PARP inhibitor and PRMT5 inhibitor synergy is independent of BRCA1/2 and MTAP status in breast cancer cells

PARP inhibitor and PRMT5 inhibitor synergy is independent of BRCA1/2 and MTAP status in breast cancer cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article PARP inhibitor and PRMT5 inhibitor synergy is independent of BRCA1/2 and MTAP status in breast cancer cells Samyuktha Suresh, Lisa McPherson, James M. Ford This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6959243/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Purpose : PARP inhibitors have been approved for treating a subset of breast cancer patients harboring BRCA1/2 mutations. However, TNBC patients with wildtype BRCA1/2 have limited targeted therapeutic options. Dysregulation of protein arginine methyltransferase 5 (PRMT5) has been implicated in the progression of various cancers, including breast cancer. This study investigates the effects of two classes of PRMT5 inhibitors, GSK3326595 and TNG908, on breast cancer cell lines with different BRCA1/2 statuses to evaluate their therapeutic potential and synergy with PARP inhibitors. Methods : A panel of seven breast cancer cell lines was treated with PRMT5 and PARP inhibitors, followed by cell viability measurements using an MTT assay. Drug interactions were analyzed using the Loewe method on the Combenefit software. Additionally, RT-qPCR was conducted to measure the expression of known DNA damage response genes Results : Synergy was observed in all cell lines, independent of the BRCA1/2 and/or MTAP status. Mechanistically, the PRMT5 inhibition did not alter the gene expression of known DNA damage response genes as measured by RT-qPCR. Notably, short-term PRMT5 inhibition was sufficient to sensitize cells to subsequent PARP inhibition. Conclusions : These findings highlight the potential of combining PRMT5 inhibitors with PARP inhibitors in a wide range of cancers beyond BRCA1/2 and MTAP mutants. Further investigation is warranted to elucidate the underlying mechanisms of sensitization and the timing of cellular responses to PRMT5 inhibition. Biological sciences/Cancer/Breast cancer Health sciences/Oncology/Cancer/Cancer therapy/Targeted therapies Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Poly (ADP-ribose) polymerase (PARP) is an enzyme involved in DNA repair, particularly in the detection and repair of single-strand breaks ( 1 ). PARP inhibitors block these enzymes and are approved for cancer therapy, especially in tumors with homologous recombination (HR) repair defects, such as BRCA1 and BRCA2 mutations ( 23 ). By inhibiting PARP, these drugs induce synthetic lethality in HR-deficient tumors like BRCA1/2 mutant breast cancers. However, PARP inhibitors are limited to patients with BRCA1/2 alterations, which represent around 5–10% of all breast cancer cases ( 2 ). ER/PR/Her2-negative, triple-negative breast cancer (TNBC), an aggressive subtype with the worst prognosis, represents around 15–20% of all breast cancers ( 3 ). Around 20% of TNBC patients harbor a BRCA1/2 mutation and can benefit from targeted therapy using PARP inhibitors, while the remaining 80% rely on standard-of-care chemotherapy and/or surgery ( 1 , 2 , 4 ). Thus, there is an urgent clinical need to identify alternative targeted treatment options for TNBC patients. Protein arginine methyltransferase 5 (PRMT5 is a promising therapeutic target for various cancers, particularly breast cancer, due to its overexpression ( 5 – 10 ). High PRMT5 levels correlate with poor prognosis in breast cancer ( 8 ), including TNBC ( 11 , 12 ). Reducing PRMT5 or inhibiting its activity decreases cell proliferation and tumor growth, and induces apoptosis ( 7 , 8 , 11 ). Several small-molecule inhibitors targeting PRMT5 activity have emerged in the past five years ( 13 ). Most operate as S-Adenosyl-Methionine (SAM)-competitive/uncompetitive or substrate-competitive inhibitors that don't require specific genetic changes for effectiveness. Recently, a new class of PRMT5 inhibitors, called MTA-cooperative, exploits the vulnerability of cells lacking MTAP (methylthioadenosine phosphorylase), making them more prone to PRMT5 inhibition ( 14 ). MTAP deletion occurs in 10–15% of all cancers ( 15 ), and estimated to be less than 5% in breast cancer ( 16 ). These inhibitors have shown promise in early-stage clinical trials for MTAP -deficient tumors ( 14 ), including melanoma, gallbladder adenocarcinoma, and mesothelioma. PRMT5 regulates various cellular functions such as transcriptional regulation, pre-mRNA splicing, and DNA damage responses ( 5 ). PRMT5 promotes HR-mediated DNA repair by directly methylating RUVBL1, a coactivator of the histone acetyltransferase TIP60 ( 17 ) or by controlling the alternative splicing of TIP60 ( 18 ). PRMT5 also regulates DNA repair via non-homologous end-joining (NHEJ) by methylating and stabilizing 53BP1, a key player in the NHEJ pathway ( 19 ). Consequently, PRMT5 inhibition increases DNA damage and triggers genomic instability in cancer cells ( 11 , 18 , 20 – 22 ). Thus, targeting PRMT5 may sensitize cancer cells to DNA-repair modulating agents like PARP inhibitors. In this study, we evaluated this hypothesis and tested the efficacy of combining two potent PRMT5 inhibitors, GSK3326595 (SAM-uncompetitive and substrate-competitive), and TNG908 (MTA-cooperative) with an FDA-approved PARP inhibitor, Talazoparib, in a panel of BRCA1/2 and/or MTAP wildtype and mutant/null breast cancer cell lines. We demonstrate synergy between these drugs in all cell lines tested and show that the synergy is independent of the BRCA1/2 and/or the MTAP status of these cell lines. Further, we show that PRMT5 inhibition does not alter the mRNA expression of key genes involved in HR and NHEJ mediated DNA repair mechanisms. Altogether, this work demonstrates the clinical potential of expanding the PARP-PRMT5 combination to cancers beyond BRCA1/2 mutation and/or MTAP deficiency. Materials and Methods Cell lines Human breast cancer cell lines (HCC1806; RRID:CVCL_1258, MDA-MB-468; RRID:CVCL_0419, MDA-MB-436; RRID:CVCL_0623, HCC1428; RRID:CVCL_1252, MDA-MB-231; RRID:CVCL_0062, and MCF7; RRID:CVCL_0031) were purchased from American Type Culture Collection (ATCC). SUM149PT (RRID:CVCL_3422) was purchased from Bio-IVT and MX-1 cells were purchased from Cell line Services. Ovarian cancer cell lines (UWB1.289; RRID:CVCL_B079 and UWB1.289+BRCA1; RRID:CVCL_B078) were a kind gift from Dr. Karlene Cimprich, Stanford University. All cells were routinely tested for mycoplasma infection using the MycoAlert Mycoplasma Detection Kit (Lonza Biosciences) and cultured in the recommended medium in a 37°C humidified incubator with 5% carbon dioxide. Drugs and Antibodies GSK3326595 and TNG908 were purchased from MedChem Express and Talazoparib was purchased from ApexBio. All primary and secondary antibodies used in this study can be found in Supplementary Table 1. MTT Assays Cells were plated in 96-well plates at 750 – 6400 cells per well to achieve 60% confluency 48 hours later. They were treated in triplicates with varying doses of GSK3326595 (0 – 100 µM), TNG908 (0 – 10 µM), and Talazoparib (0 – 50 µM) or equivalent amounts of DMSO as a vehicle control for 5 days (SUM149PT and MDA-MB-231) or 7 days (all other cell lines), chosen to reflect the time required for 4 cell divisions. Additionally, we replicated several MTT conditions using 10 – 15 day clonogenic assays to confirm our conclusions. After treatment, MTT was added to the plates and incubated for 3 hours, and the formazan crystals were solubilized by adding DMSO for 1 hour. Absorbance was measured at 570 nm using a plate reader, and the data was analyzed to determine IC 50 values and their corresponding 95% confidence intervals for each drug using non-linear regression curves on GraphPad Prism from three independent biological replicates per drug per cell line. For drug combinations, the maximum dose for each drug was chosen as approximately twice the IC 50 value and serially diluted two- or four-fold. Viability was measured using MTT as described above. Drug interactions were analyzed using the Loewe model in Combenefit Software to determine interaction types. The SYN_MAX metric was used to compare drug synergy strength between cell lines. For the sequential drug treatment in UWB1.289 and UWB1.289+BRCA1 cell lines, cells were plated in a 96-well plate at 2000 cells/well for both cell lines. After 24 hours, cells were treated with 4 µM (2 x IC 50 dose for UWB1.289 cells from a 7-day assay) of GSK3326595 or equivalent amounts of DMSO for 4, 8, or 24 hours. After each time point, GSK3326595 was removed and fresh media with talazoparib (0 – 50 µM for UWB1.289+BRCA1 and 0 – 50 nM for UWB1.289) or DMSO was added for 7 days. Viability was measured, and IC 50 was calculated as described. Western Blotting Cells were plated in 6-well plates at 2 x 10 5 cells/well for drug treatments and lysed on ice for 30 mintues using RIPA lysis buffer. Extracted proteins were quantified using the Pierce BCA kit (Thermo Fisher). 15-20 µg of protein was resolved by SDS-PAGE, and symmetric dimethylarginine was detected using a 1:1000 anti-SDMA antibody (RRID: AB_2714013), PRMT5 with a 1:1000 anti-PRMT5 antibody (RRID: AB_2904201), and MTAP with a 1:1000 anti-MTAP antibody (RRID: AB_2147094). Equal protein loading was confirmed by detecting β-actin using a 1:5000 anti- β-actin antibody (RRID: AB_2687938). Three independent biological replicates were performed for each western blot. Immunofluorescence UWB1.289 and UWB1.289+BRCA1 cells were plated in 24-well plates on coverslips at 2 x 10 5 cells/well and treated with 4 mM GSK3326595 for 4, 8, or 24 hours. After treatment, cells were fixed with 4% paraformaldehyde for 15 minutes and permeabilized with 0.1% triton-X-100 in Phosphate-buffered saline (PBS) for 5 minutes. Following a PBS wash, cells were blocked for 30 minutes with 1% bovine serum albumin (BSA) in PBS with 0.1% Tween detergent (PBST). Coverslips were incubated overnight at 4°C with anti-gH2AX (1:1000 dilution; RRID: AB_2118009), anti-RAD51 (1:1000 dilution; RRID: AB_10002131), or anti-53BP1 (1:200 dilution; AB_1290520) antibodies. After washing thrice with 1% BSA in PBST, coverslips were incubated with Goat anti-rabbit Alexa Fluor 488 (RRID: AB_2534069) or Goat anti-mouse Alexa Fluor 594 (RRID: AB_2534079) secondary antibodies for 1 hour at room temperature. Excess antibodies were washed thrice with 1% BSA in PBST and then incubated with 1 mg/ml DAPI for 10 minutes. After a PBS wash, coverslips were mounted on slides using Prolong Gold Antifade mounting medium. Imaging was done at 40X magnification on a Leica Fluorescent microscope (Leica DMI6000; RRID:SCR_018713). RNA isolation, cDNA synthesis and Quantitative PCR (qPCR) MDA-MB-468 and HCC1806 cells were plated in 6-well plates at 3 x 10 5 cells/well and treated with 40 nM or 5 mM of GSK3326595 or equivalent DMSO for 24 hours. Total RNA was extracted using Qiagen’s’ RNeasy extraction kit following manufacturer’s protocol. cDNA was synthesized from 1.5 mg of RNA using Qiagen’s Quantitect Reverse transcription kit following manufacturer’s protocol. 0.075 mg of cDNA was used in triplicate for qPCR using Maxima SYBR green on ABI’s QuantStudio 12K Flex Real-Time PCR System. Gene expression was measured using the comparative CT method with values normalized to GAPDH and DMSO control. Data presented are from three independent experiments. Primers used in this study can be found in Supplemental Table 1. Results Breast cancer cell lines with differing BRCA1/2 status demonstrate differential sensitivity to PRMT5 inhibition We treated a panel of seven breast cancer cell lines with different BRCA1/2 status (wildtype or mutant) (Table 1) with the PRMT5 inhibitor, GSK3326595, at doses ranging from 0 – 50 µM for five (MDAMB231 and SUM149PT) or seven (HCC1806, MDAMB468, MDAMB436, MX-1, HCC1428) days and measured cell viability using an MTT assay. We observed that there was a difference in sensitivity to PRMT5 inhibition ( Figure 1A ) with the BRCA1/2 wildtype cell lines HCC1806, MDAMB468, and MDAMB231 being the most sensitive with IC 50 values less than 2 µM. MDAMB436 ( BRCA1 mutant) and MX-1 ( BRCA1/2 mutant) lines were less sensitive to PRMT5 inhibition with IC50 values between 12.6 µM and 16.2 µM, respectively, while SUM149PT ( BRCA1 mutant) and HCC1428 ( BRCA2 mutant) were resistant to PRMT5 inhibition with IC 50 values greater than 50 µM. This difference in sensitivity to PRMT5 inhibition was not related to the protein expression level of PRMT5 in these cell lines ( Supplemental Figure 1 ). Next, we sought to test the efficacy of GSK3326595 on PRMT5 enzyme inhibition in our panel of seven cell lines treated with a low concentration (40 nM) and a high concentration (5 µM) of GSK3326595 for five (MDAMB231 and SUM149PT) or seven (HCC1806, MDAMB468, MDAMB436, MX-1, HCC1428) days. We measured the global protein levels of symmetric dimethylarginine (SDMA) in whole cell protein lysates using western blotting ( Figure 1B ) and observed that SDMA levels were reduced in all the cell lines starting at the low concentration of 40 nM. A difference of 21-fold was noted between the dose required to inhibit PRMT5 enzyme activity and the dose required to induce cytotoxicity for the most sensitive BRCA1/2 wildtype cell line (HCC1806), while a difference of 315 - 1250-fold was noted for the BRCA1/2 mutant lines. Overall, this indicated that though the cell lines showed differential sensitivity to PRMT5 inhibition, the most sensitive cell lines were BRCA1/2 wildtype. Table 1: Half-maximal inhibitory concentrations (IC 50 ) for PRMT5i (GSK3326595, TNG908) and PARPi (Talazoparib), BRCA1/2 status, MTAP status, and TNBC subtype in cell lines used in this study . Cell line GSK3326595 IC 50 (in µM) CI for IC50 (in µM) Talazoparib IC 50 (in nM) CI for IC50 (in nM) TNG908 IC 50 (in µM) CI for IC50 (in µM) BRCA1/2 status MTAP Status TNBC subtype HCC1806 0.84 0.48-1.68 28.02 11.32-57.51 8.65 6.16-13.8 BRCA1/2 WT MTAP WT BL2 MDA-MB-468 1.12 0.68-2.12 11.84 9.53-14.63 3.15 2.27-4.63 BRCA1/2 WT MTAP WT BL1 MDA-MB-231 1.75 1.07-3.04 112.6 65.39-179.8 1.83 1.24-2.90 BRCA1/2 WT MTAP null MSL MDA-MB-436 12.6 6.55-35.8 0.39 0.31-0.49 >10 10.67-31.74 BRCA1 mut MTAP WT MSL Mx-1 16.2 5.31-47.52 34.63 18.58-64.40 ND ND BRCA1/2 mut MTAP WT Unknown SUM149PT >50 NA 4.49 3.76-5.39 9 6.11-15.27 BRCA1 mut MTAP null BL2 HCC1428 >50 NA >50 NA ND ND BRCA2 mut MTAP WT ER+ BC cancer line UWB1.289 2.38 1.69-3.65 35.37 22.25-55.46 ND ND BRCA1 mut MTAP WT Ovarian UWB1.289+BRCA1 18.53 11.55-35.25 2393 1575-3642 ND ND BRCA1 complemented MTAP WT Ovarian Abbreviations: CI – 95% confidence intervals, BRCA1/2 WT – BRCA1 and 2 wildtype, BRCA1 mut – BRCA1 mutant, BRCA2 mut – BRCA2 mutant, MTAP WT - MTAP wildtype, BL2 – Basal-like 2, BL1 – Basal-like 1, MSL – Mesenchymal stem-like, ER+ BC - Estrogen receptor positive breast cancer, NA – not applicable, ND – not determined. PRMT5 inhibition synergizes with PARP inhibition in breast cancer cell lines Since the most sensitive cell lines to PRMT5 inhibition in our panel were BRCA1 wildtype, we sought to evaluate whether combining the PRMT5 inhibitor (GSK3326595) with a potent PARP inhibitor (Talazoparib) would be cytotoxic. First, we determined the IC 50 values of Talazoparib in our panel of cell lines by treating the cells with a range of 0 – 50 µM for five (MDAMB231 and SUM149PT) or seven (HCC1806, MDAMB468, MDAMB436, MX-1, HCC1428) days and measured cell viability using an MTT assay. As expected, the BRCA1 mutant cell lines, MDAMB436 and SUM149PT were the most sensitive to PARP inhibition with IC 50 of 0.39 and 4.49 nM, respectively ( Table 1 ). BRCA1 /2 wildtype (HCC1806, MDAMB468 and MDAMB231) and BRCA1/2 mutant (MX-1) cell lines were less sensitive to PARP inhibition (IC 50 between 11 – 34 nM) while the BRCA2 mutant cell line (HCC1428) was resistant to PARP inhibition with an IC 50 greater than 50 µM ( Table 1 ). Next, to evaluate the efficacy of combining GSK3326595 and Talazoparib, we treated our panel of seven cell lines with a maximum dose of approximately twice the IC50 dose for each drug and serially diluted it two-fold (MDAMB468, HCC1806, MDAMB436, SUM149PT, HCC1428) or four-fold (MDAMB231, MX-1). At the end of the five (MDAMB231, SUM149PT) or seven (MDAMB468, HCC1806, MDAMB436, HCC1428, MX-1) day treatment, cell viability was measured using an MTT assay and synergy was calculated using Combenefit software. We observed significant synergy between GSK3326595 and Talazoparib in all the cell lines tested, independent of the BRCA status of the cell lines ( Figure 2A ). Interestingly, when we used the SYN_MAX metric from the Combenefit Software to compare the strength of maximum synergy between the cell lines, we observed that the highest synergy scores were obtained for the BRCA1/2 wildtype cell lines, MDAMB468 and MDAMB231, while the lowest synergy scores were obtained for the BRCA1 mutant SUM149PT and MDAMB436 cell lines ( Figure 2B ). Since the BRCA1 wildtype cell lines were also the most sensitive to PRMT5 inhibition alone, we wanted to determine if that could be an indicator for synergy with PARP inhibitors. To determine this, we performed a Pearson correlation between the maximum synergy score for each cell line and their respective IC 50 values for GSK3326595 ( Figure 2C ). The Pearson correlation coefficient was negative (r = -0.5666) but was not statistically significant (p-value = 0.18), suggesting that there is a trend indicating higher PRMT5 sensitivity correlates with obtaining a higher synergy with PARP inhibitors. To understand mechanistically whether PRMT5 inhibition was altering expression of genes involved in DNA damage response, we examined the gene expression of several known genes involved in homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways upon PRMT5 inhibition in the two most sensitive BRCA1 /2 wildtype cell lines, HCC1806 and MDAMB468. Levels of ATM, BRCA1 , and BRCA2 mRNA were not consistently altered across cell lines (Supplementary Figure 2A). The only HR gene that was consistently upregulated in a dose-dependent manner and in both cell lines after PRMT5 inhibition was RAD50 ( Supplementary Figure 2A ). However, we did not observe a statistically significant increase in RAD50 protein level ( Supplementary Figure 2B, C ). These results suggested that the higher synergies observed between PRMT5i and PARPi in BRCA1/2 wildtype cells were not mediated through a consistent dysregulation of any of the known genes in the HR and NHEJ pathways. Pre-treating BRCA1 mutant and BRCA1 wildtype cell lines with PRMT5i sensitize them to PARP inhibition In our panel of breast cancer cell lines, we observed that there was a difference in sensitivity to PRMT5 inhibition based on BRCA1/2 status ( Figure 1A , Table 1 ). To determine whether BRCA1 expression was responsible for this difference in sensitivity, we tested a pair of isogenic ovarian cancer cell lines, UWB1.289 (BRCA1 mutant) and UWB1.289+BR1 (BRCA1 complemented). Upon treating these cells with a range of 0 – 50 µM GSK3326595 for seven days, we observed that the parental cell line, UWB1.289, was more sensitive to PRMT5 inhibition with an IC50 of 2.38 µM compared to the BRCA1 complemented cell line, whose IC50 was 18.53 µM ( Figure 3A ). Additionally, we treated both cell lines with two different doses of GSK3326595 (40nM and 5uM) and observed that the SDMA mark, indicative of PRMT5 enzyme activity, was decreased at the low concentration of 40nM in both cell lines ( Figure 3B ), similar to the panel of breast cancer cell lines. Next, we evaluated the effect of combining PRMT5i with PARPi in this pair of isogenic cell lines, to determine whether BRCA1 expression was contributing to the synergy between these two inhibitors. We treated both cell lines with a maximum drug dose of approximately twice the IC50 dose for each drug and serially diluted it two-fold. At the end of a seven-day treatment, we measured cell viability using an MTT assay and calculated drug synergy using the Combenefit software. Combining PRMT5i and PARPi in these cell lines demonstrated synergy ( Figure 3C ) in both cell lines, similar to our observation in the panel of breast cancer cell lines ( Figure 2A ). However, when we used the SYN_MAX metric to compare the strength of synergy between the UWB1.289 and UWB1.289+BRCA1 cell line, we observed that UWB1.289 had a higher maximum synergy score compared to UWB1.289+BRCA1 ( Figure 3D ). This is in contrast to our panel of breast cancer cell lines, where the highest synergies were observed in BRCA1 WT cell lines ( Figure 2A ). In order to determine whether sequential treatment of PRMT5i and PARPi would be more efficacious than combination treatment, we pre-treated the cells with 4 µM of GSK3326595 (equivalent to 2 X IC50 dose in UWB1.289) for 4 hours, 8 hours, and 24 hours and subsequently treated with Talazoparib for seven days. We observed that the IC 50 of PARPi was decreased by two-fold in both UWB1.289 and UWB1.289+BRCA1 cells when pre-treated with GSK3326595 ( Figure 3E ). This indicated that inhibiting PRMT5 first was sufficient to sensitize the cells to subsequent PARP inhibition, independent of their BRCA1/2 status. The similar antiproliferative and synergistic effects seen for short (4 hr) non-overlapping exposure as that after 7 days of combined exposure suggests that a non-canonical mechanism may be involved in the early stages, which could ultimately lead to the changes in DDR gene expression observed at later time points. In order to determine which DNA damage repair pathway was being regulated by GSK3326595 treatment to sensitize these cells to PARP inhibition, we analyzed γ-H2AX, RAD51 and 53BP1 foci as markers for DNA double strand breaks, HR-mediated repair, and NHEJ-mediated repair, respectively. We observed that there was no significant increase in γ-H2AX foci after PRMT5 inhibition in either UWB1.289 or UWB1.289+BRCA1 at any of the time points tested ( Supplementary Figure 3A, B and Supplementary Figure 4, 5 ). Additionally, we did not observe any significant changes in RAD51 ( Supplementary Figure 3C, D and Supplementary Figure 4, 5 ) or 53BP1 foci ( Supplementary Figure 3E, F and Supplementary Figure 4, 5 ), except a modest decrease in RAD51 foci after 4h treatment in UWB1.289 cells ( Supplementary Figure 3C ) and an increase in 53BP1 after 24h treatment in UWB1.289+BRCA1 ( Supplementary Figure 3F ). Together, these results suggested that although short-term inhibition of PRMT5 was sufficient to sensitize both UWB1.289 and UWB1.289+BRCA1 to PARP inhibition, mechanistically, this was not mediated by inducing DNA damage as measured with known markers at these early time points. MTA-cooperative PRMT5 inhibitor synergizes with PARPi in both MTAP null and MTAP WT cell lines Recent evidence suggests that cancers with MTAP deficiency are vulnerable to PRMT5 inhibition, leading to the development of a new class of PRMT5 inhibitors that are MTA-cooperative and are in clinical trials for MTAP -deleted cancers. Therefore, we sought to evaluate the effect of a potent MTA-cooperative PRMT5 inhibitor, TNG908 (15) in combination with PARP inhibitors in MTAP WT and MTAP null breast cancer cell lines. We first evaluated the sensitivity of our panel of breast cancer cell lines by treating them with a range of 0 – 10 µM of TNG908. As expected, MDA-MB-231, an MTAP null cell line ( Supplementary Figure 1 ), was the most sensitive to TNG908 with an IC 50 of 1.83 µM and MDA-MB-436, an MTAP WT cell line ( Supplementary Figure 1 ), was the least sensitive with an IC 50 > 10 µM ( Figure 4A ). Surprisingly, SUM149PT cells were not as sensitive (IC50 = 9 µM) to TNG908 despite being an MTAP null cell line ( Supplementary Figure 1 ). Then, we examined the efficacy of TNG908 to inhibit PRMT5 activity in all cell lines by measuring global SDMA levels and observed that both 40 nM and 5 µM treatments decreased SDMA levels in all cell lines ( Figure 4B ). When we combined TNG908 with Talazoparib and evaluated the efficacy of this combination, we saw that similar to the GSK-Talazoparib combination, TNG908 also synergized with Talazoparib in all cell lines ( Figure 4C ) and the maximum synergy was observed in the MDA-MB-468 and MDA-MB-231 cell lines ( Figure 4D ). To determine whether sensitivity to TNG908 could be an indicator of synergy with PARP inhibition, we performed a Pearson Correlation using the maximum synergy score (SYN_MAX metric) for each cell line with their respective IC 50 values of TNG908. Similar to the correlation between IC 50 values of GSK3326595 and the maximum synergy scores ( Figure 2C ), we obtained a negative correlation coefficient of r = -0.8990, that was also statistically significant (p-value < 0.05, Figure 3E ). This suggested that cell lines that were more sensitive to PRMT5 inhibition using TNG908 were indicative of obtaining high synergy with PARP inhibitors. Together, these results demonstrated that though MTAP null cells are more sensitive to PRMT5 inhibition using the MTA-cooperative inhibitor, the synergy between PRMT5i and PARPi does not depend on the MTAP status of the cells. Discussion Exploiting specific genetic vulnerabilities in cancer cells has led to the development and clinical use of potent therapies in cancer research. PARP inhibitors were the first drugs designed to exploit the vulnerabilities of tumors with BRCA1/2 mutations ( 23 ) and are approved for breast, ovarian, prostate, and pancreatic cancer treatment. Another cancer vulnerability being targeted is PRMT5 inhibition in MTAP -deleted tumors ( 24 ). MTAP deletion has variable frequency in different cancers and is less than 5% in breast cancer ( 16 ), but a higher incidence of TNBC is observed in tumors with MTAP loss ( 16 ). In this study, we combined an FDA-approved PARP inhibitor with two classes of PRMT5 inhibitors and demonstrated synergy in breast cancer cell lines, independent of their BRCA1/2 or MTAP status. While recent reports showed synergy between PARP and PRMT5 inhibitors, these were limited to BRCA1/2 wild-type breast and ovarian cancer models (20–22, 25) or acute myeloid leukemia models ( 18 ). Our findings suggest that the PARP-PRMT5 combination strategy (i) can serve as a novel targeted treatment for TNBC patients and (ii) can be expanded to a wider range of cancers than those with BRCA1/2 mutations and/or MTAP mutations. Thus, it would be highly clinically relevant to test this combination, in vitro and in vivo , in other cancer models where PARP inhibitors are approved, such as ovarian, prostate, and pancreatic cancer. Additionally, MTA-cooperative PRMT5 inhibitors are currently in clinical trials for MTAP -deleted solid and liquid tumors. Our data indicate that combining the MTA-cooperative PRMT5 inhibitor with PARP inhibitors is also efficient in MTAP wildtype tumors, suggesting this combination should be tested in larger clinical trials for all patients, regardless of MTAP status. The current understanding of the synergy between PARP and PRMT5 inhibitors is attributed to increased DNA damage (20,25), and/or downregulation of genes responsible for DNA damage response (21,22,25) after PRMT5 inhibition. However, we did not observe any significant downregulation of key genes in DNA damage response or an increase in DNA damage upon PRMT5 inhibition in our models. One possible explanation for this apparent discrepancy is in the timing of experiments. We studied the early response to DNA damage and related gene expression by testing 24 hours after PRMT5 inhibition, while previous studies demonstrated a late effect of PRMT5 inhibition on the DNA damage response pathway 4–7 days after treatment (20–22,25). Therefore, it is possible that the effects previously observed on DNA damage were indirectly mediated by PRMT5. Furthermore, we have shown that short-term exposure of cells, as little as 4 hours, to a PRMT5 inhibitor is sufficient to sensitize them to PARP inhibitors, suggesting that early changes are occurring within the cells that are yet to be fully understood. Thus, further studies are needed to identify these key early genes regulated by PRMT5 either through alternative splicing or direct methylation to understand this observed synergy. In conclusion, we have shown that the PARP-PRMT5 drug combination can be a beneficial treatment strategy for a larger cohort of cancers and not limited to a subset with specific genetic mutations. Further mechanistic studies will help broaden our understanding of PRMT5-mediated DNA damage response and help identify predictive biomarkers for this combination. Declarations Conflict of Interest: The authors declare no potential conflicts of interest Funding: This work was supported by grants to JMF from the BRCA Foundation and the Breast Cancer Research Foundation. Author Contribution SS made substantial contributions to the conception and design of the work. SS and LAM contributed to the acquisition, analysis, or interpretation of data and drafted the work or revised it critically for important intellectual content. JMF supervisd the work and contributed to the analysis and writing of the manuscriopt. All authors approved the version to be published. 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Proliferative role of TRAF4 in breast cancer by upregulating PRMT5 nuclear expression. Tumour Biol. 2015 Aug;36(8):5901–11. DOI: 10.1007/s13277-015-3262-0 Rengasamy M, Zhang F, Vashisht A, Song WM, Aguilo F, Sun Y, et al. The PRMT5/WDR77 complex regulates alternative splicing through ZNF326 in breast cancer. Nucleic Acids Res. 2017 Nov 2;45(19):11106–20. doi: 10.1093/nar/gkx727 Vinet M, Suresh S, Maire V, Monchecourt C, Nemati F, Lesage L, et al. Protein arginine methyltransferase 5: A novel therapeutic target for triple-negative breast cancers. Cancer Med. 2019 May;8(5):2414–28. doi: 10.1002/cam4.2114 Wu Y, Wang Z, Zhang J, Ling R. Elevated expression of protein arginine methyltransferase 5 predicts the poor prognosis of breast cancer. Tumour Biol. 2017 Apr;39(4):1010428317695917. Guo C, Wu L, Zheng X, Zhao L, Hou X, Wang Z, et al. Research Progress on Small-molecule Inhibitors of Protein Arginine Methyltransferase 5 (PRMT5) for Treating Cancer. Curr Top Med Chem. 2023;23(21):2048–74. DOI: 10.1177/1010428317695917 Hu M, Chen X. A review of the known MTA-cooperative PRMT5 inhibitors. RSC Adv. 2024 Dec 10;14(53):39653–91. DOI https://doi.org/10.1039/D4RA05497K Briggs KJ, Cottrell KM, Tonini MR, Tsai A, Zhang M, Whittington DA, et al. TNG908 is a brain-penetrant, MTA-cooperative PRMT5 inhibitor developed for the treatment of MTAP-deleted cancers. Transl Oncol. 2025 Jan 3;52:102264. DOI: 10.1016/j.tranon.2024.102264 Bou Zerdan M, Ashok Kumar P, Haroun E, Srivastava N, Ross J, Sivapiragasam A. Genomic landscape of metastatic breast cancer (MBC) patients with methylthioadenosine phosphorylase (MTAP) loss. Oncotarget. 2023 Mar 11;14:178–87. DOI: 10.18632/oncotarget.28376 Clarke TL, Sanchez-Bailon MP, Chiang K, Reynolds JJ, Herrero-Ruiz J, Bandeiras TM, et al. PRMT5-Dependent Methylation of the TIP60 Coactivator RUVBL1 Is a Key Regulator of Homologous Recombination. Mol Cell. 2017 Mar 2;65(5):900-916 e7. DOI: 10.1016/j.molcel.2017.01.019 Hamard PJ, Santiago GE, Liu F, Karl DL, Martinez C, Man N, et al. PRMT5 Regulates DNA Repair by Controlling the Alternative Splicing of Histone-Modifying Enzymes. Cell Rep. 2018 Sep 4;24(10):2643–57. DOI: 10.1016/j.celrep.2018.08.002 Hwang JW, Kim SN, Myung N, Song D, Han G, Bae GU, et al. PRMT5 promotes DNA repair through methylation of 53BP1 and is regulated by Src-mediated phosphorylation. Commun Biol. 2020 Aug 5;3(1):428. DOI: 10.1038/s42003-020-01157-z O’Brien S, Butticello M, Thompson C, Wilson B, Wyce A, Mahajan V, et al. Inhibiting PRMT5 induces DNA damage and increases anti-proliferative activity of Niraparib, a PARP inhibitor, in models of breast and ovarian cancer. BMC Cancer. 2023 Aug 18;23(1):775. DOIhttps://doi.org/10.1186/s12885-023-11260 Carter J, Hulse M, Sivakumar M, Burtell J, Thodima V, Wang M, et al. PRMT5 Inhibitors Regulate DNA Damage Repair Pathways in Cancer Cells and Improve Response to PARP Inhibition and Chemotherapies. Cancer Res Commun. 2023 Nov 6;3(11):2233–43. doi.org/10.1158/2767-9764.CRC-23-0070 Li Y, Dobrolecki LE, Sallas C, Zhang X, Kerr TD, Bisht D, et al. PRMT blockade induces defective DNA replication stress response and synergizes with PARP inhibition. Cell Rep Med. 2023 Dec 19;4(12):101326. DOI: 10.1016/j.xcrm.2023.101326 Lord CJ, Ashworth A. PARP Inhibitors: The First Synthetic Lethal Targeted Therapy. Science. 2017 Mar 17;355(6330):1152–8. doi: 10.1126/science.aam7344 Kryukov GV, Wilson FH, Ruth JR, Paulk J, Tsherniak A, Marlow SE, et al. MTAP deletion confers enhanced dependency on the PRMT5 arginine methyltransferase in cancer cells. Science. 2016 Mar 11;351(6278):1214–8.  DOI: 10.1126/science.aad5214 Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterialRevisedforSciRep.pdf Cite Share Download PDF Status: Published Journal Publication published 21 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 21 Jul, 2025 Reviews received at journal 18 Jul, 2025 Reviews received at journal 10 Jul, 2025 Reviewers agreed at journal 09 Jul, 2025 Reviewers agreed at journal 09 Jul, 2025 Reviewers agreed at journal 09 Jul, 2025 Reviewers invited by journal 09 Jul, 2025 Editor assigned by journal 04 Jul, 2025 Editor invited by journal 01 Jul, 2025 Submission checks completed at journal 26 Jun, 2025 First submitted to journal 26 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6959243","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":483708389,"identity":"e17d90f3-2637-47f6-9e22-d6807e0f29d3","order_by":0,"name":"Samyuktha Suresh","email":"","orcid":"","institution":"Stanford University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Samyuktha","middleName":"","lastName":"Suresh","suffix":""},{"id":483708390,"identity":"0307ece8-3865-41ad-95a6-f728b60fdc73","order_by":1,"name":"Lisa McPherson","email":"","orcid":"","institution":"Stanford University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Lisa","middleName":"","lastName":"McPherson","suffix":""},{"id":483708391,"identity":"f3642b53-7d98-425b-87f4-a4ec9e09e4ef","order_by":2,"name":"James M. Ford","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYBACxgbGhw9ADPYGBgYJBgZmYrQwGxswJDAw8BwgVgtQkZkEaVqYpx1mq/z5wy6Ph7334Q2GCuvEBoIOm53MdpsnIbmYh+e4sQXDmXRitOQfu82QwJy4XyKNTYKx7TBxthT+SKhP7AFr+UekFgaehMNQLQ3EaWGW5kk7ntjDc4zZIuFYujFBLYazkxk//rCpTuxhb2O88aHGWpawFhQVCYSUg4A8MYpGwSgYBaNghAMAKlc6CSMdoS0AAAAASUVORK5CYII=","orcid":"","institution":"Stanford University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"James","middleName":"M.","lastName":"Ford","suffix":""}],"badges":[],"createdAt":"2025-06-23 18:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6959243/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6959243/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-20696-1","type":"published","date":"2025-10-21T16:16:44+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86757918,"identity":"ec989826-55dd-467f-ad5f-0c6b4576644d","added_by":"auto","created_at":"2025-07-15 09:46:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":139242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePRMT5 inhibition is cytotoxic at different doses in a panel of breast cancer cell lines but decreases symmetric demethylation at similar doses. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Average IC\u003csub\u003e50\u003c/sub\u003e values of PRMT5i (GSK3326595) in all cell lines tested. (\u003cstrong\u003eB\u003c/strong\u003e) Cells were treated with 0.04 or 5 µM of GSK3326595 (GSK595) for 5 (SUM149PT and MDAMB231) or 7 days (HCC1806, MDAMB468, MDAMB436, MX-1, and HCC1428) and proteins were harvested using RIPA lysis buffer. Anti-SDMA antibody was used to detect the symmetric dimethylarginine mark and anti-β-actin was used as a loading control. Presented western blot is cropped from multiple blots and is representative of three independent biological replicates. Uncropped blots can be found in Supplementary Figures 6 and 7.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6959243/v1/f813eda1d0e7523c1ab8ab13.png"},{"id":86757920,"identity":"8d8bb54f-dea3-48f6-a10b-b95e664b8402","added_by":"auto","created_at":"2025-07-15 09:46:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":673963,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePRMT5i (GSK3326595) synergizes with PARPi (Talazoparib) in a panel of breast cancer cell lines.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Drug interactions between GSK3326595 and Talazoparib are displayed as a synergy matrix using the Loewe model from the Combenefit Software. Both PRMT5i and PARPi were serially diluted two-fold in MDA-MB-468, HCC1806, HCC1428, SUM149PT and MDAMB436 cells while the drugs were serially diluted four-fold from the maximum concentration in MDAMB231 and MX-1 cells. Matrices are representative of three independent biological replicates performed in technical triplicates. (\u003cstrong\u003eB\u003c/strong\u003e) Maximum synergy metric (SYN_MAX) from the Combenefit software analysis for all cell lines. Bars represent mean ± SD from three independent biological replicates. (\u003cstrong\u003eC\u003c/strong\u003e) Pearson correlation between maximum synergy score (SYN_MAX) and IC\u003csub\u003e50\u003c/sub\u003e values of GSK3326595 in all cell lines. Gray shaded region is the 95% confidence interval of the slope.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6959243/v1/ab827a6a57febfecc848236c.png"},{"id":86757922,"identity":"8fdd6ea3-4b6c-4880-b63d-e02692d7cd68","added_by":"auto","created_at":"2025-07-15 09:46:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":247506,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePRMT5i (GSK595) sensitizes BRCA1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003emut\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e and BRCA1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eWT\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e cells to PARPi treatment. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Average IC\u003csub\u003e50 \u003c/sub\u003evalues of GSK595 in UWB1.289 (BRCA1mut) and UWB1.289+BRCA1 (BRCA1 complemented) cell lines. (\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003ePRMT5i (GSK595) decreases symmetric dimethylarginine (SDMA) mark at similar drug concentrations in both cell lines. Cells were treated with 0.04 or 5 µM of GSK595 for 7 days and proteins were harvested using RIPA lysis buffer. Anti-SDMA antibody was used to detect the symmetric dimethylarginine mark and anti-β actin was used as a loading control. Presented western blot is representative of three independent biological replicates. Uncropped blot can be found in Supplemental Figure 7. (\u003cstrong\u003eC\u003c/strong\u003e) Drug interactions between GSK595 and Talazoparib are displayed as a synergy matrix using the Loewe model from the Combenefit Software. Both PRMT5i and PARPi were serially diluted two-fold from the maximum concentration in both cell lines. Matrices are representative of three independent biological replicates performed in technical triplicates. (\u003cstrong\u003eD\u003c/strong\u003e) Maximum synergy metric (SYN_MAX) from the Combenefit software analysis for both cell lines. Bars represent mean ± SD from three independent biological replicates. (\u003cstrong\u003eE\u003c/strong\u003e) Average IC\u003csub\u003e50\u003c/sub\u003e values of PARPi with or without pre-treatment of GSK595 in UWB1.289 and UWB1.289+BRCA1 after 4h, 8h, or 24h.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6959243/v1/8a60c73ffff770f9d23e3cb0.png"},{"id":86757923,"identity":"a74b2d8b-8f21-4cb8-ae7f-31cf0674eddf","added_by":"auto","created_at":"2025-07-15 09:46:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":299983,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePRMT5i (TN908) synergizes with PARPi (Talazoparib) in a panel of breast cancer cell lines. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Average IC\u003csub\u003e50\u003c/sub\u003e values of PRMT5i (TNG908) in all cell lines tested. (B) PRMT5i (TNG908) decreases symmetric dimethylarginine (SDMA) mark at similar drug concentrations in a panel of breast cancer cell lines. Cells were treated with 0.04 or 5 µM of TNG908 for 5 (SUM149PT and MDAMB231) or 7 days (HCC1806, MDAMB468, MDAMB436) and proteins were harvested using RIPA lysis buffer. Anti-SDMA antibody was used to detect the symmetric dimethylarginine mark and anti-β actin was used as a loading control. Presented western blot is representative of three independent biological replicates. (\u003cstrong\u003eC\u003c/strong\u003e) Drug interactions between TNG908 and Talazoparib are displayed as a synergy matrix using the Loewe model from the Combenefit Software. Both PRMT5i and PARPi were serially diluted four-fold from the maximum concentration in all cell lines. Matrices are representative of three independent biological replicates performed in technical triplicates. (\u003cstrong\u003eD\u003c/strong\u003e) Maximum synergy metric (SYN_MAX) from the Combenefit software analysis for all cell lines. Bars represent mean ± SD from three independent biological replicates. (\u003cstrong\u003eE\u003c/strong\u003e) Pearson correlation between maximum synergy score (SYN_MAX) and IC50 values of TNG908 in all cell lines. Gray shaded region is the 95% confidence interval of the slope.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6959243/v1/fd9bc6fbd0002af67fe24821.png"},{"id":94490003,"identity":"c6fc3339-6e2e-4011-9771-392d2f83df85","added_by":"auto","created_at":"2025-10-27 17:06:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2585156,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6959243/v1/e4658de2-3bba-4c58-8ab0-1d9be617f049.pdf"},{"id":86759399,"identity":"448bffb1-f5db-4340-addf-7daec4a7fbc7","added_by":"auto","created_at":"2025-07-15 10:02:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1073012,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialRevisedforSciRep.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6959243/v1/819474f270c437767fcd20ca.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"PARP inhibitor and PRMT5 inhibitor synergy is independent of BRCA1/2 and MTAP status in breast cancer cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePoly (ADP-ribose) polymerase (PARP) is an enzyme involved in DNA repair, particularly in the detection and repair of single-strand breaks (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). PARP inhibitors block these enzymes and are approved for cancer therapy, especially in tumors with homologous recombination (HR) repair defects, such as \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e mutations (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). By inhibiting PARP, these drugs induce synthetic lethality in HR-deficient tumors like \u003cem\u003eBRCA1/2\u003c/em\u003e mutant breast cancers. However, PARP inhibitors are limited to patients with \u003cem\u003eBRCA1/2\u003c/em\u003e alterations, which represent around 5\u0026ndash;10% of all breast cancer cases (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eER/PR/Her2-negative, triple-negative breast cancer (TNBC), an aggressive subtype with the worst prognosis, represents around 15\u0026ndash;20% of all breast cancers (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Around 20% of TNBC patients harbor a \u003cem\u003eBRCA1/2\u003c/em\u003e mutation and can benefit from targeted therapy using PARP inhibitors, while the remaining 80% rely on standard-of-care chemotherapy and/or surgery (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Thus, there is an urgent clinical need to identify alternative targeted treatment options for TNBC patients.\u003c/p\u003e\u003cp\u003eProtein arginine methyltransferase 5 (PRMT5 is a promising therapeutic target for various cancers, particularly breast cancer, due to its overexpression (\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). High PRMT5 levels correlate with poor prognosis in breast cancer (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), including TNBC (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Reducing PRMT5 or inhibiting its activity decreases cell proliferation and tumor growth, and induces apoptosis (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Several small-molecule inhibitors targeting PRMT5 activity have emerged in the past five years (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Most operate as S-Adenosyl-Methionine (SAM)-competitive/uncompetitive or substrate-competitive inhibitors that don't require specific genetic changes for effectiveness. Recently, a new class of PRMT5 inhibitors, called MTA-cooperative, exploits the vulnerability of cells lacking MTAP (methylthioadenosine phosphorylase), making them more prone to PRMT5 inhibition (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). \u003cem\u003eMTAP\u003c/em\u003e deletion occurs in 10\u0026ndash;15% of all cancers (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), and estimated to be less than 5% in breast cancer (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). These inhibitors have shown promise in early-stage clinical trials for \u003cem\u003eMTAP\u003c/em\u003e-deficient tumors (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), including melanoma, gallbladder adenocarcinoma, and mesothelioma.\u003c/p\u003e\u003cp\u003ePRMT5 regulates various cellular functions such as transcriptional regulation, pre-mRNA splicing, and DNA damage responses (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). PRMT5 promotes HR-mediated DNA repair by directly methylating RUVBL1, a coactivator of the histone acetyltransferase TIP60 (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) or by controlling the alternative splicing of TIP60 (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). PRMT5 also regulates DNA repair via non-homologous end-joining (NHEJ) by methylating and stabilizing 53BP1, a key player in the NHEJ pathway (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Consequently, PRMT5 inhibition increases DNA damage and triggers genomic instability in cancer cells (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Thus, targeting PRMT5 may sensitize cancer cells to DNA-repair modulating agents like PARP inhibitors.\u003c/p\u003e\u003cp\u003eIn this study, we evaluated this hypothesis and tested the efficacy of combining two potent PRMT5 inhibitors, GSK3326595 (SAM-uncompetitive and substrate-competitive), and TNG908 (MTA-cooperative) with an FDA-approved PARP inhibitor, Talazoparib, in a panel of \u003cem\u003eBRCA1/2\u003c/em\u003e and/or \u003cem\u003eMTAP\u003c/em\u003e wildtype and mutant/null breast cancer cell lines. We demonstrate synergy between these drugs in all cell lines tested and show that the synergy is independent of the \u003cem\u003eBRCA1/2\u003c/em\u003e and/or the \u003cem\u003eMTAP\u003c/em\u003e status of these cell lines. Further, we show that PRMT5 inhibition does not alter the mRNA expression of key genes involved in HR and NHEJ mediated DNA repair mechanisms. Altogether, this work demonstrates the clinical potential of expanding the PARP-PRMT5 combination to cancers beyond \u003cem\u003eBRCA1/2\u003c/em\u003e mutation and/or \u003cem\u003eMTAP\u003c/em\u003e deficiency.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cu\u003eCell lines\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eHuman breast cancer cell lines (HCC1806;\u003cem\u003e\u0026nbsp;\u003c/em\u003eRRID:CVCL_1258, MDA-MB-468;\u003cem\u003e\u0026nbsp;\u003c/em\u003eRRID:CVCL_0419, MDA-MB-436;\u003cem\u003e\u0026nbsp;\u003c/em\u003eRRID:CVCL_0623, HCC1428;\u003cem\u003e\u0026nbsp;\u003c/em\u003eRRID:CVCL_1252, MDA-MB-231;\u003cem\u003e\u0026nbsp;\u003c/em\u003eRRID:CVCL_0062, and MCF7; RRID:CVCL_0031) were purchased from American Type Culture Collection (ATCC). SUM149PT (RRID:CVCL_3422) was purchased from Bio-IVT and MX-1 cells were purchased from Cell line Services. Ovarian cancer cell lines (UWB1.289; RRID:CVCL_B079 and UWB1.289+BRCA1; RRID:CVCL_B078) were a kind gift from Dr. Karlene Cimprich, Stanford University. All cells were routinely tested for mycoplasma infection using the MycoAlert Mycoplasma Detection Kit (Lonza Biosciences) and cultured in the recommended medium in a 37\u0026deg;C humidified incubator with 5% carbon dioxide.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eDrugs and Antibodies\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eGSK3326595 and TNG908 were purchased from MedChem Express and Talazoparib was purchased from ApexBio. All primary and secondary antibodies used in this study can be found in Supplementary Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eMTT Assays\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eCells were plated in 96-well plates at 750 \u0026ndash; 6400 cells per well to achieve 60% confluency 48 hours later. They were treated in triplicates with varying doses of GSK3326595 (0 \u0026ndash; 100 \u0026micro;M), TNG908 (0 \u0026ndash; 10 \u0026micro;M), and Talazoparib (0 \u0026ndash; 50 \u0026micro;M) or equivalent amounts of DMSO as a vehicle control for 5 days (SUM149PT and MDA-MB-231) or 7 days (all other cell lines), chosen to reflect the time required for 4 cell divisions. Additionally, we replicated several MTT conditions using 10 \u0026ndash; 15 day clonogenic assays to confirm our conclusions.\u003c/p\u003e\n\u003cp\u003eAfter treatment, MTT was added to the plates and incubated for 3 hours, and the formazan crystals were solubilized by adding DMSO for 1 hour. Absorbance was measured at 570 nm using a plate reader, and the data was analyzed to determine IC\u003csub\u003e50\u003c/sub\u003e values and their corresponding 95% confidence intervals for each drug using non-linear regression curves on GraphPad Prism from three independent biological replicates per drug per cell line.\u003c/p\u003e\n\u003cp\u003eFor drug combinations, the maximum dose for each drug was chosen as approximately twice the IC\u003csub\u003e50\u003c/sub\u003e value and serially diluted two- or four-fold. Viability was measured using MTT as described above. Drug interactions were analyzed using the Loewe model in Combenefit Software to determine interaction types. The SYN_MAX metric was used to compare drug synergy strength between cell lines.\u003c/p\u003e\n\u003cp\u003eFor the sequential drug treatment in UWB1.289 and UWB1.289+BRCA1 cell lines, cells were plated in a 96-well plate at 2000 cells/well for both cell lines. After 24 hours, cells were treated with 4 \u0026micro;M (2 x IC\u003csub\u003e50\u003c/sub\u003e dose for UWB1.289 cells from a 7-day assay) of GSK3326595 or equivalent amounts of DMSO for 4, 8, or 24 hours. After each time point, GSK3326595 was removed and fresh media with talazoparib (0 \u0026ndash; 50 \u0026micro;M for UWB1.289+BRCA1 and 0 \u0026ndash; 50 nM for UWB1.289) or DMSO was added for 7 days. Viability was measured, and IC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003ewas\u003csub\u003e\u0026nbsp;\u003c/sub\u003ecalculated as described.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eWestern Blotting\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eCells were plated in 6-well plates at 2 x 10\u003csup\u003e5\u003c/sup\u003e cells/well for drug treatments and lysed on ice for 30 mintues using RIPA lysis buffer. Extracted proteins were quantified using the Pierce BCA kit (Thermo Fisher). 15-20 \u0026micro;g of protein was resolved by SDS-PAGE, and symmetric dimethylarginine was detected using a 1:1000 anti-SDMA antibody (RRID: AB_2714013), PRMT5 with a 1:1000 anti-PRMT5 antibody (RRID: AB_2904201), and MTAP with a 1:1000 anti-MTAP antibody (RRID: AB_2147094). Equal protein loading was confirmed by detecting \u0026beta;-actin using a 1:5000 anti- \u0026beta;-actin antibody (RRID: AB_2687938). Three independent biological replicates were performed for each western blot.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eImmunofluorescence\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eUWB1.289 and UWB1.289+BRCA1 cells were plated in 24-well plates on coverslips at 2 x 10\u003csup\u003e5\u003c/sup\u003e cells/well and treated with 4 mM GSK3326595 for 4, 8, or 24 hours. After treatment, cells were fixed with 4% paraformaldehyde for 15 minutes and permeabilized with 0.1% triton-X-100 in Phosphate-buffered saline (PBS) for 5 minutes. Following a PBS wash, cells were blocked for 30 minutes with 1% bovine serum albumin (BSA) in PBS with 0.1% Tween detergent (PBST). Coverslips were incubated overnight at 4\u0026deg;C with anti-gH2AX (1:1000 dilution; RRID: AB_2118009), anti-RAD51 (1:1000 dilution; RRID: AB_10002131), or anti-53BP1 (1:200 dilution; AB_1290520) antibodies. After washing thrice with 1% BSA in PBST, coverslips were incubated with Goat anti-rabbit Alexa Fluor 488 (RRID: AB_2534069) or Goat anti-mouse Alexa Fluor 594 (RRID: AB_2534079) secondary antibodies for 1 hour at room temperature. Excess antibodies were washed thrice with 1% BSA in PBST and then incubated with 1 mg/ml DAPI for 10 minutes. After a PBS wash, coverslips were mounted on slides using Prolong Gold Antifade mounting medium. Imaging was done at 40X magnification on a Leica Fluorescent microscope (Leica DMI6000; RRID:SCR_018713).\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eRNA isolation, cDNA synthesis and Quantitative PCR (qPCR)\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eMDA-MB-468 and HCC1806 cells were plated in 6-well plates at 3 x 10\u003csup\u003e5\u003c/sup\u003e cells/well and treated with 40 nM or 5 mM of GSK3326595 or equivalent DMSO for 24 hours. Total RNA was extracted using Qiagen\u0026rsquo;s\u0026rsquo; RNeasy extraction kit following manufacturer\u0026rsquo;s protocol. cDNA was synthesized from 1.5 mg of RNA using Qiagen\u0026rsquo;s Quantitect Reverse transcription kit following manufacturer\u0026rsquo;s protocol. 0.075 mg of cDNA was used in triplicate for qPCR using Maxima SYBR green on ABI\u0026rsquo;s QuantStudio 12K Flex Real-Time PCR System. Gene expression was measured using the comparative CT method with values normalized to GAPDH and DMSO control. Data presented are from three independent experiments. Primers used in this study can be found in Supplemental Table 1.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cu\u003eBreast cancer cell lines with differing BRCA1/2 status demonstrate differential sensitivity to PRMT5 inhibition\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe treated a panel of seven breast cancer cell lines with different \u003cem\u003eBRCA1/2\u003c/em\u003e status (wildtype or mutant) (Table 1) with the PRMT5 inhibitor, GSK3326595, at doses ranging from 0 \u0026ndash; 50 \u0026micro;M for five (MDAMB231 and SUM149PT) or seven (HCC1806, MDAMB468, MDAMB436, MX-1, HCC1428) days and measured cell viability using an MTT assay. We observed that there was a difference in sensitivity to PRMT5 inhibition (\u003cstrong\u003eFigure 1A\u003c/strong\u003e) with the \u003cem\u003eBRCA1/2\u003c/em\u003e wildtype cell lines HCC1806, MDAMB468, and MDAMB231 being the most sensitive with IC\u003csub\u003e50\u003c/sub\u003e values less than 2 \u0026micro;M. MDAMB436 (\u003cem\u003eBRCA1\u003c/em\u003e mutant) and MX-1 (\u003cem\u003eBRCA1/2\u003c/em\u003e mutant) lines were less sensitive to PRMT5 inhibition with IC50 values between 12.6 \u0026micro;M and 16.2 \u0026micro;M, respectively, while SUM149PT (\u003cem\u003eBRCA1\u003c/em\u003e mutant) and HCC1428 (\u003cem\u003eBRCA2\u003c/em\u003e mutant) were resistant to PRMT5 inhibition with IC\u003csub\u003e50\u003c/sub\u003e values greater than 50 \u0026micro;M. This difference in sensitivity to PRMT5 inhibition was not related to the protein expression level of PRMT5 in these cell lines (\u003cstrong\u003eSupplemental Figure 1\u003c/strong\u003e). Next, we sought to test the efficacy of GSK3326595 on PRMT5 enzyme inhibition in our panel of seven cell lines treated with a low concentration (40 nM) and a high concentration (5 \u0026micro;M) of GSK3326595 for five (MDAMB231 and SUM149PT) or seven (HCC1806, MDAMB468, MDAMB436, MX-1, HCC1428) days. We measured the global protein levels of symmetric dimethylarginine (SDMA) in whole cell protein lysates using western blotting (\u003cstrong\u003eFigure 1B\u003c/strong\u003e) and observed that SDMA levels were reduced in all the cell lines starting at the low concentration of 40 nM. A difference of 21-fold was noted between the dose required to inhibit PRMT5 enzyme activity and the dose required to induce cytotoxicity for the most sensitive \u003cem\u003eBRCA1/2\u003c/em\u003e wildtype cell line (HCC1806), while a difference of 315 - 1250-fold was noted for the \u003cem\u003eBRCA1/2\u003c/em\u003e mutant lines.\u003c/p\u003e\n\u003cp\u003eOverall, this indicated that though the cell lines showed differential sensitivity to PRMT5 inhibition, the most sensitive cell lines were \u003cem\u003eBRCA1/2\u0026nbsp;\u003c/em\u003ewildtype.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1: Half-maximal inhibitory concentrations (IC\u003csub\u003e50\u003c/sub\u003e) for PRMT5i (GSK3326595, TNG908) and PARPi (Talazoparib), BRCA1/2 status, MTAP status, and TNBC subtype in cell lines used in this study\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCell line\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGSK3326595 IC\u003csub\u003e50\u003c/sub\u003e (in \u0026micro;M)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCI for IC50 (in \u0026micro;M)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTalazoparib IC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003e(in nM)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCI for IC50 (in nM)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTNG908 IC\u003csub\u003e50\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(in \u0026micro;M)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCI for IC50 (in \u0026micro;M)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eBRCA1/2 status\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eMTAP Status\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTNBC subtype\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHCC1806\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e0.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e0.48-1.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e28.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e11.32-57.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e8.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e6.16-13.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cem\u003eBRCA1/2\u003csup\u003e\u0026nbsp;WT\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cem\u003eMTAP\u003csup\u003eWT\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003eBL2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMDA-MB-468\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e0.68-2.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e11.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e9.53-14.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e3.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e2.27-4.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cem\u003eBRCA1/2\u003csup\u003e\u0026nbsp;WT\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cem\u003eMTAP\u003csup\u003eWT\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003eBL1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMDA-MB-231\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e1.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e1.07-3.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e112.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e65.39-179.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e1.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e1.24-2.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cem\u003eBRCA1/2\u003csup\u003e\u0026nbsp;WT\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cem\u003eMTAP\u003csup\u003enull\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003eMSL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMDA-MB-436\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e12.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e6.55-35.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e0.31-0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026gt;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e10.67-31.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cem\u003eBRCA1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cem\u003eMTAP\u003csup\u003eWT\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003eMSL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMx-1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e16.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e5.31-47.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e34.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e18.58-64.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cem\u003eBRCA1/2\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cem\u003eMTAP\u003csup\u003eWT\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003eUnknown\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSUM149PT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e4.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e3.76-5.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e6.11-15.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cem\u003eBRCA1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cem\u003eMTAP\u003csup\u003enull\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003eBL2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHCC1428\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cem\u003eBRCA2\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cem\u003eMTAP\u003csup\u003eWT\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003eER+ BC cancer line\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUWB1.289\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e2.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e1.69-3.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e35.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e22.25-55.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cem\u003eBRCA1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cem\u003eMTAP\u003csup\u003eWT\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003eOvarian\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUWB1.289+BRCA1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e18.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e11.55-35.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e2393\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e1575-3642\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003eBRCA1 complemented\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cem\u003eMTAP\u003csup\u003eWT\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003eOvarian\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviations: CI \u0026ndash; 95% confidence intervals, BRCA1/2\u003csup\u003eWT\u003c/sup\u003e \u0026ndash; BRCA1 and 2 wildtype, BRCA1\u003csup\u003emut\u003c/sup\u003e \u0026ndash; BRCA1 mutant, BRCA2\u003csup\u003emut\u0026nbsp;\u003c/sup\u003e\u0026ndash; BRCA2 mutant, MTAP\u003csup\u003eWT\u003c/sup\u003e- MTAP wildtype, BL2 \u0026ndash; Basal-like 2, BL1 \u0026ndash; Basal-like 1, MSL \u0026ndash; Mesenchymal stem-like, ER+ BC - Estrogen receptor positive breast cancer, NA \u0026ndash; not applicable, ND \u0026ndash; not determined.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003ePRMT5 inhibition synergizes with PARP inhibition in breast cancer cell lines\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince the most sensitive cell lines to PRMT5 inhibition in our panel were \u003cem\u003eBRCA1\u003c/em\u003e wildtype, we sought to evaluate whether combining the PRMT5 inhibitor (GSK3326595) with a potent PARP inhibitor (Talazoparib) would be cytotoxic. First, we determined the IC\u003csub\u003e50\u003c/sub\u003e values of Talazoparib in our panel of cell lines by treating the cells with a range of 0 \u0026ndash; 50 \u0026micro;M for five (MDAMB231 and SUM149PT) or seven (HCC1806, MDAMB468, MDAMB436, MX-1, HCC1428) days and measured cell viability using an MTT assay. As expected, the \u003cem\u003eBRCA1\u003c/em\u003e mutant cell lines, MDAMB436 and SUM149PT were the most sensitive to PARP inhibition with IC\u003csub\u003e50\u003c/sub\u003e of 0.39 and 4.49 nM, respectively (\u003cstrong\u003eTable 1\u003c/strong\u003e). \u003cem\u003eBRCA1\u003c/em\u003e/2 wildtype (HCC1806, MDAMB468 and MDAMB231) and \u003cem\u003eBRCA1/2\u003c/em\u003e mutant (MX-1) cell lines were less sensitive to PARP inhibition (IC\u003csub\u003e50\u003c/sub\u003e between 11 \u0026ndash; 34 nM) while the \u003cem\u003eBRCA2\u003c/em\u003e mutant cell line (HCC1428) was resistant to PARP inhibition with an IC\u003csub\u003e50\u003c/sub\u003e greater than 50 \u0026micro;M (\u003cstrong\u003eTable 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eNext, to evaluate the efficacy of combining GSK3326595 and Talazoparib, we treated our panel of seven cell lines with a maximum dose of approximately twice the IC50 dose for each drug and serially diluted it two-fold (MDAMB468, HCC1806, MDAMB436, SUM149PT, HCC1428) or four-fold (MDAMB231, MX-1). At the end of the five (MDAMB231, SUM149PT) or seven (MDAMB468, HCC1806, MDAMB436, HCC1428, MX-1) day treatment, cell viability was measured using an MTT assay and synergy was calculated using Combenefit software. We observed significant synergy between GSK3326595 and Talazoparib in all the cell lines tested, independent of the \u003cem\u003eBRCA\u003c/em\u003e status of the cell lines (\u003cstrong\u003eFigure 2A\u003c/strong\u003e). Interestingly, when we used the SYN_MAX metric from the Combenefit Software to compare the strength of maximum synergy between the cell lines, we observed that the highest synergy scores were obtained for the \u003cem\u003eBRCA1/2\u003c/em\u003e wildtype cell lines, MDAMB468 and MDAMB231, while the lowest synergy scores were obtained for the \u003cem\u003eBRCA1\u003c/em\u003e mutant SUM149PT and MDAMB436 cell lines (\u003cstrong\u003eFigure 2B\u003c/strong\u003e). Since the \u003cem\u003eBRCA1\u003c/em\u003e wildtype cell lines were also the most sensitive to PRMT5 inhibition alone, we wanted to determine if that could be an indicator for synergy with PARP inhibitors. To determine this, we performed a Pearson correlation between the maximum synergy score for each cell line and their respective IC\u003csub\u003e50\u003c/sub\u003e values for GSK3326595 (\u003cstrong\u003eFigure 2C\u003c/strong\u003e). The Pearson correlation coefficient was negative (r = -0.5666) but was not statistically significant (p-value = 0.18), suggesting that there is a trend indicating higher PRMT5 sensitivity correlates with obtaining a higher synergy with PARP inhibitors.\u003c/p\u003e\n\u003cp\u003eTo understand mechanistically whether PRMT5 inhibition was altering expression of genes involved in DNA damage response, we examined the gene expression of several known genes involved in homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways upon PRMT5 inhibition in the two most sensitive \u003cem\u003eBRCA1\u003c/em\u003e/2 wildtype cell lines, HCC1806 and MDAMB468. Levels of \u003cem\u003eATM,\u003c/em\u003e \u003cem\u003eBRCA1\u003c/em\u003e, and \u003cem\u003eBRCA2\u003c/em\u003e mRNA were not consistently altered across cell lines (Supplementary Figure 2A). The only HR gene that was consistently upregulated in a dose-dependent manner and in both cell lines after PRMT5 inhibition was \u003cem\u003eRAD50\u003c/em\u003e (\u003cstrong\u003eSupplementary Figure 2A\u003c/strong\u003e). However, we did not observe a statistically significant increase in RAD50 protein level (\u003cstrong\u003eSupplementary Figure 2B, C\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThese results suggested that the higher synergies observed between PRMT5i and PARPi in \u003cem\u003eBRCA1/2\u003c/em\u003e wildtype cells were not mediated through a consistent dysregulation of any of the known genes in the HR and NHEJ pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003ePre-treating \u003cem\u003eBRCA1\u003c/em\u003e mutant and \u003cem\u003eBRCA1\u003c/em\u003e wildtype cell lines with PRMT5i sensitize them to PARP inhibition\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn our panel of breast cancer cell lines, we observed that there was a difference in sensitivity to PRMT5 inhibition based on \u003cem\u003eBRCA1/2\u003c/em\u003e status (\u003cstrong\u003eFigure 1A\u003c/strong\u003e, \u003cstrong\u003eTable 1\u003c/strong\u003e). To determine whether BRCA1 expression was responsible for this difference in sensitivity, we tested a pair of isogenic ovarian cancer cell lines, UWB1.289 (BRCA1 mutant) and UWB1.289+BR1 (BRCA1 complemented). Upon treating these cells with a range of 0 \u0026ndash; 50 \u0026micro;M GSK3326595 for seven days, we observed that the parental cell line, UWB1.289, was more sensitive to PRMT5 inhibition with an IC50 of 2.38 \u0026micro;M compared to the BRCA1 complemented cell line, whose IC50 was 18.53 \u0026micro;M (\u003cstrong\u003eFigure 3A\u003c/strong\u003e). Additionally, we treated both cell lines with two different doses of GSK3326595 (40nM and 5uM) and observed that the SDMA mark, indicative of PRMT5 enzyme activity, was decreased at the low concentration of 40nM in both cell lines (\u003cstrong\u003eFigure 3B\u003c/strong\u003e), similar to the panel of breast cancer cell lines.\u003c/p\u003e\n\u003cp\u003eNext, we evaluated the effect of combining PRMT5i with PARPi in this pair of isogenic cell lines, to determine whether BRCA1 expression was contributing to the synergy between these two inhibitors. We treated both cell lines with a maximum drug dose of approximately twice the IC50 dose for each drug and serially diluted it two-fold. At the end of a seven-day treatment, we measured cell viability using an MTT assay and calculated drug synergy using the Combenefit software. Combining PRMT5i and PARPi in these cell lines demonstrated synergy (\u003cstrong\u003eFigure 3C\u003c/strong\u003e) in both cell lines, similar to our observation in the panel of breast cancer cell lines (\u003cstrong\u003eFigure 2A\u003c/strong\u003e). However, when we used the SYN_MAX metric to compare the strength of synergy between the UWB1.289 and UWB1.289+BRCA1 cell line, we observed that UWB1.289 had a higher maximum synergy score compared to UWB1.289+BRCA1 (\u003cstrong\u003eFigure 3D\u003c/strong\u003e). This is in contrast to our panel of breast cancer cell lines, where the highest synergies were observed in BRCA1 WT cell lines (\u003cstrong\u003eFigure 2A\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eIn order to determine whether sequential treatment of PRMT5i and PARPi would be more efficacious than combination treatment, we pre-treated the cells with 4 \u0026micro;M of GSK3326595 (equivalent to 2 X IC50 dose in UWB1.289) for 4 hours, 8 hours, and 24 hours and subsequently treated with Talazoparib for seven days. We observed that the IC\u003csub\u003e50\u003c/sub\u003e of PARPi was decreased by two-fold in both UWB1.289 and UWB1.289+BRCA1 cells when pre-treated with GSK3326595 (\u003cstrong\u003eFigure 3E\u003c/strong\u003e). This indicated that inhibiting PRMT5 first was sufficient to sensitize the cells to subsequent PARP inhibition, independent of their BRCA1/2 status. The similar antiproliferative and synergistic effects seen for short (4 hr) non-overlapping exposure as that after 7 days of combined exposure suggests that a non-canonical mechanism may be involved in the early stages, which could ultimately lead to the changes in DDR gene expression observed at later time points.\u003c/p\u003e\n\u003cp\u003eIn order to determine which DNA damage repair pathway was being regulated by GSK3326595 treatment to sensitize these cells to PARP inhibition, we analyzed \u0026gamma;-H2AX, RAD51 and 53BP1 foci as markers for DNA double strand breaks, HR-mediated repair, and NHEJ-mediated repair, respectively. We observed that there was no significant increase in \u0026gamma;-H2AX foci after PRMT5 inhibition in either UWB1.289 or UWB1.289+BRCA1 at any of the time points tested (\u003cstrong\u003eSupplementary Figure 3A, B and Supplementary Figure 4, 5\u003c/strong\u003e). Additionally, we did not observe any significant changes in RAD51 (\u003cstrong\u003eSupplementary Figure 3C, D and Supplementary Figure 4, 5\u003c/strong\u003e) or 53BP1 foci (\u003cstrong\u003eSupplementary Figure 3E, F and Supplementary Figure 4, 5\u003c/strong\u003e), except a modest decrease in RAD51 foci after 4h treatment in UWB1.289 cells (\u003cstrong\u003eSupplementary Figure 3C\u003c/strong\u003e) and an increase in 53BP1 after 24h treatment in UWB1.289+BRCA1 (\u003cstrong\u003eSupplementary Figure 3F\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTogether, these results suggested that although short-term inhibition of PRMT5 was sufficient to sensitize both UWB1.289 and UWB1.289+BRCA1 to PARP inhibition, mechanistically, this was not mediated by inducing DNA damage as measured with known markers at these early time points.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eMTA-cooperative PRMT5 inhibitor synergizes with PARPi in both MTAP\u003csup\u003enull\u003c/sup\u003e and MTAP\u003csup\u003eWT\u003c/sup\u003e cell lines\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecent evidence suggests that cancers with \u003cem\u003eMTAP\u003c/em\u003e deficiency are vulnerable to PRMT5 inhibition, leading to the development of a new class of PRMT5 inhibitors that are MTA-cooperative and are in clinical trials for \u003cem\u003eMTAP\u003c/em\u003e-deleted cancers. Therefore, we sought to evaluate the effect of a potent MTA-cooperative PRMT5 inhibitor, TNG908 (15) in combination with PARP inhibitors in \u003cem\u003eMTAP\u003csup\u003eWT\u003c/sup\u003e\u0026nbsp;\u003c/em\u003eand \u003cem\u003eMTAP\u003csup\u003enull\u003c/sup\u003e\u003c/em\u003e breast cancer cell lines.\u003c/p\u003e\n\u003cp\u003eWe first evaluated the sensitivity of our panel of breast cancer cell lines by treating them with a range of 0 \u0026ndash; 10 \u0026micro;M of TNG908. As expected, MDA-MB-231, an \u003cem\u003eMTAP\u003csup\u003enull\u003c/sup\u003e\u003c/em\u003e cell line (\u003cstrong\u003eSupplementary Figure 1\u003c/strong\u003e), was the most sensitive to TNG908 with an IC\u003csub\u003e50\u003c/sub\u003e of 1.83 \u0026micro;M and MDA-MB-436, an \u003cem\u003eMTAP\u003csup\u003eWT\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003ecell\u003csup\u003e\u0026nbsp;\u003c/sup\u003eline (\u003cstrong\u003eSupplementary Figure 1\u003c/strong\u003e), was the least sensitive with an IC\u003csub\u003e50\u003c/sub\u003e \u0026gt; 10 \u0026micro;M (\u003cstrong\u003eFigure 4A\u003c/strong\u003e). Surprisingly, SUM149PT cells were not as sensitive (IC50 = 9 \u0026micro;M) to TNG908 despite being an \u003cem\u003eMTAP\u003csup\u003enull\u003c/sup\u003e\u003c/em\u003e cell line (\u003cstrong\u003eSupplementary Figure 1\u003c/strong\u003e). Then, we examined the efficacy of TNG908 to inhibit PRMT5 activity in all cell lines by measuring global SDMA levels and observed that both 40 nM and 5 \u0026micro;M treatments decreased SDMA levels in all cell lines (\u003cstrong\u003eFigure 4B\u003c/strong\u003e). When we combined TNG908 with Talazoparib and evaluated the efficacy of this combination, we saw that similar to the GSK-Talazoparib combination, TNG908 also synergized with Talazoparib in all cell lines (\u003cstrong\u003eFigure 4C\u003c/strong\u003e) and the maximum synergy was observed in the MDA-MB-468 and MDA-MB-231 cell lines (\u003cstrong\u003eFigure 4D\u003c/strong\u003e). To determine whether sensitivity to TNG908 could be an indicator of synergy with PARP inhibition, we performed a Pearson Correlation using the maximum synergy score (SYN_MAX metric) for each cell line with their respective IC\u003csub\u003e50\u003c/sub\u003e values of TNG908. Similar to the correlation between IC\u003csub\u003e50\u003c/sub\u003e values of GSK3326595 and the maximum synergy scores (\u003cstrong\u003eFigure 2C\u003c/strong\u003e), we obtained a negative correlation coefficient of r = -0.8990, that was also statistically significant (p-value \u0026lt; 0.05, \u003cstrong\u003eFigure 3E\u003c/strong\u003e). This suggested that cell lines that were more sensitive to PRMT5 inhibition using TNG908 were indicative of obtaining high synergy with PARP inhibitors.\u003c/p\u003e\n\u003cp\u003eTogether, these results demonstrated that though \u003cem\u003eMTAP\u003csup\u003enull\u003c/sup\u003e\u003c/em\u003e cells are more sensitive to PRMT5 inhibition using the MTA-cooperative inhibitor, the synergy between PRMT5i and PARPi does not depend on the \u003cem\u003eMTAP\u003c/em\u003e status of the cells.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eExploiting specific genetic vulnerabilities in cancer cells has led to the development and clinical use of potent therapies in cancer research. PARP inhibitors were the first drugs designed to exploit the vulnerabilities of tumors with \u003cem\u003eBRCA1/2\u003c/em\u003e mutations (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) and are approved for breast, ovarian, prostate, and pancreatic cancer treatment. Another cancer vulnerability being targeted is PRMT5 inhibition in \u003cem\u003eMTAP\u003c/em\u003e-deleted tumors (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). \u003cem\u003eMTAP\u003c/em\u003e deletion has variable frequency in different cancers and is less than 5% in breast cancer (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), but a higher incidence of TNBC is observed in tumors with \u003cem\u003eMTAP\u003c/em\u003e loss (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study, we combined an FDA-approved PARP inhibitor with two classes of PRMT5 inhibitors and demonstrated synergy in breast cancer cell lines, independent of their \u003cem\u003eBRCA1/2\u003c/em\u003e or \u003cem\u003eMTAP\u003c/em\u003e status. While recent reports showed synergy between PARP and PRMT5 inhibitors, these were limited to \u003cem\u003eBRCA1/2\u003c/em\u003e wild-type breast and ovarian cancer models (20\u0026ndash;22, 25) or acute myeloid leukemia models (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Our findings suggest that the PARP-PRMT5 combination strategy (i) can serve as a novel targeted treatment for TNBC patients and (ii) can be expanded to a wider range of cancers than those with \u003cem\u003eBRCA1/2\u003c/em\u003e mutations and/or \u003cem\u003eMTAP\u003c/em\u003e mutations. Thus, it would be highly clinically relevant to test this combination, \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, in other cancer models where PARP inhibitors are approved, such as ovarian, prostate, and pancreatic cancer. Additionally, MTA-cooperative PRMT5 inhibitors are currently in clinical trials for \u003cem\u003eMTAP\u003c/em\u003e-deleted solid and liquid tumors. Our data indicate that combining the MTA-cooperative PRMT5 inhibitor with PARP inhibitors is also efficient in \u003cem\u003eMTAP\u003c/em\u003e wildtype tumors, suggesting this combination should be tested in larger clinical trials for all patients, regardless of \u003cem\u003eMTAP\u003c/em\u003e status.\u003c/p\u003e\u003cp\u003eThe current understanding of the synergy between PARP and PRMT5 inhibitors is attributed to increased DNA damage (20,25), and/or downregulation of genes responsible for DNA damage response (21,22,25) after PRMT5 inhibition. However, we did not observe any significant downregulation of key genes in DNA damage response or an increase in DNA damage upon PRMT5 inhibition in our models. One possible explanation for this apparent discrepancy is in the timing of experiments. We studied the early response to DNA damage and related gene expression by testing 24 hours after PRMT5 inhibition, while previous studies demonstrated a late effect of PRMT5 inhibition on the DNA damage response pathway 4\u0026ndash;7 days after treatment (20\u0026ndash;22,25). Therefore, it is possible that the effects previously observed on DNA damage were indirectly mediated by PRMT5. Furthermore, we have shown that short-term exposure of cells, as little as 4 hours, to a PRMT5 inhibitor is sufficient to sensitize them to PARP inhibitors, suggesting that early changes are occurring within the cells that are yet to be fully understood. Thus, further studies are needed to identify these key early genes regulated by PRMT5 either through alternative splicing or direct methylation to understand this observed synergy.\u003c/p\u003e\u003cp\u003eIn conclusion, we have shown that the PARP-PRMT5 drug combination can be a beneficial treatment strategy for a larger cohort of cancers and not limited to a subset with specific genetic mutations. Further mechanistic studies will help broaden our understanding of PRMT5-mediated DNA damage response and help identify predictive biomarkers for this combination.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interest:\u003c/h2\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest\u003c/p\u003e\n\u003ch2\u003eFunding:\u003c/h2\u003e\n\u003cp\u003eThis work was supported by grants to JMF from the BRCA Foundation and the Breast Cancer Research Foundation.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eSS made substantial contributions to the conception and design of the work. SS and LAM contributed to the acquisition, analysis, or interpretation of data and drafted the work or revised it critically for important intellectual content. JMF supervisd the work and contributed to the analysis and writing of the manuscriopt. All authors approved the version to be published.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChehade CH, Gebrael G, Sayegh N, Ozay ZI, Narang A, Crispino T, et al. A pan-tumor review of the role of poly(adenosine diphosphate ribose) polymerase inhibitors. CA Cancer J Clin. 2025 Mar-Apr;75(2):141-167. doi: 10.3322/caac.21870. \u003c/li\u003e\n\u003cli\u003eBarchiesi G, Roberto M, Verrico M, Vici P, Tomao S, Tomao F. Emerging Role of PARP Inhibitors in Metastatic Triple Negative Breast Cancer. Current Scenario and Future Perspectives. Front Oncol. 2021;11:769280. PMID: \u003cstrong\u003e34900718\u003c/strong\u003e DOI: 10.3389/fonc.2021.769280 \u003c/li\u003e\n\u003cli\u003eMandapati A, Lukong KE. Triple negative breast cancer: approved treatment options and their mechanisms of action. J Cancer Res Clin Oncol. 2022 149(7):3701-3719. doi: 10.1007/s00432-022-04189-6. \u003c/li\u003e\n\u003cli\u003eHartman AR, Kaldate RR, Sailer LM, Painter L, Grier CE, Endsley RR, et al. Prevalence of BRCA mutations in an unselected population of triple-negative breast cancer. Cancer. 2012 Jun 1;118(11):2787\u0026ndash;95. DOI: 10.1002/cncr.26576 \u003c/li\u003e\n\u003cli\u003eChen Y, Shao X, Zhao X, Ji Y, Liu X, Li P, et al. Targeting protein arginine methyltransferase 5 in cancers: Roles, inhibitors and mechanisms. Biomed Pharmacother. 2021 Dec;144:112252. https://doi.org/10.1016/j.biopha.2021.112252\u003c/li\u003e\n\u003cli\u003eLi WJ, He YH, Yang JJ, Hu GS, Lin YA, Ran T, et al. Profiling PRMT methylome reveals roles of hnRNPA1 arginine methylation in RNA splicing and cell growth. Nat Commun. 2021 Mar 29;12(1):1946. DOI: 10.1038/s41467-021-21963-1 \u003c/li\u003e\n\u003cli\u003eShailesh H, Siveen KS, Sif S. Protein arginine methyltransferase 5 (PRMT5) activates WNT/beta-catenin signalling in breast cancer cells via epigenetic silencing of DKK1 and DKK3. J Cell Mol Med. 2021 Feb;25(3):1583\u0026ndash;600. DOI: 10.1111/jcmm.16260 \u003c/li\u003e\n\u003cli\u003eHan X, Wei L, Wu B. PRMT5 Promotes Aerobic Glycolysis and Invasion of Breast Cancer Cells by Regulating the LXRalpha/NF-kappaBp65 Pathway. Onco Targets Ther. 2020;13:3347\u0026ndash;57. doi: 10.2147/OTT.S239730\u003c/li\u003e\n\u003cli\u003eYang F, Wang J, Ren HY, Jin J, Wang AL, Sun LL, et al. Proliferative role of TRAF4 in breast cancer by upregulating PRMT5 nuclear expression. Tumour Biol. 2015 Aug;36(8):5901\u0026ndash;11. DOI: 10.1007/s13277-015-3262-0 \u003c/li\u003e\n\u003cli\u003eRengasamy M, Zhang F, Vashisht A, Song WM, Aguilo F, Sun Y, et al. The PRMT5/WDR77 complex regulates alternative splicing through ZNF326 in breast cancer. Nucleic Acids Res. 2017 Nov 2;45(19):11106\u0026ndash;20. doi: 10.1093/nar/gkx727\u003c/li\u003e\n\u003cli\u003eVinet M, Suresh S, Maire V, Monchecourt C, Nemati F, Lesage L, et al. Protein arginine methyltransferase 5: A novel therapeutic target for triple-negative breast cancers. Cancer Med. 2019 May;8(5):2414\u0026ndash;28. doi: 10.1002/cam4.2114\u003c/li\u003e\n\u003cli\u003eWu Y, Wang Z, Zhang J, Ling R. Elevated expression of protein arginine methyltransferase 5 predicts the poor prognosis of breast cancer. Tumour Biol. 2017 Apr;39(4):1010428317695917. \u003c/li\u003e\n\u003cli\u003eGuo C, Wu L, Zheng X, Zhao L, Hou X, Wang Z, et al. Research Progress on Small-molecule Inhibitors of Protein Arginine Methyltransferase 5 (PRMT5) for Treating Cancer. Curr Top Med Chem. 2023;23(21):2048\u0026ndash;74. DOI: 10.1177/1010428317695917 \u003c/li\u003e\n\u003cli\u003eHu M, Chen X. A review of the known MTA-cooperative PRMT5 inhibitors. RSC Adv. 2024 Dec 10;14(53):39653\u0026ndash;91. DOI https://doi.org/10.1039/D4RA05497K\u003c/li\u003e\n\u003cli\u003eBriggs KJ, Cottrell KM, Tonini MR, Tsai A, Zhang M, Whittington DA, et al. TNG908 is a brain-penetrant, MTA-cooperative PRMT5 inhibitor developed for the treatment of MTAP-deleted cancers. Transl Oncol. 2025 Jan 3;52:102264. DOI: 10.1016/j.tranon.2024.102264 \u003c/li\u003e\n\u003cli\u003eBou Zerdan M, Ashok Kumar P, Haroun E, Srivastava N, Ross J, Sivapiragasam A. Genomic landscape of metastatic breast cancer (MBC) patients with methylthioadenosine phosphorylase (MTAP) loss. Oncotarget. 2023 Mar 11;14:178\u0026ndash;87. DOI: 10.18632/oncotarget.28376 \u003c/li\u003e\n\u003cli\u003eClarke TL, Sanchez-Bailon MP, Chiang K, Reynolds JJ, Herrero-Ruiz J, Bandeiras TM, et al. PRMT5-Dependent Methylation of the TIP60 Coactivator RUVBL1 Is a Key Regulator of Homologous Recombination. Mol Cell. 2017 Mar 2;65(5):900-916 e7. DOI: 10.1016/j.molcel.2017.01.019 \u003c/li\u003e\n\u003cli\u003eHamard PJ, Santiago GE, Liu F, Karl DL, Martinez C, Man N, et al. PRMT5 Regulates DNA Repair by Controlling the Alternative Splicing of Histone-Modifying Enzymes. Cell Rep. 2018 Sep 4;24(10):2643\u0026ndash;57. DOI: 10.1016/j.celrep.2018.08.002 \u003c/li\u003e\n\u003cli\u003eHwang JW, Kim SN, Myung N, Song D, Han G, Bae GU, et al. PRMT5 promotes DNA repair through methylation of 53BP1 and is regulated by Src-mediated phosphorylation. Commun Biol. 2020 Aug 5;3(1):428. DOI: 10.1038/s42003-020-01157-z \u003c/li\u003e\n\u003cli\u003eO\u0026rsquo;Brien S, Butticello M, Thompson C, Wilson B, Wyce A, Mahajan V, et al. Inhibiting PRMT5 induces DNA damage and increases anti-proliferative activity of Niraparib, a PARP inhibitor, in models of breast and ovarian cancer. BMC Cancer. 2023 Aug 18;23(1):775. DOIhttps://doi.org/10.1186/s12885-023-11260\u003c/li\u003e\n\u003cli\u003eCarter J, Hulse M, Sivakumar M, Burtell J, Thodima V, Wang M, et al. PRMT5 Inhibitors Regulate DNA Damage Repair Pathways in Cancer Cells and Improve Response to PARP Inhibition and Chemotherapies. Cancer Res Commun. 2023 Nov 6;3(11):2233\u0026ndash;43. doi.org/10.1158/2767-9764.CRC-23-0070 \u003c/li\u003e\n\u003cli\u003eLi Y, Dobrolecki LE, Sallas C, Zhang X, Kerr TD, Bisht D, et al. PRMT blockade induces defective DNA replication stress response and synergizes with PARP inhibition. Cell Rep Med. 2023 Dec 19;4(12):101326. DOI: 10.1016/j.xcrm.2023.101326 \u003c/li\u003e\n\u003cli\u003eLord CJ, Ashworth A. PARP Inhibitors: The First Synthetic Lethal Targeted Therapy. Science. 2017 Mar 17;355(6330):1152\u0026ndash;8. doi: 10.1126/science.aam7344\u003c/li\u003e\n\u003cli\u003eKryukov GV, Wilson FH, Ruth JR, Paulk J, Tsherniak A, Marlow SE, et al. MTAP deletion confers enhanced dependency on the PRMT5 arginine methyltransferase in cancer cells. Science. 2016 Mar 11;351(6278):1214\u0026ndash;8.  DOI: 10.1126/science.aad5214 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6959243/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6959243/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e: PARP inhibitors have been approved for treating a subset of breast cancer patients harboring \u003cem\u003eBRCA1/2\u003c/em\u003e mutations. However, TNBC patients with wildtype \u003cem\u003eBRCA1/2 \u003c/em\u003ehave limited targeted therapeutic options. Dysregulation of protein arginine methyltransferase 5 (PRMT5) has been implicated in the progression of various cancers, including breast cancer. This study investigates the effects of two classes of PRMT5 inhibitors, GSK3326595 and TNG908, on breast cancer cell lines with different \u003cem\u003eBRCA1/2\u003c/em\u003e statuses to evaluate their therapeutic potential and synergy with PARP inhibitors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: A panel of seven breast cancer cell lines was treated with PRMT5 and PARP inhibitors, followed by cell viability measurements using an MTT assay. Drug interactions were analyzed using the Loewe method on the Combenefit software. Additionally, RT-qPCR was conducted to measure the expression of known DNA damage response genes\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Synergy was observed in all cell lines, independent of the \u003cem\u003eBRCA1/2\u003c/em\u003eand/or \u003cem\u003eMTAP\u003c/em\u003e status. Mechanistically, the PRMT5 inhibition did not alter the gene expression of known DNA damage response genes as measured by RT-qPCR. Notably, short-term PRMT5 inhibition was sufficient to sensitize cells to subsequent PARP inhibition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: These findings highlight the potential of combining PRMT5 inhibitors with PARP inhibitors in a wide range of cancers beyond BRCA1/2 and MTAP mutants. Further investigation is warranted to elucidate the underlying mechanisms of sensitization and the timing of cellular responses to PRMT5 inhibition.\u003c/p\u003e","manuscriptTitle":"PARP inhibitor and PRMT5 inhibitor synergy is independent of BRCA1/2 and MTAP status in breast cancer cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-15 09:46:21","doi":"10.21203/rs.3.rs-6959243/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-21T11:45:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-18T12:57:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-10T21:11:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"135654323576951271799428277167153202187","date":"2025-07-09T19:49:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"173873992127284153853329653946239790443","date":"2025-07-09T19:24:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"327681405341667279791497853996121682352","date":"2025-07-09T18:55:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-09T18:53:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-04T14:57:46+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-01T05:31:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-27T02:13:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-27T02:10:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"45ad6577-d659-4bf8-9198-b81654a7e403","owner":[],"postedDate":"July 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51361112,"name":"Biological sciences/Cancer/Breast cancer"},{"id":51361113,"name":"Health sciences/Oncology/Cancer/Cancer therapy/Targeted therapies"}],"tags":[],"updatedAt":"2025-10-27T16:33:22+00:00","versionOfRecord":{"articleIdentity":"rs-6959243","link":"https://doi.org/10.1038/s41598-025-20696-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-10-21 16:16:44","publishedOnDateReadable":"October 21st, 2025"},"versionCreatedAt":"2025-07-15 09:46:21","video":"","vorDoi":"10.1038/s41598-025-20696-1","vorDoiUrl":"https://doi.org/10.1038/s41598-025-20696-1","workflowStages":[]},"version":"v1","identity":"rs-6959243","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6959243","identity":"rs-6959243","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}