK1/2 activation by phosphorylation in tumor tissues, accompanied by angiogenesis reduction in tumor and lung tissues. A reduction of Cenpf mRNA levels in liver and lung tissues strongly suggested anti-invasive cancer activity of CphE. CONCLUSION: CphE inhibited 4T1 cell signal pathways that play a key role in cell growth and invasion. The potential for in vitro results to be translated in vivo was confirmed. A complete animal study is a guarantee to confirm the CphE anticancer and antimetastatic activity in vivo.

Targeting PARP1 to Enhance Anticancer Checkpoint Immunotherapy Response: Rationale and Clinical Implications.

Reinvigorating the antitumor immune response using immune checkpoint inhibitors (ICIs) has revolutionized the treatment of several malignancies. However, extended use of ICIs has resulted in a cancer-specific response. In tumors considered to be less immunogenic, the response rates were low or null. To overcome resistance and improve the beneficial effects of ICIs, novel strategies focused on ICI-combined therapies have been tested. In particular, poly ADP-ribose polymerase inhibitors (PARPi) are a class of agents with potential for ICI combined therapy. PARPi impairs single-strand break DNA repair; this mechanism involves synthetic lethality in tumor cells with deficient homologous recombination. More recently, novel evidence indicated that PAPRi has the potential to modulate the antitumor immune response by activating antigen-presenting cells, infiltrating effector lymphocytes, and upregulating programmed death ligand-1 in tumors. This review covers the current advances in the immune effects of PARPi, explores the potential rationale for combined therapy with ICIs, and discusses ongoing clinical trials.

Olaparib en mantenimiento de cáncer de ovario epitelial / Olaparib in cancer maintenance ovarian epithelial

INTRODUCCIÓN: En mujeres las neoplasias malignas de origen epitelial ovárico, de las trompas de Falopio y el peritoneo presentan características clínicas y comportamiento similares.1 Debido a esto, suelen combinarse estas entidades y se definen conjuntamente como cáncer de ovario epitelial (COE). El cáncer de ovario seroso de alto grado (COSAG) representa aproximadamente entre el 75% y 90% de todos los COE. Aproximadamente el 17% de los cánceres de ovario son atribuibles a mutaciones germinales deletéreas de los genes BRCA 1 y 2 (gBRCAm). A su vez, se presentan mutaciones somáticas de estos genes hasta en un 7%, y hasta un 50% muestran deficiencia de la recombinación homóloga que afecta los mecanismos de reparación y replicación del ADN. Los síntomas más comunes de la patología son molestias gastrointestinales y ascitis, aunque también pueden presentarse otros síntomas al inicio de la patología como la distención abdominal, dolor abdominal o pélvico, y fatiga. Debido a que los síntomas suelen aparecer tardíamente o ser atribuidos a otras enfermedades, aproximadamente el 75% de los COE son diagnosticados en estadios avanzados (III y IV), siendo en estos casos la sobrevida a 5 años del diagnóstico de aproximadamente 46%. OBJETIVO: El objetivo del presente informe será evaluar la eficacia, seguridad y el impacto presupuestario de olaparib como terapia de mantenimiento en pacientes con cáncer de ovario epitelia. METODOLOGÍA: Se buscó en los sitios públicos de Pubmed, LILACS, BRISA/REDETSA-, CRD (del inglés, Centre for Reviews and Dissemination- University of York), Cochrane; en "buscadores genéricos de internet" y sociedades científicas. En lo que respecta a agencias de ETS, se buscó́ en: Base de datos internacional para las ETS de INHATA (su sigla del inglés, International Network of Agencies for Health Technology Assessment), Base Regional de Informes de Evaluación de Tecnologías en Salud de las Américas (BRISA), NICE (del inglés, National Institute for Health and Clinical Excellence) del Reino Unido; PBAC (del inglés, The Pharmaceutical Benefits Advisory Committee) de Australia; CADTH (del inglés, Canadian Agency for Drugs and Technologies in Health) de Canadá y CONITEC (del portugués, Comissão Nacional de Incorporação de Tecnologías no SUS) de Brasil. RESULTADOS: Con la estrategia de búsqueda mencionada, se identificaron tres ensayos clínicos controlados y aleatorizados (siete registros) y tres revisiones sistemáticas (RS) que evaluaron la efectividad de olaparib en cáncer de ovario. También fueron incluidos cinco informes de evaluación de tecnologías sanitarias (ETS), tres guías de práctica clínica (GPC), 12 políticas de cobertura y siete evaluaciones económicas. CONCLUSIONES: En pacientes con COE avanzado seroso o endometroide de alto grado con respuesta completa o parcial tras una primera línea de quimioterapia en base a platino, y sean portadoras de mutaciones en los genes BRCA 1/2, evidencia de moderada calidad sugiere que olaparib como terapia de mantenimiento frente al cuidado estándar no modificaría la sobrevida global o la calidad de vida, mientras que evidencia de alta calidad muestra que produce un beneficio mayor en la sobrevida libre de progresión. Su utilización también produciría una mayor cantidad de efectos adversos severos y graves. En mujeres recaídas cuya enfermedad se haya manifestado como sensible a la quimioterapia en base a platino, evidencia de alta calidad muestra que olaparib como terapia de mantenimiento, genera un impacto mayor en la sobrevida global y libre de progresión con una mayor cantidad de efectos adversos severos y graves, respecto al estándar de cuidado. Los beneficios clínicos parecen estar limitados aquellas pacientes portadoras de mutaciones deletéreas en los genes BRCA 1/2. Evidencia de muy baja calidad proveniente de comparaciones indirectas, no permite concluir sobre la eficacia y seguridad comparativa de olaparib frente a niraparib. No se hallaron evaluaciones económicas para nuestro país o Latinoamérica. Estudios realizados para Singapur y Estados Unidos mostraron que la tecnología es costoefectiva tras la primera línea de tratamiento, pero no en el tratamiento de pacientes platino sensible recurrentes. Dos análisis de impacto presupuestario realizados en España y Estados Unidos sugieren que el impacto de la tecnología seria moderado. Para Argentina el impacto presupuestario estimado fue alto para la implementación de olaparib como terapia de mantenimiento luego de la primera línea de quimioterapia, al igual que en pacientes platino sensibles recaídas. Guías de práctica clínica de Argentina, Estados Unidos y la Red Nacional Integral del Cáncer recomiendan el uso de olaparib como terapia de mantenimiento tanto en pacientes platino sensible recurrentes como tras una primera línea de tratamiento. De las políticas de cobertura relevadas Alemania, Estados Unidos, Francia y el Reino Unido cubren olaparib como terapia de mantenimiento luego de la primera línea de tratamiento, mientras que países como Argentina, Australia, Brasil, Canadá, Chile, Colombia y Uruguay no mencionan su cobertura. En el caso de pacientes platino sensible, Brasil, Canadá y Francia contemplan su cobertura en pacientes con mutación BRCA, mientras que Estados Unidos y Reino Unido consideran su cobertura sin discriminar el estado de la mutación.