Allogeneic hematopoietic cell transplantation (HCT) can provide a curative option for patients with high-risk hematological malignancies and several non-malignant hematological disorders . In addiThe most important and well-studied immune effectors are donor T cells. They play a crucial role in the transplant process, and the extent to which the sensitive balance of their effector functions is maintained is a primary determinant of clinical outcomes , 6. Dono+ T cells or positive selection of CD34+ cells, both of which lead to profound depletion of all donor T-cell subsets. These approaches were generally successful, but recipients were plagued by severely delayed immune reconstitution and consequently high rates of serious viral infections and disease relapse  and effector memory T-cell (TEM) subsets, among others  antigen–mismatched chronic GVHD model, they asked whether effector memory CD62L−CD44+CD4+ T cells were more or less likely to induce GVHD than were unfractionated or naïve CD62L+CD44−CD4+ T cells. Impressively, they found that effector memory CD4+ T cells induced neither clinical nor histologic GVHD. To ensure that the lack of GVHD was not related to an increase in CD4+CD25+ regulatory T cells (Tregs), they performed additional experiments in which they depleted Tregs before a T-cell infusion and observed that the Treg-depleted naïve T cells still caused GVHD whereas the Treg-depleted memory T cells did not. Furthermore, memory CD4+ T cells engrafted and could mount strong proliferative recall responses when challenged in vivo, highlighting the potential to retain responses against pathogens and tumors. These findings have been confirmed in several preclinical murine models of GVHD  repertoire and showed that a reduction in the alloreactive T-cell repertoire among Tuse GVHD . The autuse GVHD , 28, 32,y expand , 34, they expand , and they expand  when comin vitro  antigens in vitro . They sh− memory cells that are responsible for aiding B-cell differentiation and antibody production.Importantly, the elimination of naïve T cells from an infused cell product would not significantly affect responses to infection, as memory T cells can transfer immunity to pathogens and retain anti-leukemic properties , 29, 31.CM cells induce milder GVHD, and that TEM cells do not cause GVHD at all  was a second primary endpoint, and although its incidence was not reduced by this procedure, it was mostly limited to the upper gastrointestinal tract without skin or liver involvement. Importantly, there were no cases of steroid-refractory aGVHD in transplant recipients; in most patients, aGVHD resolved rapidly and completely within 7 days of steroid initiation. However, the most notable finding was the remarkably low rate of chronic GVHD (cGVHD), which was comparable to that reported after pan–T-cell depletion and was significantly lower than that reported with other forms of T-cell–replete MSD transplants.The first-in-human trial of naïve T-cell depleted grafts was reported by Bleakley et al. . They pe+ T cells in patients in response to CMV reactivation, before the emergence of T-cell receptor excision circles (TRECs), which is consistent with the transfer of functional anti-CMV cellular immunity from the infused graft. This translated to low rates of non-relapse mortality (NRM). In addition, despite the reduction in cGVHD, there was no adverse impact on disease-free survival (DFS) and rates of relapse. The 2-year DFS of 70% compares favorably to the 2-year DFS rates seen after pan–T cell–depleted or T-cell–replete MSD transplants. Furthermore, the relapse rate of 21% at 2 years in this cohort compares favorably to published relapse incidences for patients with acute leukemia using different HCT approaches, implying that the GVL effect of memory T cells is probably not abrogated by this approachImmune reconstitution was rapid and robust when compared to that observed with other methods of T-cell depletion and was accompanied by the transfer of protective virus-specific immunity. There were no cases of Epstein–Barr virus–associated post-transplant lymphoproliferative disease (EBV-PTLD), and the authors demonstrated expansion of cytomegalovirus (CMV)-specific CD8Bleakley and colleagues also recently published data from three phase II trials of T-naïve–depleted peripheral blood stem cell (PBSC) grafts, including the outcomes in 138 recipients who had underlying acute leukemia or myelodysplastic syndrome (MDS) . The fircGVHD is associated with long-term morbidity and mortality and has a detrimental effect on the quality of life of survivors. If these low rates of cGVHD can be obtained without increasing the rates of relapse, this strategy has the potential to substantially change the landscape of transplantation. Furthermore, these studies also confirm that naïve T-cell depletion is feasible in the setting of MUD transplants and that lower-intensity regimens are adequate to ensure engraftment.As Bleakley et al. note, randomized trials will be needed to confirm these findings and to ensure that there is no negative impact on relapse and on composite endpoints, such as GRFS, in recipients of naïve T-cell–depleted HCTs, compared to patients treated with standard HCT or using other GVHD-reduction strategies. The same group of investigators has, therefore, initiated two phase II randomized control trials: NCT03970096, which compares naïve T-cell–depleted PBSC transplantation to T-cell–replete PBSC transplantation with Tac/MTX or with PTCy, and NCT03779854, which is a Pediatric Transplantation and Cellular Therapy Consortium trial comparing naïve T-cell–depleted PBSC transplantation to T-cell–replete bone marrow transplantation with Tac/MTX.+ cells in their graft. Here again, immune reconstitution was rapid, there was clearance of all viruses detected before HCT, and there were no new clinically relevant viral infections. The investigators noted expansion of CD8+ CMV-specific T cells that coincided with the clearance of CMV viremia. There were no cases of grade III or IV GVHD. Taken together, these data suggest that this approach would be effective for patients with CIDs or other immune deficiencies who are at high risk for GVHD and infection when receiving transplants from MMUDs.Touzot and colleagues used the approach of naïve T-cell depletion in the MMUD setting in five patients with combined immune deficiencies (CIDs) . Naïve T+-depleted progenitor grafts derived from haplo donors. The results obtained with the CD45RA+ depletion strategy compare very favorably with those obtained with the other methods of T-cell depletion previously employed at our institution. We previously reported the outcomes of 143 pediatric and young adult patients with hematologic malignancies who received a first allogeneic HCT on one of six consecutive ex vivo T-cell–depleted haplo transplant protocols over the past 15 years at our institution, including the first 50 patients treated on the CD45RA+-depleted haplo transplant study described below . This improvement was primarily due to a reduction in NRM. Historical haplo transplant cohorts had increased rates of aGVHD and cGVHD when the infused T-cell dose was >0.1 million/kg. In comparison, 1000-fold higher T-cell doses were infused without a significant increase in GVHD when CD45RA+ depletion was used. However, a skewing towards more severe aGVHD was noted in these patients. Although preclinical studies and other reported clinical studies have shown lower rates of GVHD with CD45RA+-depleted T cells, these results may indicate cross-reactivity of pathogen-specific CD45RA+-depleted T cells against allo antigens. Importantly, although the rates of grade II–IV GVHD were higher (cumulative incidence of 32%) than reported in other studies involving CD45RA+ depletion, the 2-year OS and cumulative incidence of NRM calculated from time of onset of severe grade III-IV GVHD compared favorably with the recent historical haploHCT cohort (2-year OS from the onset of severe GVHD was 76.2% versus 31.2% and the NRM was 14.3% versus 56.3% in the CD45RA-depleted and historic haploHCT cohorts respectively). This suggests that severe aGVHD may be relatively more treatment sensitive following CD45RA+depletion compared to other transplant approaches. Of note, the dose of CD45RA+-depleted T cells infused was approximately one log higher than that used in studies using MSD/MUD grafts.At our institution, we have used CD45RAed below . Our dat+-depleted haplo transplants followed by donor NK cell addback (NCT0180761) in 72 children with high-risk hematologic malignancies  was 88% for patients in CR1, 70.8% for patients in CR2, and 21.1% for patients with refractory disease (P < 0.0001). Despite the inclusion of patients with high-risk and refractory disease, the cumulative incidences of relapse and non-relapse mortality for the entire cohort were 26.5% and 11.5%, respectively. Although the rates of GVHD were higher than those observed with other T-cell–depleted grafts, this approach was associated with promising rates of LFS, relapse, and NRM in this very-high-risk patient population.We recently analyzed updated data from our prospective clinical trial that used CD45RAgnancies . Patientde novo T-cell production mediated by thymopoiesis and more closely resembles that seen in age-matched controls than that in the donor graft . The early post-transplant T-lymphocyte subset distribution recapitulated the composition of the CD45RA+-depleted graft, confirming the successful adoptive transfer of balanced memory T-cell populations. Both TEM cells and TCM cells were present and were sustained for up to 6 months after transplantation in both the CD4+ and CD8+ T-cell populations before progenitor cell–derived thymic output. At day 100 after transplant, patients uniformly exhibited low-level TREC production, but all of them (including those with no evidence of thymic output) demonstrated substantial Vβ spectratyping diversity. These TCR data, in combination with the predominant CD45RO+ phenotypes, support the hypothesis that the T-cell immunity in the first months after HCT was provided by successful adoptive transfer of diverse memory T-cell populations from the CD45RA+-depleted grafts. In addition to the rapid phenotypic reconstitution, early functional recovery was confirmed. Proliferative responses to mitogen and antigens uniformly recovered between day +30 and day +60.A major challenge with T-cell–depleted haplo transplants is the prolonged period of lymphopenia in the first 100 days after transplant, which places patients at increased risk of infection and relapse . Long-teor graft . Howevercipients . An asse+-depleted grafts and serotherapy-free conditioning would have better virus control when compared with recipients of CD3+-depleted grafts. Consistent with this hypothesis, the incidence of viremia, the peak viral loads observed, and the duration of viremia were all substantially decreased when compared with those in recipients of CD3+-depleted grafts. Importantly, recipients of CD45RA+-depleted grafts who developed viremia generally required less aggressive antiviral treatment  contributed to the high rate of HHV-6 infections in both transplant settings. Indeed, in our institutional cohort of pediatric patients who underwent haplo transplants using CD45RA+ depletion followed by NK cell addback, we did not observe a high incidence of HHV-6 encephalitis. In keeping with the in vitro findings and encouraged by the results of our study, the same group of investigators in Spain performed a follow-up study wherein they incorporated a NK-cell infusion within 10 days after a CD45RA+-depleted transplant   Gasior Kabat and colleagues evaluated the use of CD45RA+depletion has provided a therapeutic option for patients lacking matched donors, there is much work to be done to improve outcomes. Several potential strategies could be considered. Critically, the optimal dose of CD45RA+depleted T-cells that can safely be infused in the haplo-identical setting is yet to be determined and it is possible that lowering the dose will be associated with lower rates of GVHD. In addition, while avoiding the use of GVHD prophylaxis is the goal, a short course of immune suppression following HCT might be required to mitigate the severity of GVHD. Additional strategies might consist of co-infusing T-regulatory cell with a lower dose of CD45RA+depleted T-cells in an effort to tilt the balance towards lowering the risk of GVHD. Future studies must be designed to continue to interrogate these and other strategies with the goal of optimizing the therapeutic potential of CD45RA+-depleted haplo-identical donor grafts.Although the use of CD45RA+-depleted T cells are associated with low rates of alloreactivity and, therefore, with low rates of GVHD but retain specificity for leukemic and viral antigens, they have been used by several investigators as donor lymphocyte infusions after transplant in an attempt to enhance immune reconstitution. Data are summarized in The adoptive transfer of donor T cells has been used to optimize immune reconstitution after transplant but is associated with an increased risk of GVHD. As CD45RA+-depleted) donor lymphocyte infusion (mDLI) given early after HCT with TCRαβ T-cell–depleted grafts (P = 0.8). The rates of OS, EFS, NRM, and relapse were similar for the two study arms, demonstrating the safety of this approach. The incidence of CMV viremia, which was the endpoint used to define efficacy in this study, was comparable in the two arms, being 45% in the experimental arm and 55% in the control arm (P = 0.4). However, mDLI was associated with improved recovery of CMV-specific T-cell responses in a subcohort of CMV IgG–seropositive recipients, highlighting the key role of antigen exposure in the expansion and persistence of virus-specific T cells derived from mDLI.Dunaikina and colleagues evaluated the safety and efficacy of low-dose memory  after transplant. No dose-limiting grade III or IV aGVHD was observed, suggesting again that prophylactic mDLI is safe and is not associated with high rates of aGVHD or cGHVD. The sample size was too small to draw conclusions regarding the GVL effect or the impact on functional immunologic recoveryMaung and colleagues assessed the safety of CD45RA of GVHD . The tra+-depleted T cells as addback after TCRαβ-depleted haplo transplant to enhance immune reconstitution in pediatric patients with high-risk acute leukemia . There was also significant expansion of virus-specific T cells (VSTs) directed towards CMV, adenovirus, BK virus, or HHV-6, as shown by enzyme-linked immune absorbent spot (ELISpot) assays (P < 0.01). The TCR repertoire, as assessed by Vβ-spectratyping, was broad and comparable to that of the donor by month 6 post-transplant. The incidences of aGVHD within 28 days after infusion of CD45RA+-depleted T cells at dose levels 1, 2, and 3 were 0%, 20%, and 10%, respectively , suggesting that the infusion of T-cell doses as high as 1 × 107 T-cells/kg was safe. There was no chronic GVHD; however, the follow-up was short. This interim analysis suggests that the approach is safe at the dose levels used without high rates of concurrent GVHD. Comparative studies will be needed to assess the impact of CD45RA+-depleted T cells to enhance immune reconstitution.At our institution, we implemented a prospective trial (NCT03849651) using escalating doses of CD45RAleukemia . We perf+-depleted DLI after haplo transplantation with post-transplant cyclophosphamide for patients with hematologic malignancies  to 32% . While these findings are promising, there are inherent limitations of landmark comparison with retrospective control groups. Future strategies could include randomized control trials to determine whether CD45RA+-depleted DLIs can indeed lower infectious complications.Recently, Castagna et al. evaluated the role of CD45RAgnancies . CD45RA++ memory T cells into adults with relapsed disease after allogeneic HCT (NCT01523223). This was based on preclinical work showing that CD8+ memory T cells, but not CD4+ memory T cells, mediated potent antitumor effects and could eradicate tumors in mouse models as effectively as could unmanipulated DLIs but without causing GVHD. CD8+ T cells were isolated after non-mobilized donor apheresis by using a tandem immunomagnetic selection strategy of CD45RA+ depletion followed by CD8+ enrichment. Fifteen patients received CD8+ memory T cells at escalating doses. Ten of these 15 patients (67%), of whom four had active disease at the time of infusion, responded to the treatment  for at least 3 months after infusion. Five patients did not respond . Of the nine patients with active disease at the time of infusion of CD8+CD45RA-depleted DLI, five had progressive disease at 3 months, two had stable disease at 3 months and progressive disease by six months, one had a partial response at 3 months and progressive disease by six months, and one had a durable CR. Only one patient developed GVHD (grade 2 liver disease that was steroid responsive), thereby confirming the preclinical findings and highlighting the safety of this approach as compared with conventional DLI.Muffly and colleagues  evaluateex vivo T cell depletion strategy currently undergoing clinical evaluation is the use of TCRαβ+ T cell-depletion. While not the primary focus of this review, we briefly discuss here the development and use of this approach as it has several important conceptual and practical similarities to naïve T cell depletion through the targeting of CD45RA.The other major ex vivo activated and expanded TCRγδ+ T cells, mice exhibited successful engraftment but showed no evidence of GVHD  were compared with T cell-replete transplants from MUD (n= 127) or MMUD (n= 118) in pediatric patients with acute leukemias . Again, rd received serotherapy with ATG. Four-year OS was 69% and LFS was 64%. Three-year cumulative incidence of relapse and NRM was 21% and 15% respectively. Despite the lack post-transplant immune suppression and avoidance of ATG in 2/3rd of recipients, grade III to IV aGVHD was seen in only 13% of patients and 11% developed extensive cGVHD. Nonpermissive DP mismatch was associated with higher likelihood of aGVHD but not with the development of cGVHD. Overall, these data demonstrate that suggested that TCRαβ+/CD19+ depleted MUD transplant was a safe and effective approach to transplantation for patients with leukemia.Based on the success of these studies using haplo-identical donors, the use of TCRαβ+ T cell depletion has been extended to the unrelated donor setting. Leahy et al. reported on a multi-center study of sixty pediatric and young adult patients with hematologic malignancies who lacked a matched-related donor . All patP = .7). The OS was 86% in the MUD group and 87% in the MMRD group (P = .95) suggesting there was no difference in outcomes of HCT performed from MUD and MMRD using this approach for patients with PID.Maschan et al. evaluated the use of TCRαβ+/CD19+ depleted grafts using MUD and mismatched related donors (MMRDs) in patients with primary immunodeficiency (PID) . OutcomeSeveral groups have since reported on successful use of TCRαβ+/CD19+ depleted transplant in various settings including for non-malignant diseases  includinThese promising results have led to the adoption of more widespread use of TCRαβ+ T cell depletion. In general, there is more published data available in the pediatric setting using the TCRαβ+ T cell depletion approach and with the use of haplo-identical donors where very low rates of grade III-IV GVHD have been reported. In contrast, the naive T cell depletion platform has been employed more frequently in adults in the in the MSD and MUD setting. While data for haplo trials exists using naïve T-cell depletion, the rates of GVHD appear higher than those reported using TCRαβ+ T cell depleted haplo grafts, perhaps due to the higher number of T cells infused. The naïve depleted graft contains more T-cells, specifically memory T-cells, than the TCRαβ+ T cell depleted graft. This could theoretically translate into a) improved immune reconstitution, reduced opportunistic infections and infectious deaths and b) possibly an improved GVL effect. However, these advantages remain to be proven in prospective studies. Nevertheless, the use of TCRαβ+ T cell depletion and naïve T cell depletion have advanced the field of transplantation and serve as excellent platforms to enhance the safety and efficacy of allogeneic transplantation. At this time, the use of either approach is based on center experience and institutional preference, and there are currently no randomized trials comparing these techniques. Future trials comparing the approaches should evaluate key variables, besides GVHD, such as relapse and NRM with the goal to provide further insight into advantages and disadvantages of each approach. As described above, investigators are also combining these two approaches by using TCRαβ+/CD19+ depleted progenitor cell grafts and naïve T-cell depleted DLI in an attempt to incorporate the advantages of each strategy into one transplant platform.+ depletion. Data from ongoing and completed clinical studies detailed in this review highlight the safety of infusing CD45RA+-depleted T cells and demonstrate the association of this approach with rapid and robust immune reconstitution and reduced viral disease. Future steps include optimizing the number of CD45RA– T cells that can be infused, particularly in the haplo donor setting, and determining the most optimal conditioning regimens and GVHD prophylaxis to maximize the benefit of these cells. Lastly, although this approach has been more extensively studied in patients with leukemias, it could be extended to patients undergoing HCT for nonmalignant conditions.With more advanced graft engineering techniques and the development of “designer grafts” that can enhance immune reconstitution and at least partially separate the GVL effect from GVHD, there is potential for much broader use of CD45RAAll authors contributed to the article and approved the submitted version.


BAP1 loss and monosomy of chromosome 3 are present in patients with high risk of metastasis. MEK-inhibitors do not significantly block UM progression. Combinations of the MEK inhibitor trametinib and different classes of drugs targeting YAP/TAZ were used to overcome resistance. Combination of trametinib and cerivastatin were synergistic in vitro and in vivo in BAP1 mutated and chromosome 3 monosomic uveal melanoma cell lines.Uveal melanoma is a rare and aggressive disease. Gα-proteins GNAQ and GNA11 are driver mutations that activate MAP kinase and YAP/TAZ pathways. BAP1-mutated UPMM3 cell line. The combined treatment reduced ERK and AKT phosphorylation, increased the inactive, cytoplasmatic form of YAP and significantly impaired the growth of UM cells with monosomy of chromosome 3 in NSG mice. Conclusion: Statins can potentiate the efficacy of MEK inhibitors in the therapy of UM.Background: Metastatic uveal melanoma (MUM) is a highly aggressive, therapy-resistant disease. Driver mutations in Gα-proteins GNAQ and GNA11 activate MAP-kinase and YAP/TAZ pathways of oncogenic signalling. MAP-kinase and MEK-inhibitors do not significantly block MUM progression, likely due to persisting YAP/TAZ signalling. Statins inhibit YAP/TAZ activation by blocking the mevalonate pathway, geranyl-geranylation, and subcellular localisation of the Rho-GTPase. We investigated drugs that affect the YAP/TAZ pathway, valproic acid, verteporfin and statins, in combination with MEK-inhibitor trametinib. Methods: We established IC50 values of the individual drugs and monitored the effects of their combinations in terms of proliferation. We selected trametinib and cerivastatin for evaluation of cell cycle and apoptosis. Synergism was detected using isobologram and Chou–Talalay analyses. The most synergistic combination was tested in vivo. Results: Synergistic concentrations of trametinib and cerivastatin induced a massive arrest of proliferation and cell cycle and enhanced apoptosis, particularly in the monosomic,  B-Raf Proto-Oncogene (BRAF), NRAS Proto-Oncogene (NRAS), and Neurofibromin 1 (NF1) [G Protein Subunit Alpha Q (GNAQ) and 11 (GNA11), and BRCA1- associated Protein 1 (BAP1) [Protein Tyrosine Kinase 2 Beta (PTK2B) have been identified [alpha G-protein subunits GNAQ and GNA11, are found in most primary UM in a mutually exclusive pattern [BAP1, Splicing Factor 3b Subunit 1 (SF3B1), and Eukaryotic Translation Initiation Factor 1A X-Linked (EIF1AX) are associated with high, intermediate, and low metastatic risk, respectively [Polycomb gene repression [BAP1 allele.Uveal melanoma (UM), the most common primary intraocular tumour in adults, is characterized by marked variability in its ability to metastasize. Up to half of the patients with UM develop distant metastases, most commonly in the liver . Unlike 1 (NF1)  in CM; a1 (BAP1) ,7,8 in Uentified . Mutatio pattern . Mutatiopression ,13, actiGNAQ and GNA11 activate the classical G-protein signalling cascade via inositol-3-phosphate, diacyl-glycerol, and cyclic AMP leading to the stimulation of mitogen-activated protein (MAP) kinases, protein kinase B (Akt), and C (PKC), phosphoinositide 3-kinase (PI3K) and the mechanistic target of rapamycin (mTOR) . DespiteYAP/TAZ activity is under the direct control of cell shape and polarity dictated by the cytoskeletal structure . YAP/TAZStatins are potent inhibitors of the mevalonate pathway and are used in clinical practice to prevent hypercholesterolemia-derived cardiovascular and coronary heart diseases . The mevStatins, used to inhibit the HMGCR activity, also antagonise isoprenylation and inhibit nuclear localisation and transcriptional activity of YAP/TAZ, mediating a potential anti-cancer activity ,27. A poBAP1 mutation [The main oncogenic functions of YAP/TAZ have been attributed to their interaction with the TEAD transcription factor. The ophthalmological drug verteporfin, an inhibitor of the YAP/TEAD interaction, has shown efficacy in vitro on UM cell lines, except on those bearing mutation .BAP1-mutated cells [Histone acetylation controls gene expression by modulating the access of transcription factor components to genomic regions. The histone deacetylases (HDACs) levels are increased in cancer . Target ed cells ,33. A phBAP1 allele, as a model.With the aim to identify drug combinations that can be used in the treatment of metastatic UM, we analysed those expected to inhibit both MAP-kinase and YAP-signalling and to block UM proliferation. In particular, we focused on the effects of statins and used verteporfin and valproic acid drugs that potentially block YAP-signalling in parallel for comparison. Verteporfin is used to treat macular degeneration and valproic acid is under clinical investigation for adjuvant therapy of UM. The most potent statin and trametinib combination, cerivastatin and trametinib, was tested in vivo using UPMM3, a cell line with monosomy of chromosome 3 (Chr3) and a loss-of-function mutation of the remaining 2.The human UM cell lines 92.1 , MEL270 Trametinib  was dissolved in DMSO at a final concentration of 40 mM, aliquoted, and kept at −80 °C. Atorvastatin, simvastatin, cerivastatin, and verteporfin  powders were dissolved with DMSO at a final concentration of 10 mM, aliquoted, and kept at −20 °C. Valproic acid  was dissolved in deionised water at a final concentration of 1 M and kept at −20 °C.Cells were seeded in 96-well, flat-bottom plates . The following day, increasing drug concentrations and their combinations were added to the cell culture and incubated for 72 h. Cell viability was tested using a 3--2, 5-diphenyl tetrazolium bromide (MTT) assay, . Optical density (OD) was read on a multiwell scanning spectrophotometer  at 570 nm. All experiments were performed three times. The inhibitory concentration that led to 50% growth reduction (IC50) was calculated using a wide range of drug concentrations by the Graph Pad Prism v 9.4 software . The synergism evaluation was performed using the MTT cell viability results obtained after single and combined treatments. The MTT assay was used to measure cellular metabolic activity as an indicator of cell viability, proliferation and cytotoxicity. Since none of the drugs were expected to directly affect mitochondrial activity, the formation of formazan can be used as a proxy for proliferation in UM cell lines. Drug combinations were evaluated following the method established by Chou and Talalay  and the 5 cells were washed in PBS, permeabilised in cold 70% ethanol, and incubated overnight at 4 °C in the dark. After washing with PBS, the cells were incubated in 1 ml of propidium iodide (PI) staining solution for 30 min at RT in the dark. The cell cycle was determined using a flow cytometer  and analysed by ModFit LT v3.0  software.UM cells were seeded in 24-well plates and treated with either trametinib and cerivastatin at IC50 or different synergistic concentrations alone or in combination for 72 hrs. Cells were harvested and 1 × 10Apoptosis was detected with Annexin V-fluorescein isothiocyanate (FITC) and PI, accordingly to the manufacturer’s instructions , and analysed by FACScan Flow cytometer and CellQuest  software.Total proteins were extracted using Complete Lysis buffer containing, proteases inhibitors (04719956001 Roche) and phosphatase inhibitors (04906845001 Roche). Preparation of nuclear extracts was performed by using NE-PER™ nuclear and cytoplasmic extraction reagents according to the manufacturer’s instructions. The Western blot of cell lysates was performed as previously described . Antibodscid Il2rgtm1wjl/SzJ mice were obtained from Charles River . The animals were housed in pathogen-free conditions, and experiments were performed according to the National Regulation on Animal Research Resources and approved by the Institutional Review Board (Aut n°190/2021-PR (risp prot.22418.151)). UPMM3 cells (107) were injected subcutaneously in 16 mice that were randomised in 4 groups of 4 mice each. Trametinib and cerivastatin were initially dissolved in DMSO and diluted into an aqueous pooled dose containing a final concentration of 0.5% hypromellose (Sigma-Aldrich) and 0.05% Tween-80 (Sigma), in saline. Control animals received the vehicle. Final maximal DMSO concentration was 8.8% v/v. Mice weight and tumours were measured weekly. Tumour volume was calculated as follows: V = ½ × L × W × H.Six-weeks old NOD.Cg-Prkdchttp://www.ncbi.nlm.nih.gov/geo/, accessed on 26 January 2023), under accession number GSE212219.UPMM3 cells were treated for 24 h with trametinib 10 nM, or cerivastatin 0.125 µM or the combination of these drugs. The experiment was repeated three times. RNA was extracted from cell lines using RNeasy Plus mini kit . RNA quality was assessed with Nanodrop and BioAnalyser tools . cDNA, ds-cDNA, and cRNA synthesis and fragmentation were performed using the 3’ IVT Express Kit . Hybridisation, washing, and staining were performed using the GeneAtlas . All micro-array data are MIAME compliant. The dataset, corresponding to 12 cell files is available from the GEO database . Analyses were performed using PRISM v 9.4 .The paired or unpaired Student’s Micro-array gene expression data were analysed in R/BioConductor. Quantile normalisation was performed using RMA , differeMAPK-targeted therapy has so far not been successful in UM as a single agent. With the objective to find drugs that can be combined with inhibitors of MAPK to improve their effectiveness, we decided to evaluate the cytotoxic effects of drugs targeting the oncoprotein YAP/TAZ in UM cell lines because the YAP/TAZ pathway plays an important role in UM development . We assessed the effects of statins that target the mevalonate/cholesterol biosynthetic pathway and interfere with YAP protein activation and that inhibit YAP nuclear translocation . We alsobona fide UM cell lines with GNAQ or GNA11 mutations  and OMM2.5 (MUM) were derived from the same patient.We selected the following 6 human utations : 92.1, MCells were treated with different concentrations of each drug for 72 h. Trametinib showed an IC50 value in the nanomolar range of concentrations. The cell line MEL270 was the most sensitive cell line, with the lowest IC50 , OMM1 and OMM2.5 were the least sensitive cell lines  with OMM1 showing the highest IC50 . PrimaryWe tested the combination of trametinib with cerivastatin combining both drugs at concentrations below IC50. We used the method described by Chou and Talalay  to calcuBAP1-mutated UM cell lines (UPMM2 and UPMM3) representative of high-risk primary UM and on OMM2.5 derived from a liver metastasis , respectively, induced 40% of apoptosis. A higher percentage of cells underwent apoptosis when the IC50s of the two drugs were combined A. The ceTreatment with trametinib blocked the cell cycle in the G0/G1 phase in all UM cells tested. Synergistic combinations of trametinib and cerivastatin caused the same cell cycle arrest observed when using the IC50 concentrations of trametinib B.We next focused on the possible signal transduction pathways involved in the increased cytotoxic activity mediated by the synergistic combination of trametinib and cerivastatin treatment. Cleaved PARP-1 and active caspase3 were only expressed in the synergistic combination , suggestB4GALT5, IDH1 and RASSF3 displayed an intermediate level of expression compared with cerivastatin and trametinib alone , cerivastatin  or trametinib and cerivastatin for 57 days (until day 64). The end of treatment was followed by 15 days of observation. Strong inhibition of tumour growth was observed for trametinib-treated mice. The addition of cerivastatin determined a significantly stronger inhibition of tumour growth compared with mice treated with trametinib alone (p = 0.03, Mice showed neither signs of toxicity nor loss of body weight (mice, while it remained not detectable in trametinib and cerivastatin-treated mice. Indeed, in days 71, 77 and 81, tumours from mice treated with trametinib and cerivastatin remained significantly smaller than those from trametinib group (p = 0.02, p = 0.04, p = 0.01, respectively, mice treated with cerivastatin presented a trend to smaller tumours at the end of the treatment compared with control mice although this did not reach statistical significance.In order to validate the effects of trametinib and cerivastatin combination on UM cells in vivo we generated xenografts of a primary monosomic y weight C. DuringGNAQ or GNA11 that activate two different oncogenic signalling pathways, the MAP/MEK-kinase and the YAP/TAZ pathways. The latter, considered important for organ growth control, has only recently gained more attention in oncology. Oncogenic mutations of GNAQ and GNA11 lead to dephosphorylation of YAP and TAZ, with nuclear translocation of YAP and increased cell growth and anti-apoptotic activity [In stark contrast to what has been obtained for cutaneous melanoma, targeted therapy using MAP-kinase and MEK-inhibitors  and immuactivity .BAP1 than in cells in which BAP1 function is abolished by mutations and monosomy of chromosome 3, as is typical for UM with high metastatic risk [Given the activation of two different oncogenic pathways, the association of different classes of drugs targeting signalling downstream of GNAQ or GNA11 has been proposed to block UM cell growth ,58. Drugtic risk . In additic risk .BAP1-mutated UPMM3 cell line. HDAC inhibitors such as VPA also have inhibitory effects on UM cell growth [BAP1-mutant UM in a mouse model [In our hands, verteporfin displayed synergistic activity with trametinib in three of the six cell lines tested, including the monosomic l growth . More rel growth . Quisinose model . Though se model . We showse model ,62. In oEpidemiological studies have shown evidence of reduced cancer incidence and mortality with statins, though their anti-cancer properties remain under investigation . High thBAP1-mutated UM cell lines, models of UM at high risk of metastasis. OMM2.5 cells, derived from an UM liver metastasis, are also sensitive to the combination of trametinib and cerivastatin, although at a lower level compared with the monosomic/BAP1 mutated cells. The simultaneous exposure of UM cells to cerivastatin and the MEK inhibitor allowed us to obtain the IC50 at trametinib doses 100-times lower in the combination, than with trametinib alone. This translates into enhanced efficacy with possibly more manageable toxicity when performing clinical trials. The synergic concentration of cerivastatin was six-fold lower than the IC50 value of the drug alone, an important fact since cerivastatin has been discontinued for clinical use as a cholesterol-lowering medication due to its toxic effects [We have shown that statins have antiproliferative effects in the majority of the UM cell lines tested. The most potent effect was obtained with cerivastatin. We observed synergistic effects of trametinib and cerivastatin in UPMM3 and UPMM2 monosomic and  effects .mice led to the development of tumours in 100% of mice. The oral administration of trametinib and cerivastatin significantly reduced tumour growth compared with control and to the two drugs used as a single treatment with no evidence of toxicity. Cerivastatin alone did not significantly affect tumour growth. Trametinib alone, instead, reduced the size of tumours in mice, confirming an inhibitory effect of trametinib on UM growth in vivo [mice treated with trametinib and cerivastatin but not in the mice treated with trametinib used as a single drug.Transplantation of the UPMM3 cell line in highly immunologically deficient  in vivo . HoweverThe synergistic treatment in vitro of UPMM3 cells affected YAP translocation into the nucleus and involved inhibition of ERK1/2 activation and AKT signalling. Notably, the upregulation of AKT signalling, a potential mechanism of escape from MEK inhibition , is blocRASSF3, a paralog of RASSF1 that has already been implicated as a tumour suppressor in UM [KRAS and RAS-related genes appear upregulated probably as a consequence of the unavailability of the correct post-translationally modified proteins caused by the mevalonate block [Mice in which the gene encoding the molecular target of statins, HMG-CoA reductase, has been disrupted, develop rhabdomyolysis spontaneously [The reduced expression of or in UM , followior in UM ,71, an aor in UM . KRAS ante block ,53. All te block . Rhabdomte block . In manyte block  or gemfite block . The exate block . Mice inaneously . Cerivasaneously . TherefoWhen considering cerivastatin for treating MUM, the patient’s specific risk profile must be considered. Patients at high risk of developing statin-induced rhabdomyolysis can be identified ,79. The We propose to test the combination of trametinib and cerivastatin in clinical trials. This combination could improve the survival of metastatic uveal melanoma patients while waiting for the development of specific GNAQ and GNA11 inhibitors and in the rarer cases of GNAQ/11 wild-type metastatic uveal melanoma.


Ocular graft-versus-host-disease (GVHD) remains a significant clinical complication after allogeneic hematopoietic stem cell transplantation. Impaired visual function, pain, and other symptoms severely affect affected individuals’ quality of life. However, the diagnosis of and therapy for ocular GVHD involve a multidisciplinary approach and remain challenging for both hematologists and ophthalmologists, as there are no unified international criteria. Through an exploration of the complex pathogenesis of ocular GVHD, this review comprehensively summarizes the pathogenic mechanism, related tear biomarkers, and clinical characteristics of this disease. Novel therapies based on the mechanisms are also discussed to provide insights into the ocular GVHD treatment. With the improvements in HSCT technology, a growing number of patients with hematological diseases have longer life expectancies after allo-HSCT , 2. HoweAt present, there are two widely acknowledged international diagnostic criteria for ocular GVHD. The National Institutes of Health (NIH) revised their diagnostic criteria in consensus papers published in 2014. According to the NIH guidelines, ocular GVHD can be diagnosed by a low Schirmer’s test value, with a mean value of ≤ 5 mm at 5 minutes, or a new onset of keratoconjunctivitis sicca as determined by a slit lamp examination, with a mean Schirmer’s test value of 6 to 10 mm . In 2013. Unfortunately, many patients do not achieve adequate control despite these treatments. Thus, novel therapeutic strategies are urgently needed, particularly strategies based on the disease’s pathogenesis.Due to the wide severity spectrum and the lack of randomized studies, there is currently no unified and approved topical treatment for ocular GVHD. The common therapies for ocular GVHD are mostly empirical and consist of lubrication, autologous serum eye drops, topical cyclosporine, tacrolimus, corticosteroids, and therapeutic lenses. Generally, comprehensive treatment, such as punctual occlusion, amniotic membrane graft, cornea grafts, and tarsorrhaphy, is required for patients with severe, persistent, or complex ocular GVHD, depending on the patient’s condition , 11 , conjunctiva, cornea, and meibomian gland. T-cell infiltration of these tissues initiates an immunoinflammatory cascade. Reportedly, the cascade response primarily consists of apoptosis induction, immune cell recruitment, and the production of inflammatory cytokines and chemokines . Thus, s2.1.The LG is the primary target of ocular involvement in cGVHD. In ocular GVHD, donor CD4+ T cells and activated CD8+ T cells infiltrate the periductal area of the LG. Then, T cells caused tissue damage and generated a proinflammatory environment that is characterized by the recruitment of macrophages, Antigen-presenting cells (APCs), and additional T cells. In addition to the recruitment of immune cells, CD34+ stromal fibroblasts are also recruited and activated –15. The The efficacy of artificial tears for the first-line management of ocular GVHD patients with dry eyes has been confirmed. It has been revealed that artificial tears lubricate the ocular surface, replenish tears, and even decrease ocular surface inflammation . NeverthAs shown above, fibrosis is another prominent pathological hallmark of ocular GVHD, and HSP47-expressing fibroblasts play a critical role in LG fibrosis. Reducing collagen synthesis by inhibiting the expression of HSP47 in fibroblasts may contribute to antifibrotic effects. In animal models of ocular GVHD, various studies related to this novel strategy against LG fibrosis are in progress. In a mouse model of ocular chronic GVHD, vitamin A–coupled liposomes containing HSP47 small interfering RNA against HSP47 (VA-lip HSP47) reduced HSP47 expression in fibroblasts, decreased collagen deposition, and restored tear secretion after ocular instillation . Another2.2.Due to the severe inflammatory reaction and fibrosis in the LG, meibomian gland, and eyelid in patients with ocular GVHD, the cornea also often suffers secondary complications. Typically, patients present with pain, photophobia, vision impairment, and high-frequency application of tear substitutes, which significantly affects the patient’s activities of daily living . CornealIn addition to the use of autologous serum eye drops (ASEDs) as tear replacements, wound healing factors such as TGF-β, nerve growth factor, epidermal growth factor (EGF), and fibroblast growth factor, which are present in ASEDs in abundance, support the epithelial healing of severely impaired corneas and conjunctivae , 53. SevAdditionally, scleral lenses and bandage soft contact lenses can be used to treat ocular GVHD with a severely refractory ocular surface , 63. WheDue to the finding that NK1R is relevant to ocular GVHD, Romina et al.  discover2.3).The conjunctival involvement of patients with ocular GVHD often presents as a conjunctival injection or chronic conjunctivitis. The typical histopathological presentation is accompanied by lymphocytic infiltration of the subconjunctival stroma . TatematOcular surface chronic inflammation is essential to ocular GVHD pathogenesis, and topical anti-inflammatory drugs, including corticosteroids, cyclosporine A (CSA), and tacrolimus, have already been employed to treat patients with ocular GVHD.Topical steroids have the capacity to promote lymphocyte apoptosis and suppress cell-mediated inflammation . A complCSA is a calcineurin inhibitor that exerts immunomodulatory effects by blocking T-cell infiltration and activation and the subsequent release of inflammatory cytokines and increases goblet cell density to improve ocular surface and tear functions . TopicalAs shown above, NETs and NET-associated proteins, including eDNA, oncostatin M (OSM), neutrophil gelatinase-associated lipocalin (NGAL), and tumor necrosis factor superfamily member 14(TNFSF14) have the potential to cause ocular surface pathology in ocular GVHD patients. Hence, drugs such as deoxyribonuclease I (DNase I); a subanticoagulant dose of heparin; and antagonists of OSM, NGAL, and TNFSF14 could be potential therapies for managing ocular GVHD . Among tIn addition, the treatment for systemic GVHD based on the microbiome, in the animal model by oral various antibiotics, revealed that gentamicin significantly suppressed inflammatory cell infiltration and fibrosis in cGVHD-affected organs and attenuate the ocular manifestations of cGVHD .2.4.Meibomian gland dysfunction (MGD) is the second most frequent complication of ocular GVHD, with a prevalence of 47.8% in this disease . AlthougIn patients with meibomian gland dysfunction, which causes tear film instability and evaporative dry eye, the usual treatment lines include warm compresses, lid scrubs, and the maintenance of proper lid hygiene practices . In part2.5Several other promising novel treatments for ocular GVHD have demonstrated efficacy in reducing inflammation and halting disease progression. In a preclinical mouse model, a vascular adhesion protein-1 inhibitor , a seconAt present, the use of cell-based therapy is a potential therapeutic strategy for the treatment of various conditions . Given t.In the clinical trial phase, long-term treatment with topical rebamipide and diquafosol can improve clinical signs and symptoms by enhancing tear stability and ocular surface condition due to their mucin-inducing and secretion-promoting effects , 135. Ac3.Cellular mediators also play a crucial role in the underlying mechanism of pathophysiological processes. Consequently, it is essential to investigate the function and cellular origin of each soluble mediator in the tear film that helps to improve the ocular surface condition in GVHD-associated DED. In addition, tear testing is a simple, safe, quantifiable noninvasive screening method. Previously, various studies have identified tear cytokines/chemokines, lipid metabolites, and proteins as promising potential objective biomarkers that could serve as diagnostic, prognostic, and monitoring tools for both ocular and systemic diseases In ocular GVHD, T cells have long been recognized as a key driver of alloreactivity. Hence, various cytokines related to T helper cell-1, T helper cell-2, or T helper cell-17 are involved in this immune process. Previous studies have characterized the tear cytokine profile of patients with ocular GVHD. In particular, it was found that IL-1, IL-2, IL-6, IL-8, IL-10, IL-17, IP-10/CXCL-10, ICAM-1, TNF-α, EGF, lymphotoxin-α(LT-α), E-selectin (CD62E), B-cell activation factor (BAFF), and neuropilin-1 could be regarded as biomarkers of ocular GVHD for prediction, diagnosis, and prognosis , 150–152Profiling the proteomics and lipid metabolites of tear fluid from GVHD patients may also reveal a potential biomarker signature for the disease. Previous research has reported reduced levels of tear proteins in ocular GVHD. Gerber-Hollbach et al.  made addMa et al.  analyzed4Ocular GVHD, a common condition among patients after undergoing allo-HSCT, has a substantial negative impact on the quality of life of patients. Due to the complex pathological mechanisms and diverse clinical manifestations of ocular GVHD, it remains an obstacle to diagnosing affected patients. Moreover, there are still not enough satisfactory effective prophylactic and therapeutic strategies for ocular GVHD. Newer diagnostic methods, especially ocular surface biomarkers, could help in diagnosing the disease earlier, monitoring its response to treatment, and helping to further understand the occurrence and development of ocular GVHD. In addition, while some therapeutic strategies have demonstrated efficacy, the treatment is usually initiated during the symptomatic stage, and by that time, the damage to the LG and conjunctival tissue might be permanent. Therefore, it is particularly important to advocate prevention first, early diagnosis, and early intervention. It is also desirable to collect data on the long-term development and treatment outcomes of ocular GVHD by maintaining a regular follow−up of these patients to better support the implementation of preemptive and therapeutic strategies. Eventually, the elucidation and further study of the relevant pathogenic mechanism underlying ocular GVHD may translate into efficacious mechanism-based therapeutics to permit the development of new perspectives and targeted treatments for patients with ocular GVHD.The manuscript was conceptualized by XZ, XW, and RY. XC wrote the majority of the manuscript. XC, RH, and SH contributed equally to the manuscript. The figures were designed by RH and WF and drawn by XC and SH. XC, RH, and SH summarized the tables. All authors contributed to the article and approved the submitted version.


Esophageal squamous cell carcinoma is the most common type of esophageal cancer and accounts for 5% of malignant tumor deaths. Recent research suggests that chronic inflammation and DNA damage may drive the onset of esophageal squamous cell carcinoma, implying that lowering chronic inflammation and DNA damage compounds may provide chemo-prevention. According to epidemiological and experimental evidence, selenium is linked to a lower risk of several malignancies, including esophageal squamous cell carcinoma. However, its exact mechanism is still unclear. In the present study, we used cell lines and a 4-NQO mice model to explore the anti-cancer mechanism of four types of selenium. Our findings indicated that selenium inhibited the proliferation, colony formation, and ROS level of ESCC cell lines in a time-dependent manner. Intriguingly, selenium treatment impeded 4-NQO-induced high-grade intraepithelial neoplasia and reduced the number of positive inflammatory cells by preserving DNA from oxidative damage. In addition, selenium significantly decreased the expression of Ki-67 and induced apoptosis. This study demonstrates that selenium has a significant chemo-preventive effect on ESCC by reducing high-grade dysplasia to low-grade dysplasia. For the first time, selenium was shown to slow down the progression of esophageal cancer by lowering inflammation and oxidative DNA damage. Esophageal squamous cell carcinoma (ESCC) is the deadliest form of gastrointestinal malignancy and is the eighth most common cancer worldwide. It is the most prevalent in Asia and Africa, but North America and Europe are experiencing a rapid increase in cases ,2. This Several previous studies indicate that inflammation accelerates cell cycle progression, cell proliferation, tumor vascularization, and the evasion of apoptotic cell death, all of which play a role in cancer initiation and progression . Damage Selenium (Se) is a vital and unique trace element that helps to prevent various tumor malignancies, including ESCC ,11. PrevWe first examined three established human ESCC cell lines, KYSE 150, KYSE 520, and CSEC 216, for their sensitivity to MSA, MSC, and Se-Met in comparison with sodium selenite using CCK8 cytotoxicity assays. The cells were treated with 1–100 µM selenite, 10–1000 µM MSC or Se-Met, and 0.001–6 µM MSA for 2 and 6 days. All three cell lines were susceptible to selenite, which induced a dose- and time-dependent growth inhibition A. At theThe anti-proliferative capacity of selenium was also evaluated by colony formation assay. Three ESCC cell lines treated with 6 µM (selenite), 100 µM , and 2 µM (MSA) showed an inhibition of their colony-forming efficacy during 14 days of culture with a regular change of normal medium, every three days E–G, whicp < 0.001) and large size of lesions , compared to untreated mice, respectively. Meanwhile, mice treated with selenium compounds had a significantly lower number of lesions: selenite group , MSA group , MSC group , Se-Met group , MSA group , MSC group , Se-Met group , which is thought to be the most effective chemical for inducing in animals histological alterations similar to those seen in humans. A smooth surface, normal thickness, and flexibility were observed in the untreated esophageal tissue of mice. However, we observed obvious lesions in all 4-NQO-treated mice. The effects of selenium compounds were quite remarkable B. Upon gMoreover, we observed microscopical morphological changes in esophageal tissues. We found that the esophageal epithelium of the 4-NQO-treated group exhibited high-grade intraepithelial neoplasia (HGIN) and more inflammatory cell infiltration than the untreated group. At the same time, selenium compounds  showed low-grade intraepithelial neoplasia (LGIN). Nevertheless, the range of esophageal epithelial lesions in the selenite group was relatively closer to high-grade intraepithelial neoplasia. Similarly, the number of abnormal esophageal epithelial lesions in the MSA group was relatively lower, and inflammatory infiltration was also relatively lower E. This rTo gain insights into the possible mechanisms underlying the inhibitory effects of selenium compounds in ESCC cell lines . The present study assessed their effects on apoptosis by flow cytometry. Annexin V-positive (early and late apoptosis) cells were considered the apoptotic population. ESCC cell lines were treated with selenium compounds—selenite (6 µM), MSA (2 µM), MSC (100 µM), and Se-Met (100 µM)—for 2 and 6 days. Our results indicate that all selenium compounds significantly increased cell apoptosis in KYSE 150 A,B, KYSENext, we examined the morphology of three ESCC cell lines treated with selenium compounds for 2 and 6 days. The results show that the number of cells in an unscratched area reduced. The cells showed typical apoptotic morphology, including a detachment from the bottom of the culture dish and a change in cell shape  under a phase-contrast microscope C,D and . Our resWe identified whether selenium compounds could also reduce proliferation and induce apoptosis in the esophageal epithelium tissue and investigated the proliferative status of these esophageal lesions after selenium treatment by detecting Ki-67 expression. A weak nuclear immunoexpression was observed in mice of the untreated group. When mice were exposed to 4-NQO, cell proliferation was highly elevated in the basal cells of the esophageal epithelium. In contrast, there was a significant decrease in Ki-67 expression in epithelial cells following selenium compound treatment E,F. To f2O2 increased nuclear fluorescence regarding the control. Thus, H2O2 seems to increase inflammation in both cell lines. In contrast, the combined exposure of H2O2 and selenium compounds markedly decreased nuclear fluorescence signals to those of only H2O2 treatment  in the esophagus tissues of the 4-NQO group compared with those in the untreated group  per analyzed field were significantly higher  accumulation plays a vital part in developing human esophageal cancer since ROS produces oxidative alterations of cellular macromolecules, such as DNA, proteins, and lipids ,8. We th2O2 for 16 h. The treatment of cells with H2O2 markedly increased the number of γ-H2AX foci  . Therefo2AX foci  and nucl2AX foci  relativep < 0.001). However, a decrease in γ-H2AX immunoreactivity was observed in mice that received selenium compounds: selenite group , MSA group , MSC group , Se-Met group  (p < 0.0003), MSA group , MSC group , Se-Met group ,  I,J compa0.0001), K,L that Esophageal cancer is one of the most common malignancies. ESCC occurs at very high rates in certain regions of China . CurrentOne of the physiological changes in cancer cells is their continued development paired with their loss of apoptotic mechanisms, which is associated with increased cell proliferation . A signi2O2-treated human immortalized esophageal epithelial cell lines.Inflammation is a natural aspect of the human body’s reaction to tissue damage when inflammation becomes persistent and is thought to be a significant risk factor for various human malignancies, including cancer . Our preDNA damage can be caused by a variety of hazardous substances, including inflammatory cytokines . ReactivSodium selenite (selenite), Se-(methyl) selenocysteine hydrochloride (MSC), selenomethionine (Se-Met), and methylseleninic acid (MSA) were purchased from Sigma . Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), trypsin–EDTA Roswell Park Memorial Institute (RPMI) 1640 Medium, keratinocyte–SFM medium, and an Epilife medium were acquired from Gibco . Penicillin and streptomycin were bought from Sigma . WST-8  (CCK 8) was purchased from . The carcinogen 4-NQO was purchased from Sigma Aldrich . Pentobarbital sodium was purchased from Sigma . Annexin V-FITC/PI Apoptosis Detection kit was purchased from . 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA) was obtained from . Antifade Mounting with DAPI was obtained from Vector lab, Inc. . Antibodies against Ki67 (#12202), γH2AX (#9718), NF-ĸB (#8242), CD45 (#70257), CD4 (#25229) and CD8 (#98941) were purchased from Cell Signaling Technology . 8-OHdG (bs-1278R) was purchased from Bioss Biotechnologies, Beijing, China. Normal goat serum was bought from ZSGB-BIO, ZLI-9022, Beijing, China. Secondary antibodies (Max Vision HRP rabbit kit) were acquired from MXB Biotechnologies, Kit-5004, Fuzhou, China. Immunofluorescence secondary antibodies were obtained from . DAB Kit was obtained from MXB Biotechnologies, DAB-0031, Fuzhou, China).2. KYSE 150 and KYSE 520 were maintained in RPMI 1640 with 10% fetal bovine serum and antibiotic mixture at 37 °C in a humidified incubator containing 5% CO2. Cells were passaged upon reaching 70% to 80% confluency. Cells at passages 4–8 were used for all experiments.Three ESCC cell lines  and two human esophageal immortalized epithelial cell lines (NE2 and NE6) were included in this study. The ESCC cell lines were gifts from Shantou University Medical College . CSEC 21NE2 and NE6 cell culture: Prof. George Tsao kindly provided the human immortalized esophageal epithelial cell lines (NE2 and NE6) from Hong Kong University. NE2 and NE6 cells were grown in a defined keratinocyte–SFM medium and an Epilife medium supplemented with a defined keratinocyte–SFM growth supplement. NE2 cells were accompanied with an additional 1% FBS. Cell cultures were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The culture medium was renewed every three days. selenite, MSC, MSA, and Se-Met were prepared in double-distilled water, filter sterilized, and then stored in 100 µL aliquots and kept at −20 °C in a freezer. A fresh vial was thawed for each use. All experiments were performed with mycoplasma-free cells. All human cell lines were authenticated within the last 3 years using STR profiling.Cell viability was estimated by CCK colorimetric assay and carried out as previously described . See MetSingle-cell suspensions were generated for each ESCC cell line, and a specified numbers of cells were seeded into six-well tissue culture plates. Then, cells were exposed to different doses of Se compounds  for 2 weeks at 37 °C. Culture medium was replaced every three days until the appearance of colonies. The cells were fixed with 4% buffered formalin for 15 min and then stained with 1% crystal violet  for 30 min. The plates were gently washed with PBS. Cell clusters with >50 cells were considered as a colony, and colonies were imaged and counted by the software ImageJ.Cell apoptosis was quantified using the Annexin V-FITC/PI Apoptosis Detection kit following the manufacturer’s protocol. Briefly, after indicated treatment, cells of each ESCC cell line were collected by centrifugation, washed twice with ice-cold PBS, centrifuged at 1000 rpm for 5 min, then gently resuspended in 500 µL of binding buffer. Then, 5 µL Annexin V-FITC and 5 µL PI were added to each sample. The cell samples were incubated in the dark for 15 min at room temperature, detected using flow cytometry , and analyzed using BD Accuri C6 plus software. The apoptotic rate was calculated as the percentage of early + late apoptotic cells.The intracellular ROS level of three ESSC cell lines  was determined at culture days of 1 and 3 using a 2, 7-dichlorofluorescin-diacetate (DCFH-DA) based kit following the manufacturer’s protocol. See Immunofluorescence staining was performed against phospho-H2AX, 8-OHdG, to examine the effect of Se compounds on oxidative DNA damage in three ESSC cell lines  using a method similar to that previously described : see Metn = 30 total) were randomized into six groups: Normal group (n = 5): fed with water containing propylene glycol from the 1st week to 16th week, then fed only normal water for further 4 weeks (shaded in grey); 4-NQO group (n = 5): fed with water containing 4-NQO (100 μg/mL) from the 1st to 16th week (shaded in black) and maintained with regular drinking water for further 4 weeks; selenite group ; MSA group ; MSC group ; and Se-Met group : administered through force-feeding via intragastric intubation using 22-gauge needle size  [Six-week-old C57BL/6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China . Five mice were maintained in each plastic cage in an air-conditioned room with a 12 h light/dark cycle. The temperature and relative humidity were kept at 25 °C ± 2 °C and 50 ± 10%, respectively. All mice were fed with a basal diet  and allowed free access to deionized water. All the animal procedures and experiments conducted in this study were approved by the Animal Ethics Committee of Shantou University Medical College (Permit NO. SUMC2021-228), and all efforts were made to minimize suffering to the animals. We used 4-Nitroquinoline 1-oxide (4-NQO) to induce esophageal lesions. Stock solution (1 mg/mL) was prepared weekly in propylene glycol, diluted in drinking water to a working concentration of 100 μg/mL, and stored at 4 °C. Drinking water containing 4-NQO was freshly prepared every week using deionized water and administered to the mice in light-shielded water bottles ,39,40. Tton, MA)  at a volAfter completing the experimental period (20 weeks), all mice were euthanatized with a 1% aqueous pentobarbital sodium solution (i p). The whole esophagus and stomach were opened longitudinally, and esophagus lesions were performed under white light and carefully identified. Tissues were fixed in freshly made 4% paraformaldehyde overnight at 4 °C, embedded in paraffin, and sectioned into 4 µm sections. A histopathologic assessment of esophagus sections was performed to examine the effects of Se compounds on the incidence of esophagus lesions. In addition to histologic evaluation, esophagus sections were immunostained for the proliferation marker Ki-67, oxidative DNA damage marker 8-OHdg, γ-H2AX, inflammatory marker CD45, CD4, and CD8, and tunnel assay. The selected dose was based on a previous study that showed the inhibitory effects of selenium compounds when tested on various types of cancer in vivo ,43,44.3) = (width2 × length)/2.Two certified pathologists conducted the histological determination of squamous neoplasia at the Shantou University Medical College on the sectioned tissue samples. The esophagus sections were de-paraffinized, rehydrated, stained with H & E for histopathology, and observed by a light microscope. The ESCC precursor lesions observed were classified into the following types: Squamous dysplasia—cytological atypia  and architectural . Low grade—involvement of only the lower half of the epithelium, with only mild cytological atypia. High grade—involvement of more than half of the epithelium or severe cytological atypia  according to WHO 2019 classification and reclassified as the same lesions using the binary classification system . Lesion Apoptosis was assayed by TUNEL staining using the Yeasen Biotech Kit according to the manufacturer’s instructions. Briefly, formalin-fixed, paraffin-embedded (FFPE) tissue on the slides was washed with PBS (pH 7.4) and permeabilized with 0.1% Triton X-100 for 2 min, followed by incubation with 50 μL of TUNEL reaction mixture in a humidified chamber at 37 °C for 1 h in the dark. Then, sections were counterstained with DAPI and observed under a fluorescence microscope . We calculated the percentage of TUNEL-positive cells by dividing the number of TUNEL-positive nuclei by the total number of DAPI-positive nuclei in defined fields and multiplied by 100.Immunostaining of esophagus sections for Ki-67, γ-H2AX, 8-OHdG, CD45, CD4, and CD8 was performed on formalin-fixed paraffin-embedded (FFPE) esophagus sections using the Envision technique, Dako Real EnVision Detection System, and Peroxidase/DAB+  according to the manufacturer’s protocol. See p value ≤ 0.05 was considered statistically significant; NS, non-significant.Statistical significance in the experiments was assessed using GraphPad Prism, version 8.02 . All data were collected and analyzed, blinded, and presented as mean ± SD. Statistical evaluations of the post hoc multiple group comparisons were conducted using one-way ANOVA and two-way ANOVA , followed by Tukey contrasts. A Based on our findings, we suggest that selenium possesses a robust chemotherapeutic effect against ESCC. This effect is related to promoting apoptosis, inhibiting the proliferation of cells, preventing oxidative stress, and reducing inflammatory T cells . Selenium could diminish high-grade dysplasia to low-grade dysplasia via multi-targeted effects, including anti-oxidants and anti-inflammatory effects . These d


Breast cancer remains a leading cause of female cancer related mortality worldwide. Loss of genomic stability and dysregulation of cellular metabolism are well-recognized features of breast cancer, presenting an opportunity to study the drivers of breast cancer progression and resistance to chemotherapy. The overarching goal of this work is to perform combined analysis of DNA damage repair and cellular metabolism in response to olaparib treatment in a panel of breast cancer cell lines. By applying a combined untargeted metabolomics and molecular biology approach, our findings show dysregulation of amino acid metabolism and metabolic reprogramming from glycolysis to amino acid utilization to be a common feature in all breast cancer cell lines examined, some of which are consistent with findings from the analysis of clinical breast cancer tumours. Functional assessment of genetic alterations offers the scope to design new prognostic tools and inform the design of new chemotherapies or drug combinations.Metabolic reprogramming and genomic instability are key hallmarks of cancer, the combined analysis of which has gained recent popularity. Given the emerging evidence indicating the role of oncometabolites in DNA damage repair and its routine use in breast cancer treatment, it is timely to fingerprint the impact of olaparib treatment in cellular metabolism. Here, we report the biomolecular response of breast cancer cell lines with DNA damage repair defects to olaparib exposure. Following evaluation of olaparib sensitivity in breast cancer cell lines, we immunoprobed DNA double strand break foci and evaluated changes in cellular metabolism at various olaparib treatment doses using untargeted mass spectrometry-based metabolomics analysis. Following identification of altered features, we performed pathway enrichment analysis to measure key metabolic changes occurring in response to olaparib treatment. We show a cell-line-dependent response to olaparib exposure, and an increased susceptibility to DNA damage foci accumulation in triple-negative breast cancer cell lines. Metabolic changes in response to olaparib treatment were cell-line and dose-dependent, where we predominantly observed metabolic reprogramming of glutamine-derived amino acids and lipids metabolism. Our work demonstrates the effectiveness of combining molecular biology and metabolomics studies for the comprehensive characterisation of cell lines with different genetic profiles. Follow-on studies are needed to map the baseline metabolism of breast cancer cells and their unique response to drug treatment. Fused with genomic and transcriptomics data, such readout can be used to identify key oncometabolites and inform the rationale for the design of novel drugs or chemotherapy combinations. In a bid to develop new therapies against various cancer types, and genomic instability, its underpinning mechanisms and contribution to tumorigenesis have been extensively investigated over the past few decades. Genomic instability, a well-known contributor to cancer, presents a therapeutic vulnerability that can be targeted in the development of novel chemotherapy agents .To maintain their genomic integrity, cells are equipped with a range of DNA damage repair (DDR) pathways and responses to counteract DNA lesions formed in response to endogenous and exogenous insults . HereditPARP inhibitors, as a class of DDR inhibitors, block the activity of PARP enzymes involved in DNA damage repair, therefore leading to accumulation of DNA double-strand breaks that gives rise to genomic instability and subsequent apoptosis . SeveralWhile PARP inhibitors present a therapeutic opportunity for targeting DDR defects in breast and ovarian cancers, emerging evidence has shown a role for oncometabolites—small molecule intermediates of cellular metabolism—in determining the response to these chemotherapies. The biology of oncometabolites and their role in modulating DDR has been increasingly studied over the past few years, guiding new combination therapies and novel biological targets for drug discovery .Metabolic reprogramming—a key feature of all cancers —gives riDDR mechanisms induce cellular metabolic changes through interference with purine and pyrimidine biosynthetic pathways, amino acid metabolism, protein biosynthesis, and energy metabolism, impacting several metabolic routes . MediatoDNA repair and regulation of metabolism is critical for maintaining homeostasis in normal human cells. However, the extensive dysregulation and aberrant function of both these pathways promotes tumorigenesis. Until recently, DNA repair and metabolic pathways have routinely been researched as distinct fields within their own right, but emerging research evidence an intrinsic inter-dependency between these pathways. Here, we report the differential cellular response of breast cancer cell line models with different mutational signatures see  and DDR v/v FBS , 1% v/v non-essential amino acids (NEAA) and 1% v/v penicillin-streptomycin (Invitrogen). Corresponding cell line origins, hormone receptor status and mutational profiles are included in v/v FBS and 1% v/v penicillin-streptomycin. All cell lines were maintained at 37 °C in a pre-humidified atmosphere containing 5% v/v CO2 and used within ten passages for the purposes of this work (passage 2–10). Olaparib  was prepared as a 100 mM stock solution in DMSO, aliquoted, and stored at −20 °C until use. γH2AX, p53BP1 primary antibodies , were used for foci immunostaining alongside the Alexa Fluor® 488-conjugated secondary antibody .All cell lines used in this study were purchased from the vendor and maintained in accordance with manufacturer instructions. All cell culture reagents were obtained from Gibco . MCF7  and MDA-MB-231 cells  were purchased and maintained in Dulbecco’s Modified Eagle Medium  supplemented with 10% v/v CO2. Treatment media were replaced every three days with treatment medium. Following a seven-day incubation, cell viability was measured using CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay  -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent. The resultant absorbance at 490 nm was measured using a GM3500 Glomax® Explorer Multimode Microplate Reader (Promega).MCF-7, MDA-MB-231, and HCC1937 cells undergoing exponential growth were seeded at a density of 4,000 cells/well in 96 well plates and incubated overnight to facilitate cell attachment. On the following day, cells were exposed to either blank growth medium (control) or growth medium containing different concentrations of olaparib (treatment medium) ranging from 0.01–500 µM for seven days at 37 °C and 5% 50) was determined using a linear regression model. Statistical analysis was performed using GraphPad Prism . Three independent biological replicates (five wells per treatment concentration) were performed for each cell line.Growth curves represent percentage cell growth following treatment with different concentrations of olaparib and are plotted as a semi-log dose–response curve. The half maximal inhibitory concentration  for seven days. Briefly, cell monolayers were fixed in chilled 4% w/v formaldehyde containing 2% w/v sucrose in PBS, followed by fixation in ice-cold methanol (100% v/v). Subsequently, cells were permeabilized in 0.25% v/v Triton X-100 in PBS, blocked with 5% v/v goat serum/5% w/v BSA, immunoprobed with either a primary rabbit anti-γH2AX antibody (RRID:AB_420030) (1:1000) or primary rabbit anti-P53BP1 (1:200) antibody  overnight at 4 °C. Cell monolayers were treated with goat, anti-rabbit Alexa Fluor® 488 conjugated secondary antibody and counterstained with DAPI. Image acquisition was carried out using an Invitrogen EVOS Auto Imaging System (AMAFD1000-Thermo Fisher Scientific) with a minimum of 100 cells imaged per treatment condition. Resultant foci images were analyzed in Cell Profiler (v.4.2.1.) using a modified version of the speckle counting pipeline.Foci immunodetection for γH2AX and p53BP1 was performed in both control (growth medium) and for cells treated with olaparib . Following exposure to olaparib, the growth medium was aspirated from each well, centrifuged to remove cell debris, and stored at −80 °C. Next, treated cells were washed with pre-chilled PBS, with the metabolites quenched and extracted in a final volume of 1.5 mL pre-chilled (−80 °C) mixed solvent (Methanol:Acetonitrile:Water = 50:30:20). Resultant cell pellets were collected, and submerged in liquid nitrogen, vortexed, and sonicated for 3 min in an ice-water bath. This procedure was performed in triplicate. Resultant extracts were centrifuged at 13,000× g for 10 min at 4 °C and the pellets were retained for protein quantification using the Bradford assay. The resultant supernatant was collected and dried with a Speed vac centrifuge (Savant-SPD121P). Dried metabolite pellets were reconstituted in Acetonitrile:Water (50:50) at volumes normalized to the relative protein content. Quality control (QC) samples were prepared by pooling samples across all control and treatment groups. Solvent blank and QC samples were inserted in analytical batch after every five samples to assess the stability of detecting system.MCF-7, MDA-MB-231, and HCC1937 cells were seeded at a density of 2 × 10Metabolite separation was performed on a binary Thermo Vanquish ultra-high performance liquid chromatography system, where 5 µL of reconstituted cellular extract was injected on to a Thermo Accucore HILIC column . The temperature of the column oven was maintained at 35 °C, while the autosampler temperature was set at 5 °C. For chromatographic separation, a consistent flow rate of 500 µL/min was used where the mobile phase in positive heated electrospray ionisation mode (HESI+) was composed of buffer A  and buffer B  . Likewism/z with a MS1 resolution of 60,000. Subsequent MS/MS scans were processed with a resolution of 15,000. High-purity nitrogen was used as nebulising and as the collision gas for higher energy collisional dissociation. Further details are included in A high-resolution Exploris 240-Orbitrap mass spectrometer (Thermo Fisher Scientific) was used to perform full scan and fragmentation analyses. Global operating parameters were set as follows: spray voltages of 3900 V in HESI+ mode, and 2700 V in HESI-mode. The temperature of the transfer tube was set as 320 °C with a vaporizer temperature of 300 °C. Sheath, aux gas, and sheath gas flow rates were set at 40, 10, and 1 Arb, respectively. Data-dependent acquisitions (DDA) were performed using the following parameters: full scan range was 70–1050 TM software 4.2 were imported into Compound DiscovererTM 3.2 software where the “Untargeted Metabolomics with Statistics Detect Unknowns with ID Using Online Databases and mzLogic” feature was selected , a HMDB identification code was assigned to each selected metabolite. A joint pathway analysis was performed by integrating the genes relative to each cell line . For cell viability and immunofluorescence quantification data, the Shapiro normality test was performed . Peak areas were log10 transformed and p values were calculated for the sample group by analysis of variance (ANOVA) test. A p value < 0.05 and fold-change of 1.5 was deemed to be statistically significant.All data are presented as mean ± standard deviation  and a non-triple-negative (MCF-7) cell line.50) of 10 µM and 14 µM, respectively. However, in the case of HCC1937 cells, a higher concentration of olaparib was required to achieve the same reduction in cell viability (150 µM), indicating a lower efficacy of response to olaparib in this cell line.Our results show that exposure to olaparib caused a reduction in cell viability in all cell lines in a dose-dependent manner . We obse10, IC25, and IC50—determined from MTS assays) promotes the accumulation of DNA double strand breaks (DSBs) in MCF-7, MDA-MB-231, and HCC1937 cell lines. Key markers for DNA DSB formation include phosphorylated histone H2 variant H2AX (γH2AX) [PARP inhibition induced by olaparib exposure results in the accumulation of DNA damage in cells by compromising their DDR mechanisms. Therefore, we next investigated the extent to which olaparib exposure at various doses (IC (γH2AX) . To measBased on our results, p53BP1 and γH2AX foci levels increased in a dose-dependent manner in both MCF7 and MDA-MB-231 cells in response to ascending doses of olaparib a,b,d,e. To comprehensively measure the extent of variation induced by olaparib exposure in MCF-7, MDA-MB-231, and HCC1937 cell lines, we profiled their metabolome using an in-house untargeted liquid chromatography-mass spectrometry-based metabolomics pipeline . After d50 olaparib treatment dose across all cell lines , suggesting a strong reliance of breast cancer cell metabolism on amino acids under baseline conditions (control samples). Upon olaparib exposure, the same pathways (amino acid metabolism) were among the most significantly altered across all cell lines, while fatty acid (butanoate metabolism) and vitamin B6 metabolism were only significantly perturbed in MCF-7 cells.Next, we explored individual metabolites that were associated with significantly altered metabolic pathways in response to olaparib exposure and evaluated relative changes in their levels between control and treatment samples. These results are presented through a heatmap clustering analysis . A correMultiple amino acids  were found to be depleted following olaparib exposure (relative to control) in all cell lines examined. Arginine and proline metabolism were significantly depleted by olaparib exposure, with depletion of their derived polyamines detected in all cell lines examined. Conversely, catabolic products of arginine and proline metabolism  were enriched. Elevated levels of serine were observed in MCF7 and MDA-MB-231 cells, while depletion of serine levels was seen in HCC1937 cells.Alpha-ketoglutarate (α-KG-glutamine-derived intermediate of the TCA cycle) was enriched in MCF7 and depleted in MDA-MB-231 and HCC1937 cells. A negative correlation was observed between α-KG and glutamine levels, and a positive correlation between α-KG, and citric and fumaric acid (TCA cycle intermediates). Aspartate (a TCA cycle product) accumulated in the KRAS-mutant MDA-MB-231 cells, while aspartate depletion was observed in MCF7 and HCC1937 cells. Glucose levels were significantly elevated relative to control samples in HCC1937 cells. Asparagine (a byproduct of aspartate) was absent in MDA-MB-231 cells, while its enrichment was detected in MCF7 and HCC1937 cells. In parallel, accumulation of AMP was observed in both MCF7 and HCC1937 cell lines, while it was absent in MDA-MB-231 cells, and enrichment of PPi was detected in all cell lines examined following olaparib exposure.In the case of lipid metabolism, we observed a global depletion of phosphocholines (PC) and phosphoethanolamines (PE) in all cell lines following olaparib treatment. Acylcarnitine levels varied across the cell lines, with an overall enrichment of long (C14–C21) and very-long chain acylcarnitines (>C22) in all cell lines treated with olaparib. Moreover, we observed enriched alpha-linoleic acid (a polyunsaturated fatty acid-PUFA) levels in MCF7 and MDA-MB-231 cells, which was absent in HCC1937 cells.Compared to non-treated cells, elevated levels of glucose were detected in all cell lines studied following olaparib treatment, while downregulation of most nucleobases was observed. Finally, NAD+ downregulation was detected in all cell lines treated with olaparib.An overview of the metabolic features altered in response to olaparib exposure is given in PARP inhibitors have shown promising results in the treatment of metastatic breast cancers harboring germline BRCA1/2 mutations ,23. Rece50—150 µM). These findings are in agreement with previous reports of HCC1937 resistance to PARP inhibition, where the identification of predictive biomarkers of response to PARP inhibitor treatments was recommended beyond BRCA1/2 status [In this study we analyzed the sensitivity of two triple-negative (MDA-MB-231 and HCC1937) and MCF-7  cell lines to olaparib PARP inhibition (PARP1/2). The rationale for selecting these cell lines was to explore how their different genetic profiles see  define t2 status .Our analysis of γH2AX and p53BP1 DNA DSB immunolabelled foci  showed aGenetic biomarkers are routinely used in the clinical stratification of breast cancers and predicting treatment-emergent resistance . While gOur goal in the present study was to apply combined analysis of DNA damage foci formation with global untargeted mass-spectrometry based metabolomics to map the metabolic changes occurring following exposure to olaparib. We examined the baseline differences in cellular metabolism across the cell line panel and extended this evaluation to examine cell-line-dependent response to olaparib treatment. Under baseline cell culture conditions, we found overlapping metabolic features  occurring across all three breast cancer cell lines studies, and metabolic signatures that were unique to specific cell lines  .10 treatment concentration, accompanied by a decrease in NAD+ levels at ascending concentrations of olaparib. Reduced levels of NAD+ were also detected in the MDA-MB-231 and HCC1937 cells at all treatment concentrations. Recent studies have shown that in TNBC cells, olaparib enhances the signaling pathways of other NAD+-dependent deacetylase  [Our analysis of metabolites significantly altered in response to olaparib treatment correlate with reports from Bhute et al., where metabolic markers of PARP inhibition were reported as changes in amino acid metabolism , downregulation of osmolyte levels , phosphocreatine, lactate, and pyruvate in MCF7 cells . We repoirtuins) ,30. ThesGlutamine, a precursor for protein, nucleotide, and lipid biosynthesis, is a fundamental amino acid in breast cancer cell metabolism, playing a pivotal role in providing anaplerotic intermediates for the tricarboxylic acid (TCA) cycle . PreviouGlutamine is also a source of nitrogen groups for the synthesis of nucleobases and nucleotides, either directly or through a process involving the transamination of glutamate and the TCA cycle-derived oxaloacetate that generates aspartate ,39,40. OAmong the pathways of aspartate utilization, asparagine is converted through the enzyme asparagine synthetase (ASNS). The reaction requires glutamine as a substrate and consumption of adenosine triphosphate (ATP) to produce adenosine monophosphate (AMP) and pyrophosphate (PPi). Physiological levels of asparagine occur at levels of <0.05 mM in human plasma . Cancer Beyond asparagine synthesis, aspartate amidation through ASNS presents a source of amino building blocks for the synthesis of arginine in the urea cycle, which is in turn responsible for the synthesis of polyamines catalyzed by ornithine decarboxylase (ODC). Polyamine accumulation has previously been correlated with the increased proliferation of both hormone-dependent and independent breast cancer cells , and wasGlutathione (GSH) is involved in the protection against ROS and regulation of intracellular redox homeostasis. Elevated GSH levels have previously been reported in TNBC compared to luminal breast cancers, suggesting the relevance of GSH to our observations of lower sensitivity to olaparib in TNBC cell lines ,52.Lipids mediate various cellular biological functions, including energy storage, cell membrane structural composition and signal transduction, the increased biosynthesis of which is a marker of metabolic rewiring observed in malignant breast cancers ,54. Our Future targeted metabolomics studies using additional TNBC cell lines and clinical tumor clinical specimens are required to validate our observations. Validation of our findings could define prognostic biomarkers that will aid evaluation of patient prognosis in the clinical setting and enable the implementation of precision medicine in the management of breast cancer.Our data show differential sensitivity of breast cancer cell lines to olaparib treatment that was dose-dependent and demonstrated the increased sensitivity of TNBC cells to DNA damage foci accumulation. The application of metabolomics to the study of breast cancer remains in its infancy, with only a handful of studies reporting combined metabolomics and phenotypic analyses. Data acquired from metabolomics analysis can be validated against routine molecular biology and phenotypic assays, providing a powerful platform for biomarker detection or the discovery of novel actionable pathways for drug development.Our results show that fingerprinting the metabolic profile of cells can be a powerful tool for uncovering potential oncometabolites or mechanisms giving rise to chemoresistance. Findings from such studies may provide potential additional actionable targets for modulating response to drug treatment or the design of new drug combinations that will overall enhance DNA damage efficacy, ultimately improving patient response to radiotherapy and adjuvant chemotherapy.