BRCA1 Research Articles

Abstract P5-03-20: Mutations of TP53 and genes related to homologous recombination repair in breast cancer with germline BRCA1/2 mutations

Abstract Background Germline mutations of breast cancer susceptibility gene BRCA1 and BRCA2 (gBRCA1/2) are associated with elevated risk of breast cancer in young women in Asia. BRCA1 and BRCA2 proteins contribute to genomic stability through homologous recombination (HR)-mediated double strand DNA break repair (DDR) in cooperation with other HR-related proteins. In this study, we analyzed the targeted sequencing data of the breast cancer patients with gBRCA1/2 mutations to investigate the landscape of HR-related gene mutations and their clinical implications. Materials and Methods Data of the breast cancer patients with pathogenic gBRCA1/2 mutations and qualified targeted next generation sequencing, SNUH FiRST cancer panel, were analyzed. Single nucleotide polymorphisms, small insertions and deletions were analyzed with functional annotations using ANNOVAR. HR-related genes were defined as ABL1, ATM, ATR, BARD1, BRCA1, BRCA2, CDKN1A, CDKN2A, CHEK1, CHEK2, FANCA, FANCD2, FANCG, FANCI, FANCL, KDR, MUTYH, PALB2, POLE, POLQ, RAD50, RAD51, RAD51D, RAD54L, and TP53. Mismatch-repair genes were MLH1, MSH2, and MSH6. Clinical data were analyzed with cox proportional hazard models and survival analyses. Results Fifty five Korean breast cancer patients with known gBRCA1/2 mutations and qualified targeted NGS data were analyzed. Ethnically distinct mutations in gBRCA1/2 genes were noted, with higher frequencies of Val1833Ser (14.8%), Glu1210Arg (11.1%), and Tyr130Ter (11.1%) in gBRCA1 and Arg2494Ter (25.0%) and Lys467Ter (14.3%) in gBRCA2. Considering subtypes, gBRCA1 mutations were associated with triple-negative breast cancers (TNBC), while gBRCA2 mutations were more likely hormone receptor-positive breast cancers. At least one missense mutation of homologous recombination (HR)-related genes were observed in 44 cases (80.0%). The most frequently co-mutated gene was TP53 (38.1%). In patients with gBRCA1/2 mutations, however, genetic variations of TP53 occurred in locations different from the known hotspots of those with sporadic breast cancers. The patients with both gBRCA1/2 and TP53 mutations were more likely to have TNBC, high Ki-67 values, and increased genetic mutations, especially of HR-related genes. Survival benefit was observed in the TP53 mutants of patients with gBRCA2 mutations, compared to those with TP53 wildtypes. Conclusion Our study showed distinct genetic landscape of breast cancer patients with gBRCA1 and gBRCA2 mutations in the Asian populations. Further studies on precision medicine are needed for tailored treatments of patients with genetic diversity among different ethnic groups. Citation Format: Jinyong Kim, Kyeonghun Jeong, Hyeji Jun, Kwangsoo Kim, Tae-Yong Kim, Dae-Won Lee, Go-un Woo, Songyi Park, Hanbaek Yi, Kyung-Hun Lee, Seock-Ah Im. Mutations of TP53 and genes related to homologous recombination repair in breast cancer with germline BRCA1/2 mutations [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-03-20.

Rucaparib for metastatic castration-resistant prostate cancer (mCRPC): TRITON3 interim overall survival and efficacy of rucaparib vs docetaxel or second-generation androgen pathway inhibitor therapy.

18 Background: TRITON3 (NCT02975934) is a randomized, multicenter, open-label, phase 3 study of rucaparib vs physician’s choice (docetaxel [DTX], abiraterone [ABI] or enzalutamide [ENZ]) in patients (pts) with chemotherapy-naïve mCRPC with BRCA1/2 (BRCA) or ATM alterations. It was previously reported that rucaparib significantly improved the primary endpoint of radiographic progression-free survival (rPFS) vs physician’s choice in pts with BRCA or ATM alterations. We report here the interim overall survival (OS) and also report on efficacy of rucaparib compared individually with either DTX or ABI/ENZ. Methods: Pts had disease progression after 1 prior second-generation androgen pathway inhibitor therapy in any setting and were randomized 2:1 to rucaparib 600 mg BID or physician’s choice of DTX, ABI or ENZ. An ordered step-down multiple comparisons procedure was used to control the overall error rate. OS was a key secondary endpoint with rPFS as the primary endpoint tested first in the BRCA subgroup, then the intent-to-treat (ITT) population. Subgroup analyses based on physician’s choice were exploratory. Results: As of August 25, 2022, 302 pts with BRCA and 103 pts with ATM alterations were randomized. OS maturity was 54% in the BRCA subgroup and 59% in the ITT population. rPFS and OS are shown. The most frequent treatment-emergent adverse event (TEAE) in the rucaparib, DTX and ABI/ENZ groups was asthenia/fatigue (61.1%, 67.6% and 57.6%, respectively). The most frequent grade ≥3 TEAE in the rucaparib, DTX and ABI/ENZ groups was anemia (23.7%), neutropenia (14.1%), and hypertension (10.2%), respectively. Conclusions: Rucaparib significantly improved rPFS vs either DTX or ABI/ENZ; safety was consistent with prior reports. Interim OS results suggest a trend towards improvement for rucaparib vs DTX or ABI/ENZ in pts with mCRPC and BRCA alterations. Clinical trial information: NCT02975934 . [Table: see text]

Carbon ion irradiation induces DNA damage in melanoma and optimizes the tumor microenvironment based on the cGAS-STING pathway.

Increased number of studies reveal the crucial role of the Cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway in anti-tumor immunity. In this study, we aim to explore the effect of cGAS/STING on tumor immune microenvironment of melanoma after carbon ion radiotherapy (CIRT) and the underlying mechanism. C57BL/6 mouse tumor models were used to evaluate the efficacy of different treatments (X-ray, carbon ion, PD-L1 inhibitor and combination therapies) on tumor growth and process. Mass cytometry was performed to assess tumor-infiltrating lymphocytes (TILs). DNA damage response (DDR) and cGAS/STING pathway were investigated by immunofluorescence-co-localization assays, γ-H2AX, P53-binding protein 1 (53BP1), Breast Cancer 1 (BRCA1), and cGAS measurements. Carbon ion irradiation caused more DNA damages and cGAS-STING pathway activation compared with X-ray irradiation, and the former slowed the melanoma growth in syngeneic model. Although X-ray irradiation is not sensitive for melanoma treatment, carbon ion irradiation showed a significant anti-tumor effect for melanoma treatment. TILs analysis revealed that CIRT boosted the infiltration of natural killer (NK), CD4+, and CD8+ T cells, meanwhile increased the number of immune checkpoint (programmed death-1, PD-1, lymphocyte activation gene 3, LAG-3 and T-cell immunoglobulin and mucin domain-containing protein 3, TIM-3). Moreover, CIRT increased PD-L1 exposure on cell surface compared with X-ray group. Furthermore, CIRT combined with PD-L1 inhibitor therapy increased the number of T cells and NK cells in melanoma, and slowed the growth of melanoma compared with other therapies. Our findings showed that CIRT displayed biological effects by increasing DNA damages of tumor cells and improving immunity in melanoma, which indicated that CIRT might be a potential synergetic treatment for radiotherapy and radioimmunotherapy in melanoma patients. Our works put forward a new insight to provide an effective strategy for melanoma therapy. These findings may help in the design of strategies on melanoma in clinical studies.

Dry tobacco leaves: an in vivo and in silico approach to the consequences of occupational exposure.

Exposure of tobacco workers handling dried tobacco leaves has been linked to an increased risk of toxicity and respiratory illness due to the presence of nicotine and other chemicals. This study aimed to evaluate the DNA damage caused by the exposure of tobacco growers during the dry leaf classification process and the relation to cellular mechanisms. A total of 86 individuals participated in the study, divided into a group exposed to dry tobacco (n = 44) and a control group (n = 42). Genotoxicity was evaluated using the alkaline comet assay and lymphocyte micronucleus (MN) assay (CBMN-Cyt), and measurement of telomere length. The levels of oxidative and nitrosative stress were evaluated through the formation of thiobarbituric acid reactive species, and nitric oxide levels, respectively. The inorganic elements were measured in the samples using particle-induced X-ray emission method. The combination of variables was demonstrated through principal component analysis and the interactions were expanded through systems biology. Comet assay, MN, death cells, thiobarbituric acid reactive species, and nitrosative stress showed a significant increase for all exposed groups in relation to the control. Telomere length showed a significant decrease for exposed women and total exposed group in relation to men and control groups, respectively. Bromine (Br) and rubidium (Rb) in the exposed group presented higher levels than control groups. Correlations between nitrate and apoptosis; Br and MN and necrosis; and Rb and telomeres; besides age and DNA damage and death cells were observed. The systems biology analysis demonstrated that tobacco elements can increase the nuclear translocation of NFKB dimers inducing HDAC2 expression, which, associated with BRCA1 protein, can potentially repress transcription of genes that promote DNA repair. Dry tobacco workers exposed to dry leaves and their different agents showed DNA damage by different mechanisms, including redox imbalance.

Microtubule-associated proteins MAP7 and MAP7D1 promote DNA double-strand break repair in the G1 cell cycle phase

The DNA-damage response is a complex signaling network that guards genomic integrity. The microtubule cytoskeleton is involved in the repair of DNA double-strand breaks; however, little is known about which cytoskeleton-related proteins are involved in DNA repair and how. Using quantitative proteomics, we discovered that microtubule associated proteins MAP7 and MAP7D1 interact with several DNA repair proteins including DNA double-strand break repair proteins RAD50, BRCA1 and 53BP1. We observed that downregulation of MAP7 and MAP7D1 leads to increased phosphorylation of p53 after γ-irradiation. Moreover, we determined that the downregulation of MAP7D1 leads to a strong G1 arrest and that the downregulation of MAP7 and MAP7D1 in G1 arrested cells negatively affects DNA repair, recruitment of RAD50 to chromatin and localization of 53BP1 to the sites of damage. These findings describe for the first time a novel function of MAP7 and MAP7D1 in cell cycle regulation and repair of DNA double-strand breaks.

Replication gap suppression depends on the double-strand DNA binding activity of BRCA2

Replication stress (RS) is a major source of genomic instability and is intrinsic to cancer cells. RS is also the consequence of chemotherapeutic drugs for treating cancer. However, adaptation to RS is also a mechanism of resistance to chemotherapy. BRCA2 deficiency results in replication stress in human cells. BRCA2 protein’s main functions include DNA repair by homologous recombination (HR) both at induced DNA double-strand breaks (DSB) and spontaneous replicative lesions. At stalled replication forks, BRCA2 protects the DNA from aberrant nucleolytic degradation and is thought to limit the appearance of ssDNA gaps by arresting replication and via post-replicative HR. However, whether and how BRCA2 acts to limit the formation of ssDNA gaps or mediate their repair, remains ill-defined. Here, we use breast cancer variants affecting different domains of BRCA2 to shed light on this function. We demonstrate that the N-terminal DNA binding domain (NTD), and specifically, its dsDNA binding activity, is required to prevent and repair/fill-in ssDNA gaps upon nucleotide depletion but not to limit PARPi-induced ssDNA gaps. Thus, these findings suggest that nucleotide depletion and PARPi trigger gaps via distinct mechanisms and that the NTD of BRCA2 prevents nucleotide depletion-induced ssDNA gaps.

Mild photothermal therapy boosts nanomedicine antitumor efficacy by disrupting DNA damage repair pathways and modulating tumor mechanics

A variety of Poly (ADP-ribose) polymerase inhibitors (PARPi) have been approved for the clinical treatment of breast cancers. However, pre-clinical and clinical evidences indicate that PARPi only benefits patients with homologous recombination (HR)-deficient breast cancers. Besides, the abnormal mechanical microenvironment of breast cancers severely restricts drug transport to tumor cells. Leveraging efforts from nanomedicine, mesoporous polydopamine (mP) with excellent biocompatibility and large specific surface area was employed to deliver both Olaparib (Ola), an FDA approved PARPi, and Doxorubicin (Dox), a typical DNA-damaging drug. In this nano drug delivery system (NDDS), mP operates not only as a drug carrier but also as a photothermal generator. Mechanistically, we revealed that locally mild photothermal therapy (M-PTT, around 43 ℃) on tumors was capable of inhibiting the HR repair pathway via dramatically downregulating the expressions of key HR-related proteins MRE11, RAD51 and BRCA2. Concomitantly, in vivo results indicated that M-PTT could effectively repress cancer associated fibroblasts (CAFs) by relieving hypoxia, resulting in diminution of dense extracellular matrix (ECM) of breast cancer (collagen Ⅰ and fibronectin decreased by 77.1% and 36.7%, respectively). Furthermore, the reduction of ECM normalized both tumor mechanics and tumor vasculature, facilitating drug delivery and penetration. Therefore, with the aid of M-PTT, this NDDS induces potent DNA damage, thereby enhancing antitumor efficacy (tumor inhibition rate of 86.1%) while minimizing systemic side effects. This work not only highlights the great potential of M-PTT-induced on-demand HR deficiency in clinical cancer therapy, but also reveals the potential mechanisms of M-PTT in inhibiting DNA damage repair and regulating tumor mechanics.

Obesity partially potentiates dimethylbenz[a]anthracene-exposed ovotoxicity by altering the DNA damage repair response in mice†.

Obesity adversely affects reproduction, impairing oocyte quality, fecundity, conception, and implantation. The ovotoxicant, dimethylbenz[a]anthracene, is biotransformed into a genotoxic metabolite to which the ovary responds by activating the ataxia telangiectasia mutated DNA repair pathway. Basal ovarian DNA damage coupled with a blunted response to genotoxicant exposure occurs in obese females, leading to the hypothesis that obesity potentiates ovotoxicity through ineffective DNA damage repair. Female KK.Cg-a/a (lean) and KK.Cg-Ay/J (obese) mice received corn oil or dimethylbenz[a]anthracene (1mg/kg) at 9 weeks of age for 7 days via intraperitoneal injection (n = 10/treatment). Obesity increased liver weight (P < 0.001) and reduced (P < 0.05) primary, preantral, and corpora lutea number. In lean mice, dimethylbenz[a]anthracene exposure tended (P < 0.1) to increase proestrus duration and reduced (P = 0.07) primordial follicle number. Dimethylbenz[a]anthracene exposure decreased (P < 0.05) uterine weight and increased (P < 0.05) primary follicle number in obese mice. Total ovarian abundance of BRCA1, γH2AX, H3K4me, H4K5ac, H4K12ac, and H4K16ac (P > 0.05) was unchanged by obesity or dimethylbenz[a]anthracene exposure. Immunofluorescence staining demonstrated decreased (P < 0.05) abundance of γH2AX foci in antral follicles of obese mice. In primary follicle oocytes, BRCA1 protein was reduced (P < 0.05) by dimethylbenz[a]anthracene exposure in lean mice. Obesity also decreased (P < 0.05) BRCA1 protein in primary follicle oocytes. These findings support both a follicle stage-specific ovarian response to dimethylbenz[a]anthracene exposure and an impact of obesity on this ovarian response.

Functional Analyses of Rare Germline Missense BRCA1 Variants Located within and outside Protein Domains with Known Functions.

The BRCA1 protein is implicated in numerous important cellular processes to prevent genomic instability and tumorigenesis, and pathogenic germline variants predispose carriers to hereditary breast and ovarian cancer (HBOC). Most functional studies of missense variants in BRCA1 focus on variants located within the Really Interesting New Gene (RING), coiled-coil and BRCA1 C-terminal (BRCT) domains, and several missense variants in these regions have been shown to be pathogenic. However, the majority of these studies focus on domain specific assays, and have been performed using isolated protein domains and not the full-length BRCA1 protein. Furthermore, it has been suggested that BRCA1 missense variants located outside domains with known function are of no functional importance, and could be classified as (likely) benign. However, very little is known about the role of the regions outside the well-established domains of BRCA1, and only a few functional studies of missense variants located within these regions have been published. In this study, we have, therefore, functionally evaluated the effect of 14 rare BRCA1 missense variants considered to be of uncertain clinical significance, of which 13 are located outside the well-established domains and one within the RING domain. In order to investigate the hypothesis stating that most BRCA1 variants located outside the known protein domains are benign and of no functional importance, multiple protein assays including protein expression and stability, subcellular localisation and protein interactions have been performed, utilising the full-length protein to better mimic the native state of the protein. Two variants located outside the known domains (p.Met297Val and p.Asp1152Asn) and one variant within the RING domain (p.Leu52Phe) were found to make the BRCA1 protein more prone to proteasome-mediated degradation. In addition, two variants (p.Leu1439Phe and p.Gly890Arg) also located outside known domains were found to have reduced protein stability compared to the wild type protein. These findings indicate that variants located outside the RING, BRCT and coiled-coiled domains could also affect the BRCA1 protein function. For the nine remaining variants, no significant effects on BRCA1 protein functions were observed. Based on this, a reclassification of seven variants from VUS to likely benign could be suggested.

CTP synthase 2 predicts inferior survival and mediates DNA damage response via interacting with BRCA1 in chronic lymphocytic leukemia

BackgroundCytidine triphosphate synthase 2 (CTPS2) is an essential metabolic enzyme that catalyzes the biosynthesis of CTP. CTP synthases contribute to lymphocytes proliferation and tumorigenesis, but the role of CTPS2 in chronic lymphocytic leukemia (CLL) remains undefined.MethodsIn silico analysis was performed to quantified the expression and clinical analysis of CTPS2 and BRCA1. The expression was then validated on the internal sets. Loss-and gain-of-function assays were conducted to investigate the physiological phenotypes in CLL. RNA-seq was employed to probe the molecular mechanism of CTPS2.ResultsHerein, significant elevated expression of CTPS2 was observed in CLL patients compared to normal CD19 + B cells, which was verified in three independent cohorts. Furthermore, overexpression of CTPS2 was closely associated with undesired prognostic indicators, including unmutated IGHV status and chromosome 11q23 deletion. Additionally, elevated CTPS2 expression predicted adverse overall survival and treatment-free survival with independent prognostic significance. Downregulation of CTPS2 in CLL cells exhibited attenuated cell proliferation, arrested G2/M cell cycle and increased apoptosis. The addition of CTP or glutamine could reverse the above effects. Since RNA-seq showed the enrichment in DNA damage and response signaling, we subsequently found that silence of CTPS2 remarkably elevated DNA damage and decreased DNA repair. It was demonstrated that CTPS2 mediated DNA damage response via interacting with Breast Cancer 1 (BRCA1) protein in CLL through CoIP assays and rescued experiments.ConclusionsCollectively, our study generated the novel findings that CTPS2 promoted CLL progression via DNA damage response and repair pathway. Targeting nucleotide metabolism potentially became an attractive strategy for treatment against CLL.

Mutations of TP53 and genes related to homologous recombination repair in breast cancer with germline BRCA1/2 mutations

BackgroundGermline mutations of breast cancer susceptibility gene BRCA1 and BRCA2 (gBRCA1/2) are associated with elevated risk of breast cancer in young women in Asia. BRCA1 and BRCA2 proteins contribute to genomic stability through homologous recombination (HR)-mediated double-strand DNA break repair in cooperation with other HR-related proteins. In this study, we analyzed the targeted sequencing data of Korean breast cancer patients with gBRCA1/2 mutations to investigate the alterations in HR-related genes and their clinical implications.Materials and methodsData of the breast cancer patients with pathogenic gBRCA1/2 mutations and qualified targeted next-generation sequencing, SNUH FiRST cancer panel, were analyzed. Single nucleotide polymorphisms, small insertions, and deletions were analyzed with functional annotations using ANNOVAR. HR-related genes were defined as ABL1, ATM, ATR, BARD1, BRCA1, BRCA2, CDKN1A, CDKN2A, CHEK1, CHEK2, FANCA, FANCD2, FANCG, FANCI, FANCL, KDR, MUTYH, PALB2, POLE, POLQ, RAD50, RAD51, RAD51D, RAD54L, and TP53. Mismatch-repair genes were MLH1, MSH2, and MSH6. Clinical data were analyzed with cox proportional hazard models and survival analyses.ResultsFifty-five Korean breast cancer patients with known gBRCA1/2 mutations and qualified targeted NGS data were analyzed. Ethnically distinct mutations in gBRCA1/2 genes were noted, with higher frequencies of Val1833Ser (14.8%), Glu1210Arg (11.1%), and Tyr130Ter (11.1%) in gBRCA1 and Arg2494Ter (25.0%) and Lys467Ter (14.3%) in gBRCA2. Considering subtypes, gBRCA1 mutations were associated with triple-negative breast cancers (TNBC), while gBRCA2 mutations were more likely hormone receptor-positive breast cancers. At least one missense mutation of HR-related genes was observed in 44 cases (80.0%). The most frequently co-mutated gene was TP53 (38.1%). In patients with gBRCA1/2 mutations, however, genetic variations of TP53 occurred in locations different from the known hotspots of those with sporadic breast cancers. The patients with both gBRCA1/2 and TP53 mutations were more likely to have TNBC, high Ki-67 values, and increased genetic mutations, especially of HR-related genes. Survival benefit was observed in the TP53 mutants of patients with gBRCA2 mutations, compared to those with TP53 wild types.ConclusionOur study showed genetic heterogeneity of breast cancer patients with gBRCA1 and gBRCA2 mutations in the Korean populations. Further studies on precision medicine are needed for tailored treatments of patients with genetic diversity among different ethnic groups.

Abstract C028: The differential burden of BRCA2 gene mutation in prevalent cancers in Nigeria: Any difference between Africa and the world?

Abstract Introduction: The high mortality rate among cancer patients in Africa has been attributed to the high rate of late-stage presentation, aggressiveness, and metastatic nature of the tumors. The manifestation of cancer has also been linked with mutation of DNA repair genes including BRCA1, evidenced by the lack of expression of the tumor suppressors in the affected tissue. However, in Africa, there is a paucity of studies on the frequency of BRCA2 gene mutation in prevalent cancers. This study determined the prevalence of BRCA2 mutation in common cancers in Nigeria in a bid to compare the disparity of the mutation between Africa and elsewhere. Methods: This retrospective study included 51, 54, 58, 48, and 55 viable formalin-fixed Paraffin-embedded tissues of the breast, colorectal, endometrial, ovarian, and prostate cancers, respectively diagnosed between 2017 and 2019 in two tertiary health care facilities in Southern Nigeria. Tissues were stained for BRCA2 protein using the immunohistochemical technique, scored based on staining intensity, and documented as 0, +1, +2, and +3. Tissues with scores of 0 and +1 were considered negative whereas those with scores of +2 and +3 were considered positive. Descriptive, Chi-square test and Pearson’s correlation were carried out and significance was set at ≤0.05. Results: The mean age of women diagnosed with endometrial cancer was lower (41.34 ± 9.16 years) compared with breast, colorectal, ovarian, and prostate cancers (51.00 ± 10.98 years, 47.34 ± 14.73 years, 50.15 ± 12.29 years, and 72.66 ± 8.67 years, respectively). The prevalence of BRCA2 mutation was higher in endometrial cancer (78.4%) than in breast, colorectal, ovarian, and prostate cancers (41.5%, 25%, 54.2%, and 65.1%, respectively) at p&amp;lt; 0.001. There was a direct relationship between age and BRCA2 mutation among women with endometrial cancer (p= 0.005). Conclusion: This study suggests that the prevalence of BRCA2 mutation is high in Southern Nigeria and could be linked with the poor survival rate observed among cancer patients in West Africa. Citation Format: Jude Ogechukwu Okoye, Chiemeka Michael Emeka, Okechi Obioma. The differential burden of BRCA2 gene mutation in prevalent cancers in Nigeria: Any difference between Africa and the world? [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C028.

Next-generation DNA sequences identifies mutated FANCD2 Gene as a novel Biomarker for early disease progression in Chronic Myeloid Leukemia patients

Next-generation DNA sequences identifies mutated FANCD2 Gene as a novel Biomarker for early disease progression in Chronic Myeloid Leukemia patients

Next-generation DNA sequencing identifies mutated FANCD2 Gene as a novel Biomarker for monitoring early disease progression and timely therapeutic interventions in advanced phase Chronic Myeloid Leukemia patients

Next-generation DNA sequencing identifies mutated FANCD2 Gene as a novel Biomarker for monitoring early disease progression and timely therapeutic interventions in advanced phase Chronic Myeloid Leukemia patients

Selection of MicroRNAs Associated between Neural Stem Cells and Multiple Sclerosis

Background: Diagnosis and treatment of multiple sclerosis (MS) in its advanced state have been one of the medical community's concerns so far. Cell therapy has been a modern and successful treatment. However, it has not yet been effective enough to treat MS. This study aimed to find the relationship between neural stem cells (NSCs) and MS, and by considering important signaling pathways of pathogenesis, the most important microRNAs (miRNAs) for its diagnosis and treatment were investigated. Materials and Methods: Using the bioinformatics approaches and appropriate databases, the relationship between NSCs and MS were recognized, and after obtaining common genes between them, the protein products by them were evaluated. Finally, after nominating essential genes, we isolated and analyzed the microarrays involved in these pathways. Results: In the first step, 76 upregulated and 1600 down-regulated common genes between NSCs and MS were recognized. Upregulated genes obtained axon guidance, NCAM, and RHO signaling pathways, and the cell cycle, RNA metabolism, and DNA repair signaling pathways by down-regulated genes. Then, high-expression PAK3, ROBO2, and LIMK2, and low-expression AURKA, BIRC5, BLM, and BRCA1 proteins were identified. Accordingly, high-expression miRNAs included hsa-miR-4790-5p, hsa-miR-4281, and hsa-miR-4327, but low-expression miRNAs included hsa-miR-103b, hsa-miR-638, and hsa-miR-4537 were recognized. Conclusion: Our study indicated that the abovementioned important miRNAs have a major role in diagnosing and treating MS.

Combination with vorinostat enhances the antitumor activity of cisplatin in castration-resistant prostate cancer by inhibiting DNA damage repair pathway and detoxification of GSH.

Like DNA methylation, histone modifications are considered important processes for epigenetic alterations in gene function, and abnormally high expression of histone deacetylases (HDACs) plays a key role in many human diseases. In addition to regulating the acetylation levels of histone and non-histone proteins and gene transcription, HDAC inhibitors as antitumor drugs can also affect the DNA damage repair (DDR) pathway in tumor cells. Prostate cancer (PCa) is one of the most heritable malignancies in which DDR pathway defects can be detected in a considerable proportion of cases. Such defects are more prevalent in castration-resistant prostate cancer (CRPC) and are highly enriched in metastatic lesions. There is currently evidence that DDR pathway-deficient PCa is associated with high-risk biological behaviors and response sensitivity to platinum-based chemotherapy. Platinum-based drugs have been used in multiple clinical trials as monotherapy or in combination with other chemotherapeutic agents for the treatment of CRPC. This study evaluated the combined anticancer effect of (cisplatin) CDDP and the HDAC inhibitors vorinostat (SAHA) on three androgen-dependent cell lines PC-3, DU-145, and C4-2B in vitro. The efficacy and safety of SAHA combined with CDDP in the treatment of CRPC were further verified through animal experiments. The combination of the two drugs increases cytotoxic effects by increasing DNA damage. Our results showed that the SAHA could not only reduce the expression of homologous recombinant repair proteins BRCA2, BRCA1, PARP1, and RAD51, but also decrease enzymes that Reduce the key enzymes of GSH biosynthesis, GSS and GCLC, and GSTP1 which can catalyze the binding of GSH to cisplatin. The intracellular GSH level also decreased with the increase of SAHA concentration, at the same time, the content of intracellular Pt element. The combination of CDDP and SAHA can produce synergistic anticancer effects in androgen-independent PCa cells in vitro and in vivo. Our results open up a new avenue for the effective treatment of CRPC. To optimize the chemotherapy regimen for patients with advanced PCa, it is necessary to further study the molecular mechanism of platinum drugs, HDAC inhibitors, and their combined action.

Tumor-infiltrating lymphocytes and levels of PD-L1 and BRCA protein expression may identify patients with breast cancer with a higher rate of BRCA1 mutations

Introduction. Breast cancer (BC) is a heterogeneous disease, treated as per the predictive role of immunohistoche­mistry (IHC) identifiers as estrogen/progesterone and HER2 receptor proteins. Deeper molecular classification (MC) identifies molecular subtypes according to the gene-expression profiles, with different molecular genetic alterations and biological features, present in the different subtype. An overlap between IHC and MC exists, even if somewhat incomplete. We aimed to identify the overlap between IHC and MC, and identify patients with basal-like subtype of BC. We hypothesized that the rates of tumor expression of breast cancer-related protein 1 (BRCA1), the type of tumor-infiltrating lymphocytes, and the expression of programmed death ligand 1 (PD-L1) by immune cells vary among different subtypes of BC. Material and methods. Parafin-embedded samples from 100 patients with primary invasive BC were analyzed and expression levels of estrogen and progesterone receptors, HER2 status, and Ki-67 were assessed via IHC, defining four groups – luminal A-like, luminal B-like (LumA, LumB), HER2-positive non-luminal, and triple negative (TN). The pri­mary endpoint of our study was to identify via IHC with CK5/6 and 17 basal-like subtypes of BC amongst others, and to describe specific clinicopathological features together with protein expression of BRCA1 and PD-L1 and tumor-infiltrating lymphocytes, using CD20, CD3, CD4, CD8, and FoxP3. Results. Basal-like BC were predominantly characterized as triple negative by IHC (p &lt; 0.05) and were more frequently seen among special BC subtypes as compared to no special type (NST), with p = 0.036. Their immune response was represented mostly by high concentration of intratumoral cytotoxic CD8 (+) T-lymphocytes (p &lt; 0.05) and stromal PD-L1-positive immune cells (p = 0.008). In these tumors, absence of expression of BRCA1 protein was more frequent (p &lt; 0.001). Basal-like subtype of BC with absent expression of BRCA1 is associated with poorer &lt;5-year survival (p = 0.001 and p = 0.017, respectively). Conclusions. The use of IHC can establish basal-like BC, the type of its immune response and possible dysfunction in the BRCA -gene, reflected in the lack of expression in the BRCA-related protein.

Phytochemical Profiling, Biological Activities and In Silico Virtual Screening of Bioactive Compounds of Trichodesma Indicum (L) R. Br. Extracts

AbstractThe current study was designed to evaluate the biological potential of bioactive compounds in the selected fractions of Trichodesma indicum. The results showed that the chloroform and ethyl acetate fractions of ethanolic extract of the whole plant T. indicum were rich in TPCs (total phenolic content) i.e 499.80 and 744.65 mg of gallic acid equivalents per g, respectively, while TFCs (total flavonoid content) were 42.48, and 55.02 mg of catechin equivalents per g, respectively. HPLC analysis confirmed the presence of phenolics i. e., chlorogenic acid (252.35 ppm), ferulic acid (92.56 ppm), caffeic acid (28.72 ppm), cinnamic acid (28.97 ppm), syringic acid (23.75 ppm), gallic acid (7.28 ppm) and flavonoids i. e. myricetin (72.1 ppm), Quercetin (57.3 ppm) in a selected fraction of T. indicum ethanolic extract. The significant potential of antioxidants was observed through ABTS, DPPH, and H2O2 methods. The highest % inhibition of inflammation was observed in ethyl acetate fraction by performing in‐vitro and in‐vivo assays, 87.57 % and 79.18 % (P&lt;0.05) respectively. The thrombolytic activity of the T. indicum fractions was observed in the range from 9.85 % to 27.26. GC‐MS coupled with molecular docking depicted the inhibition of COX‐1 and COX‐2 by the GC‐MS‐identified compound 9,19‐Cyclolanost‐24‐en‐3‐ol, (3. Beta.)‐ (−9.6 kJ/mol). The in‐silico anti‐proliferative analysis was conducted by using BRCA1 and BRCA2 cancerous protein against GC‐MS identified compounds among which stigmasterol (−8.0 kJ/mol) was observed for high binding affinity.

In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls-Part 2: Adaptive Response.

Oxidized in vitro genomic DNA (gDNA) is known to launch an adaptive response in human cell cultures. The cfDNA extracted from the plasma of schizophrenic patients (sz-cfDNA) and healthy controls (hc-cfDNA) contains increased amounts of 8-oxodG, a DNA-oxidation marker. The aim of the research was answering a question: can the human cfDNA isolated from blood plasma stimulate the adaptive response in human cells? In vitro responses of ten human skin fibroblasts (HSFs) and four peripheral blood mononuclear cell (PBMC) lines after 1-24 h of incubation with sz-cfDNA, gDNA and hc-cfDNA containing different amounts of 8-oxodG were examined. Expressions of RNA of eight genes (NOX4, NFE2L2, SOD1, HIF1A, BRCA1, BRCA2, BAX and BCL2), six proteins (NOX4, NRF2, SOD1, HIF1A, γH2AX and BRCA1) and DNA-oxidation marker 8-oxodG were analyzed by RT-qPCR and flow cytometry (when analyzing the data, a subpopulation of lymphocytes (PBL) was identified). Adding hc-cfDNA or sz-cfDNA to HSFs or PBMC media in equal amounts (50 ng/mL, 1-3 h) stimulated transient synthesis of free radicals (ROS), which correlated with an increase in the expressions of NOX4 and SOD1 genes and with an increase in the levels of the markers of DNA damage γH2AX and 8-oxodG. ROS and DNA damage induced an antioxidant response (expression of NFE2L2 and HIF1A), DNA damage response (BRCA1 and BRCA2 gene expression) and anti-apoptotic response (changes in BAX and BCL2 genes expression). Heterogeneity of cells of the same HSFs or PBL population was found with respect to the type of response to (sz,hc)-cfDNA. Most cells responded to oxidative stress with an increase in the amount of NRF2 and BRCA1 proteins along with a moderate increase in the amount of NOX4 protein and a low amount of 8-oxodG oxidation marker. However, upon the exposure to (sz,hc)-cfDNA, the size of the subpopulation with apoptosis signs (high DNA damage degree, high NOX4 and low NRF2 and BRCA1 levels) also increased. No significant difference between the responses to sz-cfDNA and hc-cfDNA was observed. Sz-cfDNA and hc-cfDNA showed similarly high bioactivity towards fibroblasts and lymphocytes. Conclusion: In cultured human cells, hc-cfDNA and sz-cfDNA equally stimulated an adaptive response aimed at launching the antioxidant, repair, and anti-apoptotic processes. The mediator of the development of the adaptive response are ROS produced by, among others, NOX4 and SOD1 enzymes.

Abstract B030: Combined inhibition of BET proteins and PARP promotes impaired DNA damage repair response and cell cycle dysregulation in preclinical models of bladder cancer

Abstract Background: Bladder cancer (BC) remains a common and deadly malignancy, with a projected 81,180 new diagnoses and 17,100 deaths in the United States in 2022. According to the MSK/TCGA Bladder Cancer dataset, mutations in DNA damage response (DDR) genes that are part of the homologous recombination (HR) pathway occurred in up to 55% of patients, representing a potential therapeutic target in BC. In previous work, we used UNC’s EpiG Diamond compound library to show that, as a class, inhibition of the methyl-lysine reader bromodomain and extra-terminal domain (BET) proteins potently abrogate BC cell line viability. Here, we evaluated mechanisms related to HR inhibition that could explain pan-BET inhibitor OTX-015’s potency in BC. Methods: 5637 and J82 BC cells were treated with 1 µM OTX-015 or 0.1% DMSO control for 48-hours, total RNA was extracted, and then bulk RNA-sequencing (RNA-seq) was performed on an Illumina NovaSeq 6000 (Novogene, Sacramento, CA). Expression data was analyzed using Geneious Prime v2022.2.1 (Biomatters, San Diego, CA). Cells were then treated with 1 μM OTX-015, 5 μM of the PARP inhibitor olaparib, or combination for gene and protein expression analysis. Gene expression changes in BRCA1, BRCA2, PALB2, and RAD51, as well as MYC as a positive control, were confirmed by RT-PCR in biological triplicates after 48-hour incubation, normalized to an SDHA housekeeper and compared to a 0.1% DMSO control. Western blotting evaluated OTX-015 and olaparib effects on BRCA1, BRCA2, c-MYC, PALB2, and RAD51 protein expression after 72-hour incubation, using a GAPDH loading control and compared to a 0.1% DMSO control. Last, cells were treated with the same treatment schema for 24-72 hours, fixed with methanol, stained with propidium iodide (25 μg/mL), and flow cytometry evaluated effects on cell cycle using a ThermoFisher Attune NxT and FlowJo v10.8.1 (BD Life Sciences, Ashland, OR). Results: Bulk RNA-seq analyses revealed significantly altered expression of HR pathway genes in 5637 and J82 cells treated with OTX-015±olaparib. In 5637 cells, OTX-015 alone significantly reduced BRCA1, BRCA2, PALB2 and RAD51 expression, (n=3; P&amp;lt;0.001 for all), but only PALB2 and RAD51 expression was significantly reduced after combination treatment (n=3; P&amp;lt;0.001 for both). In J82 cells, OTX-015 alone and combination treatment significantly reduced BRCA1, PALB2 and RAD51 expression, (n=3; P&amp;lt;0.001 for all). Similarly, both OTX-015 and combination treatment resulted in reduced BRCA1, c-MYC, PALB2 and RAD51 protein expression in both cell lines. In both cell lines, combination treatment resulted in a substantially increased G2/M fraction at 48 hours compared to 0.1% DMSO (5637 cells: 62.9% vs 12.6% G2/M; J82 cells: 28.2% vs 17.8% G2/M). Conclusions: These data indicate that OTX-015-mediated inhibition of RAD51 and PALB2 could be responsible for impaired HR. Cell cycle data revealed a G2/M stall, rather than an S phase stall, suggesting mechanisms independent from impaired HR could be responsible for cell cycle dysregulation. Citation Format: Ryan M. Kemper, Manfred Meng, Daniel J. Crona. Combined inhibition of BET proteins and PARP promotes impaired DNA damage repair response and cell cycle dysregulation in preclinical models of bladder cancer. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B030.