Impact of homologous recombination deficiency on CDK4/6 inhibitor sensitivity in HR+/HER2-breast cancer

Impact of homologous recombination deficiency on CDK4/6 inhibitor sensitivity in HR+/HER2-breast cancer | 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 Impact of homologous recombination deficiency on CDK4/6 inhibitor sensitivity in HR+/HER2-breast cancer Daniela Haas, Selina Wolf, Urtė Stankutė, Ethan S. Sokol, Meagan Montesion, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7253927/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract While the combination of endocrine therapy (ET) and CDK4/6 inhibitors (CDK4/6i) improves progression-free survival (PFS) in HR+/HER2- metastatic breast cancer, resistance remains a major challenge. BRCA2 pathogenic variants have been linked to reduced PFS, potentially due to co-deletion of the neighboring RB1 gene on chromosome 13q. As RB1 is a key target of CDK4/6, its loss drives resistance. Using CRISPR/Cas9, we generated cell lines with single and combined BRCA2 and RB1 deletions. Loss of RB1 but not BRCA2 increased proliferation and conferred resistance to the CDK4/6i palbociclib and abemaciclib. Dual loss reduced proliferation but increased resistance to CDK4/6i in vitro . However, sensitivity to the PARP inhibitor olaparib was maintained. Finally, analysis of real-world clinical data revealed that RB1 mutations were more frequent in tumors exhibiting homologous recombination deficiency signatures and 13q loss. These genomic features were associated with shorter treatment duration on CDK4/6i plus ET. In conclusion, our findings suggest that RB1 loss, alone or with BRCA2 deletion, contributes to CDK4/6 inhibitor resistance and may help explain reduced efficacy in patients with BRCA2 mutations. Importantly, despite this resistance, sensitivity to PARP inhibition is retained, highlighting a potential therapeutic vulnerability in this molecular context. Biological sciences/Cancer Biological sciences/Genetics Biological sciences/Molecular biology Health sciences/Oncology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction While advancements in screening methods and innovative therapeutic strategies have significantly improved the overall survival of breast cancer (BC) patients over the past few decades 1 , metastatic BC (mBC) continues to be an incurable disease and the leading cause of cancer-related death in women 2 . The standard of care for advanced hormone-receptor positive/human epidermal growth factor receptor 2 negative (HR+/HER2-) BC involves endocrine therapy (ET) in combination with cyclin-dependent-kinase 4/6 inhibitors (CDK4/6i) such as palbociclib, abemaciclib and ribociclib 3 . These treatment combinations significantly increase progression-free survival (PFS) 4 and overall survival (OS) 5 in mBC. However, disease progression is frequently observed and acquired therapy resistance constitutes a significant clinical problem in patients with mBC 6 . Mutations impacting cell cycle genes, such as upregulation of CDK2 or FGFR1, have been described leading to CDK4/6i resistance and disease progression 7 – 9 . However, recent findings show that patients with germline pathogenic variants (PVs) in the BRCA2 gene have a shorter PFS when treated with ET combined with CDK4/6i 10 . Inactivating mutations or loss of heterozygosity (LOH) of BRCA2 have been linked to BC development and are observed in approximately 30–40% of patients with sporadic disease 11 . Yet, the precise mechanism by which BRCA2 mutations confer resistance to CDK4/6i remains unclear. A recent report suggests that BRCA2 PVs may not directly cause resistance, but instead promote CDK4/6i resistance due to the close genomic proximity of BRCA2 and retinoblastoma protein 1 ( RB1 ) on chromosome 13q, which can result in the simultaneous loss of both genes 10 . This phenomenon has previously been described in prostate cancer where it is associated with epithelial-mesenchymal transition, higher grade of invasiveness and poor clinical outcome 12 . Since RB1 is a direct downstream target of CDK4/6, its inactivation or LOH confers significant resistance against CDK4/6 inhibition 13 – 16 . Thus far, BRCA2 alterations are primarily associated with homologous recombination deficiency (HRD) 17 . HRD is associated with an elevated BC risk and an elevated mutational burden, resulting not only from BRCA2 loss but also from mutations in other genes involved in homologous recombination repair, such as BRCA1 or partner and localizer of BRCA2 (PALB2) 18 – 20 . Inactivating mutations in BRCA1 and PALB2 are more frequently observed in patients with triple-negative BC (TNBC) 21 – 23 , whereas BRCA2 mutations are associated with both TNBC and HR+/HER2- BC 23 . In BRCA -mutated ( BRCA m) triple-negative breast cancer (TNBC), PARP inhibitors (PARPi) are the preferred treatment, whereas standard care for HR+/HER2- mBC typically consists of ET alone or combined with CDK4/6i, irrespective of HRD status 3 . However, in early-stage BC, both CDK4/6i as well as PARPi have been approved for treatment and are associated with improved disease-free survival (DFS) and OS in independent studies 24 – 26 . Nevertheless, it remains uncertain whether PARPi would be a better option compared to CDK4/6i in combination with ET for HRD-positive HR+/HER2- mBC cases. Using CRISPR-engineered HR+/HER2- breast cancer cell lines, we show here that RB1 loss promoted CDK4/6i resistance, while BRCA2 -deficient cells remained CDK4/6i-sensitive. Interestingly, while associated with increased sensitivity to PARPi, dual loss of RB1 and BRCA2 led to marked resistance to CDK4/6 inhibitors. Genomic profiling revealed that not only BRCA2 loss, but HRD in general is linked to shorter time to treatment discontinuation and RB1 co-loss in real world clinical data. Our findings highlight the need for a more comprehensive understanding of how HRD – beyond BRCA2 mutations – contributes to CDK4/6i resistance and the incidence of RB1 alterations. These data also suggest the need for more tailored and optimized treatment strategies for patients with BRCA2 mutations or other forms of HRD. Materials and methods Cell culture MCF7 and T47D cells were obtained from the Tissue Culture Core Facility at the Medical University of Graz. All cell lines were authenticated prior to use and routinely checked for mycoplasma contamination. MCF7 cells were cultivated in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) and T47D cells were cultivated in high-glucose DMEM (both from Gibco™ by Thermo Fisher Scientific). Media were supplemented with 10% fetal bovine serum (Serana Europe GmbH), 1% Antibiotic-Antimycotic and 2 mM L-glutamine (both from Gibco™ by Thermo Fisher Scientific). Both cell lines were grown as a monolayer and maintained at 37°C, 5% CO 2 and 95% humidity. CRISPR/Cas9 mediated genome engineering In order to establish RB1 and BRCA2 knockout cell lines, the protocol published by Andrew J. Kueh and Marco J. Herold, WEHI, Melbourne was followed 27 . Briefly, guide RNAs targeting RB1 , BRCA2 and lacZ were designed using CRISPick and ordered from Eurofins Genomics. The guide RNAs were cloned into two different inducible lentiviral expression vectors with different fluorescent reporters. For the BRCA2 and lacZ guide RNA, Fgh1tUTG (Addgene plasmid #70183), and for the RB1 guide RNA either Fgh1tUTG (Addgene plasmid #70183) or FgH1tUTCyan (Addgene plasmid #70183) was used. Cas9 protein was expressed constitutively by the vector FUCas9mCherry (Addgene plasmid #70182). The plasmids were amplified by High Efficiency Transformation of NEB® Stable Competent E.coli (C3040H) and isolated using the Plasmid Miniprep Kit by Monarch. Correct insertion of the sgRNA in the lentiviral vector was confirmed by sequencing using the Mix2Seq Kit (Eurofins). Lentiviruses were generated using packaging vectors pMDL/pRRE (Addgene plasmid #12251), pRSV-rev (Addgene plasmid #12253) and pVSV-G (Addgene plasmid #138479), as well as the respective plasmid for guide RNA or Cas9 in 293FT cells (Tissue Culture Core Facility, Medical University of Graz). MCF7 and T47D cells were infected by adding 1 ml of lentivirus containing Cas9 and guide RNA followed by a 2 hour centrifugation (2200 rpm, 32°C). Cells were sorted by flow cytometry and the expression of the guide RNA was induced by doxycycline (Merck, Darmstadt, Germany). Bulk or single cell clones were used for further experiments. Immunoblotting Cells were harvested and lysed in cold Pierce™ RIPA Buffer containing Protease Inhibitor Cocktail (both from Thermo Fisher Scientific). Protein concentration was measured using the BCA Protein Assay Kit (EMD Millipore Corporation). The samples were diluted in RIPA Buffer to the desired concentration. Laemmli Buffer (Bio-Rad) was added and the samples were heated to 95°C at 650 rpm for 10 minutes. After separating the proteins by SDS-PAGE, the proteins were transferred onto nitrocellulose (Bio-Rad) or Hybond® PVDF (Amersham™) membranes. After the transfer, the membranes were blocked in 5% skim milk for 1h. Then, the membranes were incubated with following antibodies overnight at 4°C: Rb (4H1) Mouse mAb (#9309, Cell Signaling Technology), BRCA2 (D9S6V) Rabbit mAb (#10741, Cell Signaling Technology), Phospho-Histone H2A.X (Ser 139) (20E3) Rabbit mAB (#9718, Cell Signaling Technology), HSP70 (produced at WEHI antibody facility, Melbourne, Australia), Anti-mouse IgG, HRP-linked Antibody (#7076, Cell Signaling Technology) and Anti-rabbit IgG, HRP-linked Antibody (#7074, Cell Signaling Technology). Proteins were detected using Immobilon® Classico Western HRP substrate (Millipore). Cell cycle analysis Cells were harvested and washed two times before they were fixed by adding cold 70% ethanol dropwise on the cell pellet. After fixation for at least 30 minutes at 4°C, cells were washed two times in PBS followed by adding ribonuclease A (Thermo Fisher Scientific). After adding the propidium iodide solution (Sigma Aldrich), the suspension was incubated for 10 minutes at room temperature (RT) in the dark. The PI signal was measured at 605 nm with the CytoFLEX S (Beckman Coulter GmbH) flow cytometer. Data were analyzed using Modfit LT™ 5.0 software. Cell viability analysis To analyze apoptosis by flow cytometry, cells were harvested, washed two times in PBS and resuspended in Annexin A5 binding buffer (0.01 M HEPES, 0.14 M NaCl, 2.5 mM CaCl 2 ) containing allophycocyanine (APC) - Annexin V (Biolegend) and 7-Aminoactinomycin D (7-AAD) (Biolegend) per sample. After incubation for 15 minutes at RT in the dark, cells were analyzed by the CytoFLEX S flow cytometer (Beckman Coulter GmbH) at 660 nm. Data were analyzed using CytExpert 2.6. Comprehensive Genomic Profiling Comprehensive genomic profiling of formalin-fixed, paraffin-embedded (FFPE) tissue sections from breast cancer biopsies (local, metastatic and lymph nodes) was performed using FoundationOne® or FoundationOne®CDx in a Clinical Laboratory Improvement Amendments–certified, College of American Pathologists–accredited laboratory (Foundation Medicine Inc.). Hybrid capture was carried out on at least 324 cancer-related genes and selected introns from 34 genes frequently rearranged in cancer, as previously described 28 , 29 . For the BC cohort, HER2 status was determined from the HER2 ( ERBB2 ) amplification status based on the FoundationOne® or FoundationOne®CDx assay and ER status was derived from accompanying pathology reports, where available. Approval for this study, including a waiver of informed consent and Health Insurance Portability and Accountability Act (HIPAA) waiver of authorization, was obtained from the Western Institutional Review Board. Prevalence and co-occurrence were compared using a Fisher’s exact test implemented using the SciPy stats package in Python2. An HRD signature status (HRDsig) was calculated from the genomic profiling results. The HRDsig algorithm evaluates genome-wide copy number patterns linked to HRD using a comprehensive set of copy number features 30 . Scores range from 0 to 1, with a predefined cutoff of 0.7, determined from prior analyses demonstrating 90% sensitivity for identifying biallelic BRCA alterations in BRCA -driven tumors. Definition of BRCA1 , BRCA2 and RB1 positive status BRCA1 and BRCA2 positive status were defined based on the presence of a known or likely pathogenic alteration in the BRCA1 or BRCA2 gene. Biallelic BRCA1 and BRCA2 positive status was defined as any patient who had at least two known or likely pathogenic short variant alterations, at least one known or likely pathogenic homozygous short variant alteration, a homozygous deletion, or at least two known or likely pathogenic rearrangements in the BRCA1 or the BRCA2 gene. BRCA negative was defined as any patient without a known or likely pathogenic alteration in either the BRCA1 or the BRCA2 gene. RB1 positive status was defined as any patient with a known or likely pathogenic alteration (short variant, copy deletion, or rearrangement) in the RB1 gene. Clinicogenomic database (CGDB) Methods Real-world time to treatment discontinuation (TTD) was assessed utilizing the nationwide (US-based) Flatiron Health-Foundation Medicine clinicogenomic database (FH-FMI CGDB). All data were de-identified and originated from approximately 280 US cancer clinics (~ 800 sites of care). The CGDB analysis assessed 2,092 unique patients and only one sample per patient was used for the analysis. Clinical data were derived from electronic health records, comprised of patient-level structured and unstructured data, and were linked to genomic data derived from FoundationOne® or FoundationOne®CDx comprehensive genomic profiling results via de-identified, deterministic matching 31 , 32 . Treatment information was abstracted from oncologist-defined, rule-based lines of therapy. IRB approval of the study protocol was obtained prior to study conduct and included a waiver of informed consent. TTD was calculated from the start of first-line standard of care (receipt of a CDK4/6 inhibitor (abemaciclib, palbociclib, or ribociclib) with endocrine therapy (anastrozole, elacestrant, exemestane, fulvestrant, goserelin, letrozole, leuprolide, megestrol, tamoxifen, or toremifene) in any combination, excluding maintenance therapy) until the time of treatment discontinuation, for any reason, including death. Patients that did not have documentation of death, ending a treatment, or beginning a new treatment were subsequently censored at the date of last clinic visit or structured activity. Survival probability was estimated using the Kaplan-Meier method and survival curves were compared using a log-rank test. Biomarker status was determined from a solid tumor biopsy taken prior to start of therapy and significance was determined by a univariate Cox proportional hazards model. To mitigate bias, at-risk intervals were left-truncated and patients were treated as at risk of death only after the later of their first Foundation Medicine report date and their second visit in the Flatiron Health network, as both are requirements for inclusion in the database. Statistical analysis Data analysis and graph generation were performed using GraphPad Prism 10.5.0 and CGDB analyses were performed using R version 4.3.2. The number of biological replicates is indicated by "n". Statistical significance for in vitro studies was assessed using one-way or two-way analysis of variance (ANOVA), with multiple comparison testing, as specified in the figure legends. Adjusted p-values less than 0.05 were considered statistically significant. Significance levels are denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Data availability All relevant data are provided within the article and its accompanying Supplementary Data. Because of HIPAA requirements, we are not consented to share individualized patient genomic data, which contains potentially identifying or sensitive patient information. Foundation Medicine is committed to collaborative data analysis, and has well-established and widely utilized mechanisms by which investigators can query the core genomic database of >850,000 de-identified sequenced cancers to obtain aggregated datasets. More information and mechanisms for data access can be obtained by contacting the corresponding authors or the Foundation Medicine Data Governance Council at [email protected] Code availability Not applicable. Results Loss of RB1 alters cell proliferation in cancer cell lines To investigate the impact of RB1 or BRCA2 single loss on cell proliferation and response to CDK4/6i, we used the HR+/HER2- breast cancer cell lines MCF7 and T47D and generated RB1 - and BRCA2 -deficient (Fig. 1 a, b and Fig. S1 a, b) cells using an inducible CRISPR/Cas9 system 27 . Single-gene knockout (KO) cells were used to study competitive interactions and investigate selective growth advantages under both untreated conditions and in the presence of the CDK4/6i palbociclib. As expected, we observed that RB1 -deficient MCF7 cells showed significantly enhanced proliferation and outcompeted wild-type cells when co-cultured over a 7-day period in both untreated (Fig. 1 c) and palbociclib-treated conditions (Fig. 1 d). In contrast, loss of BRCA2 provided no growth advantage, irrespective of CDK4/6i treatment (Fig. 1 c, d). A similar trend was observed in the T47D model: while RB1 -deficient cells showed increased proliferation and a competitive advantage, particularly under CDK4/6 inhibition (Fig. S1 c-e), BRCA2 -deficient cells had no advantage under both untreated and palbociclib-treated conditions (Fig. S1 c-e). Co-alteration of RB1 / BRCA2 reduces fitness in vitro To study the interaction between the loss of both RB1 and BRCA2 in HR+/HER2- breast cancer cells, we established double-KO models for MCF7 and T47D cells. For MCF7, we selected two single-cell clones (SCCs) derived from the RB1 (Fig. 2 a) or BRCA2 KO (Fig. 2 b) bulk populations. Then, they were transduced with a doxycycline-inducible lentiviral construct for expression of the additional guide RNA targeting BRCA2 (Fig. 2 c) or RB1 (Fig. 2 d), respectively, resulting in acute induction of a RB1 and BRCA2 double KO. The inducible single- and double-KO was also introduced in the T47D cell line. Here, a RB1 KO bulk population was subsequently transduced with a lentivirus carrying the BRCA2 -specific guide RNA (Fig. S2a). Notably, acute double-KO induction of RB1 and BRCA2 resulted in impaired proliferation (Fig. 2 e and Fig. S2b). In MCF7 cells, the acute induction of the BRCA2 knockout in addition to the pre-existing RB1 -deficiency resulted in significantly decreased proliferation (Fig. 2 e). This effect could also be seen in BRCA2 -deficient MCF7 cells upon induction of the RB1 knockout, although the effect was slightly less pronounced (Fig. 2 e). We speculated that this might be due to increased DNA-damage as both genes, either directly or indirectly, play a role in DNA damage repair 17 , 33 , 34 . Indeed, double KO cells showed increased gamma-H2A.X (γH2A.X) levels (Fig. 2 f), indicating elevated DNA double-strand break (DSB) formation or DNA damage response activation. A similar impact on proliferation was observed upon induction of acute double KO of BRCA2 and RB1 in T47D cells (Fig. S2b). This suggests that the combined loss of these genes provides a growth disadvantage for the cells at steady-state, which is potentially driven, at least in part, by increased DNA damage. However, given that RB1 -deficiency is a major driver of CDK4/6i resistance, co-alteration of RB1 and BRCA2 may still increase selective advantage under therapeutic pressure. RB1 -deficiency is the major driver of CDK4/6i resistance in RB1 / BRCA2 -co-mutated cells Next, we investigated the impact of RB1 and BRCA2 co-loss on sensitivity to CDK4/6i. As expected, palbociclib treatment had no significant effect on cell cycle distribution in RB1 -deficient MCF7 cells, whereas BRCA2 -deficient cells showed an increased G1-phase arrest (Fig. 3 a) and decreased S-phase (Fig. 3 b) indicating therapy sensitivity. However, in RB1 plus BRCA2 KO cells, palbociclib failed to induce efficient G1-phase arrest (Fig. 3 c) and S-phase was significantly less reduced compared to control cells (Fig. 3 d), indicating resistance to CDK4/6 inhibition. The same effect was observed with the alternative CDK4/6i abemaciclib, where BRCA2 loss alone was not sufficient to induce therapy resistance, but only the presence of RB1 deficiency (Fig. 3 e, f). Similar results were observed in T47D cells, where G1-phase arrest and reduced S-phase occurred in control and BRCA2 -deficient cells but were absent in RB1 -deficient or double-KO cells (Fig. S3a,b). Taken together, these results suggest that BRCA2 loss alone does not confer resistance to CDK4/6 inhibition in vitro and resistance is primarily mediated by RB1 deficiency. Although RB1 / BRCA2 co-loss may reduce fitness in untreated conditions, these cells may gain a selective advantage under therapeutic pressure. PARPi are effective in RB1/BRCA2 -deficient cells PARPi, such as olaparib, are used in patients with mBC who progress on ET in combination with CDK4/6i and harbor germline mutations e.g., in BRCA1 or BRCA2 , due to their ability to induce synthetic lethality. To assess whether the PARPi olaparib is also effective in RB1 / BRCA2 -deficient cells, we evaluated their response to olaparib treatment. Upon PARPi treatment, MCF7 RB1 / BRCA2 single- and double-knockout cells exhibited significantly increased G2-phase arrest, a hallmark of PARPi sensitivity 35 , compared to control cells ( lacZ ), which primarily exhibited an increase in G1-phase arrest (Fig. 4 a, b). Notably, after PARPi treatment, RB1 / BRCA2 single- and double- knockout cells displayed a strong reduction in cell count (Fig. 4 c) and live cells (Fig. 4 d), indicating therapy sensitivity. For the p53-mutated T47D cell line, the results were similar. This cell line is already relatively sensitive to olaparib and double knockout of RB1 and BRCA2 did not significantly impact the sensitivity of the cells (Fig. S4). These findings suggest that PARPi are effective in BRCA2 -deficient tumors, regardless of the RB1 status, and might be an alternative treatment method for patients with CDK4/6i-resistance caused by loss of RB1 . RB1 mutations are associated with HRD and loss of 13q Based on our in vitro data and given that patients with BRCA2 mutations have worse prognosis on ET + CDK4/6i treatment, possibly because of the co-loss of RB1 10 , we investigated the frequency of co-alteration of RB1 and HRD-genes, such as BRCA2. Therefore, we analyzed a large genomic database of 55,747 mutational profiles from formalin-fixed paraffin-embedded (FFPE) samples. Only one sample per patient was included. In the absence of BRCA1/2 mutations, RB1 mutations were observed in 7.46% (n = 3930/52664; 95% CI [7.24; 7.69]) of all BC samples. In BRCA1 -mutated ( BRCA1 m) BC samples, the frequency of concurrent RB1 mutations was higher than in BRCA wt (13.84%; n = 289/2088; 95% CI [12.39; 15.40]; p < 0.0001) (Fig. 5 a). For BRCA2 -mutated ( BRCA2 m) samples, 12.79% (n = 341/2666; 95% CI [11.55; 14.12]; p < 0.0001) of all BC samples displayed concurrent RB1 m (Fig. 5 a). The percentage of PALB2 -mutated ( PALB2 m) BC samples with co-alteration in RB1 was 10.74% (n = 78/726; 95% CI [8.59; 13.23]; p < 0.01 v BRCA wt) (Fig. 5 a). These data indicate that mutations in RB1 and HR genes frequently co-occur in BC. Additionally, samples with biallelic loss of BRCA1 or BRCA2 loss exhibited a higher fraction of co-occurring RB1 mutations compared to tumors with monoallelic loss ( BRCA wt: 7.46%; n = 3930/52664; 95% CI [7.24;7.69]; BRCA1 monoallelic: 11.22%; n = 23/205; 95% CI [7.25; 16.36]; BRCA1 biallelic: 15.59%; n = 222/1424; 95% CI [13.74; 17.58]; BRCA2 monoallelic: 6.71%; n = 22/328; 95% CI [4.25; 9.98]; BRCA2 biallelic: 14.76%; n = 279/1890; 95% CI [13.19; 16.44]; PALB2 monoallelic: 12.11%; n = 27/223; 95% CI [8.13; 17.13]; PALB2 biallelic: 9.45%; n = 36/381; 95% CI [6.71; 12.84];) (Fig. 5 b). The most pronounced difference in the frequency of RB1 mutations could be observed between samples with monoallelic and biallelic loss of BRCA2 (Fig. 5 b). Additionally, BRCA2 m samples showed the highest tendency to gain RB1 mutations over time in patients with multiple biopsies (16.04%; n = 17/106; 95% CI [9.63;24.43]; p < 0.0001), when compared to BRCA wt (6.94%; n = 5/72; 95% CI [2.29;15.47]), PALB2 m (5.41%; n = 2/37; 95% CI [0.66; 18.19]) or BRCA1 m (5.03%; n = 124/2465; 95% CI [4.20; 5.97]) samples (Fig. S5a). This reflects possible genomic evolution during disease progression and therapy resistance. RB1 and BRCA2 are located in proximity on chromosome 13q. Analyzing samples from all BC subtypes, we observed that 7.45% (n = 2054/27566; 95% CI [7.14;7.77]) of samples without 13q loss showed mutations in RB1 whereas this was the case for 10.13% of samples with 13q loss (n = 1577/15574; 95% CI [9.66; 10.61]) (loss v no loss: p < 0.0001) (Fig. 5 c). In the HR+/HER2- subset, 4.95% (n = 59/1193; 95% CI [3.79;6.33]) of samples without 13q loss displayed RB1 mutations versus 8.65% (n = 68/786; 95% CI [6.78;10.84]) of samples with loss of 13q (loss v no loss: p < 0.01) (Fig. 5 c). Furthermore, HRD + samples based on genomic scarring (HRDsig+) exhibited a higher frequency of RB1 mutations, regardless of subtype. In all breast samples, we could observe that 6.81% (n = 3125/45907; 95% CI [6.58;7.04]) of HRDsig- samples vs. 14.68% of HRDsig + samples harbor mutations in RB1 (n = 1280/8722; 95% CI [13.94;15.44]) (HRD + vs. HRD-: p < 0.0001). Similar results were observed in the HR+/HER2- subset (HRDsig-: 5.07%; n = 103/2031 95% CI [4.16;6.12]; vs. HRDsig+: 11.71%; n = 35/299; 95% CI [8.29;15.90] p < 0.0001) (Fig. 5 d). Our analysis further revealed that BRCA2 mutations are associated with a higher percentage of mutations in RB1 , especially in recent years (Fig. S5b). Together, these data indicate that HRD and the resulting genomic instability, especially mediated by loss of BRCA2 , are linked to RB1 alterations. HRD and RB1 mutations are associated with reduced time to treatment discontinuation (TTD) We examined the outcomes of patients with HR+/HER2- metastatic breast cancer on 1st line CDK4/6i + ET in a real-world clinco-genomic database. In this setting, BRCA wt patients exhibited a median time to treatment discontinuation (TTD) of 15.0 months. TTD was significantly shorter for patients with BRCA1 m (median TTD of 5.6mo, hazard ratio (HR) relative to BRCA wt of 2.3; p < 0.001) (Fig. 5 e) or BRCA2 (Fig. 5 f) (median TTD of 11.1mo; HR relative to BRCA wt of 1.6; p < 0.001) mutations compared to BRCA wt. This effect was more pronounced when considering only biallelic BRCA1 (median TTD of 4.2mo, HR relative to BRCA wt of 3.8; p < 0.001) (Fig. 5 g) or BRCA2 (median TTD of 10.7mo; HR relative to BRCA wt of 1.8; p < 0.001) (Fig. 5 h) alterations. As a positive control, we examined outcomes in patients with RB1 mutations, which were associated with significantly shorter TTD compared to RB1 wt (median TTD 4.3mo v 15.1mo; HR = 3.2; p < 0.001) (Fig. 5 i). Analysis of TTD in patients harboring both BRCA2 and RB1 mutations revealed a marked reduction in TTD (median TTD 7.7mo v 15.4mo; HR = 3.3; p < 0.001). When considering a scar-based measure of HRD (HRDsig), patients that were HRDsig + had a worse outcome relative to HRDsig- patients (median TTD of 10.7 vs. 15.1 months; HR = 1.6; p < 0.001) (Fig. 5 k). This suggests that HRD, which induces genome-wide LOH and other scars that can induce a second hit, is associated with poor outcomes on 1st line CDK4/6i + ET. Discussion The addition of CDK4/6i to ET has led to increased PFS and OS in BC patients with advanced HR+/HER2- disease 24 , 36 , 37 and is now considered standard of care 3 , regardless of HRD-status. While patients with TNBC and inactivating mutations in HRD-associated genes are treated with PARPi 3 , it remains unclear whether this is an effective treatment option for patients with HR+/HER2- metastatic or early-stage BC. A recent study demonstrated that patients with HR+/HER2- BC and germline BRCA2 mutations experience shorter PFS and OS when treated with CDK4/6i and ET likely as a result of gained RB1 mutations 10 . Although further experimental and clinical data on RB1 / BRCA2 co-alterations HR+/HER2- BC remain limited, studies in prostate cancer have shown that co-deletions are strongly correlated 38 , 39 and linked to increased invasiveness and shorter relapse-free survival 39 . In contrast, patients with high-grade serous ovarian cancer tend to have improved survival outcomes when their tumors exhibit loss of RB1 and BRCA2 40 , highlighting a complex interaction between these genes. Therefore, to further investigate the effects of RB1 and BRCA2 single- and co-alteration in HR+/HER2 BC, we modelled these mutations in vitro . We found that RB1 loss was strongly correlated with increased proliferation, while BRCA2 KO led to decreased cell growth. Notably, loss of BRCA2 did not increase CDK4/6i resistance, while loss of RB1 , as expected, led to increased palbociclib and abemaciclib resistance. Co-loss of BRCA2 and RB1 resulted in significantly increased resistance to CDK4/6i. Given the importance of RB1 in the CDK4/6-pathway, loss of RB1 is a strong predictor of CDK4/6i resistance 16 and was dominant over BRCA2 mutations. Therefore, our data support the recent findings on BRCA2 and RB1 co-alterations in BC leading to increased resistance towards CDK4/6i 10 . To further assess the impact of HRR-associated alterations beyond BRCA2 , such as those in BRCA1 and PALB2 , on response to CDK4/6i in BC, patient data from a large real-world dataset were analyzed. These analyses demonstrated that HRD was correlated with significantly shorter TTD compared to the general cohort of CDK4/6i-treated BC patients. We observed that HRDsig + samples, particularly those harboring BRCA2 mutations, exhibited an increased frequency of RB1 alterations, a phenomenon also seen in prostate 12 and ovarian cancer 40 . Interestingly, our findings indicated that co-alteration of RB1 and BRCA2 negatively impacts cell proliferation in absence of treatment pressure. We hypothesize that this might be a result of the impaired ability to repair DNA double-strand breaks. Strikingly, the enrichment of proliferation-related hallmark gene sets, including E2F targets, G2/M checkpoint regulators and mitotic spindle targets is associated with elevated BRCA2 expression 41 . This occurrence might be due to the essential role of BRCA2 in homologous recombination repair (HRR) 42 . Additionally, RB1 has a non-canonical role in the non-homologous end joining pathway 43 and is crucial to maintain chromosomal integrity 33 , 44 . Hence, inactivation of both genes might increase the dependence of the cells on other error-prone repair pathways, such as single-strand annealing or micro-homology-mediated end joining (MMEJ, also known as alternative end joining). Olaparib, in addition to targeting other PARP family members, also inhibits PARP1 45 , a component of the MMEJ repair pathway 46 . This pathway is upregulated in RB1 -deficient cells 34 , 47 , which may explain why RB1 - and BRCA2 -deficient cells – whether single or co-mutated – exhibit similar responses to olaparib. The increased efficacy of PARPi in RB1 -deficient cells is also seen in osteosarcoma 48 , prostate cancer 49 and lung adenocarcinoma preclinical models 47 . Although to date this has not been studied thoroughly in BC, our findings suggest that PARPi may have the potential to overcome therapy failure in RB1 -deficient, CDK4/6i-resistant breast tumors. Clinical data analysis suggests that patients with HRD may benefit more from PARPi 50 , although it remains uncertain whether PARPi could be a better treatment option than CDK4/6i plus endocrine therapy for patients with HR+/HER2- mBC or early BC. The optimal approach—whether to employ PARPi instead or sequentially with CDK4/6i —requires further investigation. Currently, a phase 3 clinical trial in mBC is performed, screening patients for BRCA1/2 and PALB2 alterations and randomizing them to treatment with the PARP1i saruparib and ET versus CDK4/6i combined with ET in the first-line setting (EvoPAR-Br01; ClinicalTrials.gov identifier: NCT06380751), which may help address some of these questions. Additionally, recent studies have demonstrated that RB1 -deficient cells are sensitive to the BCL-XL inhibitor navitoclax, with co-alteration of BRCA2 further enhancing this sensitivity in preclinical models 51 . These findings are of high relevance for patients with advanced, and potentially also early-stage HR+/HER2- BC, as CDK4/6i are increasingly used for higher risk disease 24 , 25 . Additionally, the OlympiA phase III trial has demonstrated benefit of adjuvant olaparib in patients with BRCA1 / 2 mutations 26 . It is important to determine whether patients with HRD benefit less or not at all from the addition of CDK4/6i, and if there is a benefit, to establish the optimal treatment sequence. Our findings contribute to the growing body of evidence for treatment selection, nevertheless, our study has several limitations that should be considered. First, the functional experiments were conducted in only two cell line models, which may not fully capture the heterogeneity of HR+/HER2- breast cancer. Additionally, we did not include patient-derived organoid (PDO) models or in vivo studies, which are important for validating findings in more physiologically relevant systems. Finally, the analysis of real-world clinical data is inherently limited by potential biases, incomplete clinical annotations, and variability in treatment and follow-up, which may affect the robustness of the observed associations. Therefore, further research is needed to clarify the mechanisms and timing of RB1 mutations in HRD patients. In conclusion, we propose that patients with HR+/HER2- BC should be evaluated for the presence of an HRD-signature prior to initiating treatment with CDK4/6i. Our findings demonstrate that CDK4/6i are effective in BRCA2 -deficient cell line models. However, given the higher prevalence of RB1 co-alterations observed in patients with BRCA2 mutations, PARPi may represent a more effective therapeutic option for this subset of patients. Therefore, while identifying HR+/HER2- patients with these alterations at diagnosis requires extensive screening, collaborative efforts are essential to optimize treatment strategies for this highly selected BC patient population. Declarations Acknowledgements The authors thank Petra Franzmayr, BSc, Sophie Kienreich, MSc and Bettina Flasch, MSc for their technical support. Cell sorting was performed at the Flow Cytometry Core Facility of the Center for Medical Research, Medical University of Graz. D.H. is funded by the Medical University of Graz within the PhD Program “Molecular Medicine”. Author contribution D.H.: Conceptualization; acquisition, analysis, and interpretation of data; visualization; writing – original draft/review & editing. M.A.D. and M.B.: Conceptualization; project supervision; interpretation of data; writing – original draft/review & editing. E.S.S. and M.M.: Data curation; data analysis and interpretation; writing – original draft/review & editing. S.W., U.S. and M.K.: Investigation; data acquisition and analysis; writing – review & editing. S.O., A.V.L., C.E.G, P.J.J., N.D.: Data interpretation; writing – review & editing. All authors have read and agreed to the version of the manuscript. Grant support Nothing to declare. Competing interests E.S.S. and M.M. are Employees at Foundation Medicine and Shareholders in Roche. M.B. received honoraria from AstraZeneca, Daiichi Sankyo, Eli Lilly, Gilead, Menarini, MSD, Novartis, Pierre Fabre, Pfizer, Seagen, Stemline, and research funding from Astra Zeneca, Daiichi Sankyo, Novartis, and Pfizer. 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Fulvestrant plus palbociclib versus fulvestrant plus placebo for treatment of hormone-receptor-positive, HER2-negative metastatic breast cancer that progressed on previous endocrine therapy (PALOMA-3): final analysis of the multicentre, double-blind, phase 3 randomised controlled trial. Lancet Oncol. 17, 425–439 (2016). Kensler, K. H., Baichoo, S., Pathania, S. & Rebbeck, T. R. The tumor mutational landscape of BRCA2-deficient primary and metastatic prostate cancer. Npj Precis. Oncol. 6, 39 (2022). Lozano, R. et al. Impact of concurrent tumour events on the prostate cancer outcomes of germline BRCA2 mutation carriers. Eur. J. Cancer 185, 105–118 (2023). Saner, F. A. M. et al. Concurrent RB1 Loss and BRCA Deficiency Predicts Enhanced Immunologic Response and Long-term Survival in Tubo-ovarian High-grade Serous Carcinoma. Clin. Cancer Res. 30, 3481–3498 (2024). Satyananda, V., Oshi, M., Endo, I. & Takabe, K. High BRCA2 Gene Expression is Associated with Aggressive and Highly Proliferative Breast Cancer. Ann. Surg. Oncol. 28, 7356–7365 (2021). Groelly, F. J., Fawkes, M., Dagg, R. A., Blackford, A. N. & Tarsounas, M. Targeting DNA damage response pathways in cancer. Nat. Rev. Cancer 23, 78–94 (2023). Cook, R. et al. Direct Involvement of Retinoblastoma Family Proteins in DNA Repair by Non-homologous End-Joining. Cell Rep. 10, 2006–2018 (2015). Vélez-Cruz, R. & Johnson, D. The Retinoblastoma (RB) Tumor Suppressor: Pushing Back against Genome Instability on Multiple Fronts. Int. J. Mol. Sci. 18, 1776 (2017). Murai, J. et al. Trapping of PARP1 and PARP2 by Clinical PARP Inhibitors. Cancer Res. 72, 5588–5599 (2012). Mansour, W. Y., Rhein, T. & Dahm-Daphi, J. The alternative end-joining pathway for repair of DNA double-strand breaks requires PARP1 but is not dependent upon microhomologies. Nucleic Acids Res. 38, 6065–6077 (2010). Dong, Q. et al. Mutant RB1 enhances therapeutic efficacy of PARPis in lung adenocarcinoma by triggering the cGAS/STING pathway. JCI Insight 8, e165268 (2023). Zoumpoulidou, G. et al. Therapeutic vulnerability to PARP1,2 inhibition in RB1-mutant osteosarcoma. Nat. Commun. 12, 7064 (2021). Jiang, B. et al. Case Report: Two cases of chemotherapy refractory aggressive variant prostate cancer with extreme durable response to PARP inhibitor. Front. Oncol. 15, 1533627 (2025). Batalini, F. et al. Homologous Recombination Deficiency Landscape of Breast Cancers and Real-World Effectiveness of Poly ADP-Ribose Polymerase Inhibitors in Patients With Somatic BRCA1 / 2 , Germline PALB2 , or Homologous Recombination Deficiency Signature. JCO Precis. Oncol. e2300091 (2023) doi: 10.1200/PO.23.00091 . Varkaris, A. et al. BH3 mimetics targeting BCL-XL have efficacy in solid tumors with RB1 loss and replication stress. Nat. Commun. 16, 4931 (2025). Additional Declarations Competing interest reported. E.S.S. and M.M. are Employees at Foundation Medicine and Shareholders in Roche. M.B. received honoraria from AstraZeneca, Daiichi Sankyo, Eli Lilly, Gilead, Menarini, MSD, Novartis, Pierre Fabre, Pfizer, Seagen, Stemline, and research funding from Astra Zeneca, Daiichi Sankyo, Novartis, and Pfizer. C.E.G. received research funding to institutions, travel support to attend research meetings and provide lectures, honoraria for lectures from AstraZeneca and research funding to institutions and travel support to attend research meetings from Daiichi Sankyo, Genentech, Roche and Exact Sciences. Supplementary Files HaasetalSupplementary.docx Cite Share Download PDF Status: Posted Version 1 posted 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-7253927","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":498522183,"identity":"3bacbcdc-c80e-46dd-a812-96812fe6a13a","order_by":0,"name":"Daniela Haas","email":"","orcid":"","institution":"Medical University of Graz","correspondingAuthor":false,"prefix":"","firstName":"Daniela","middleName":"","lastName":"Haas","suffix":""},{"id":498522184,"identity":"c5007b06-81d0-401d-8ab9-f4c33cd932d7","order_by":1,"name":"Selina Wolf","email":"","orcid":"","institution":"Medical University of Graz","correspondingAuthor":false,"prefix":"","firstName":"Selina","middleName":"","lastName":"Wolf","suffix":""},{"id":498522185,"identity":"206cf1a2-2cdd-4dfe-ad09-a0e92af5bf40","order_by":2,"name":"Urtė Stankutė","email":"","orcid":"","institution":"Medical University of Graz","correspondingAuthor":false,"prefix":"","firstName":"Urtė","middleName":"","lastName":"Stankutė","suffix":""},{"id":498522186,"identity":"d1725ca7-e605-4951-a6ab-9fe24b185dc8","order_by":3,"name":"Ethan S. 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Changes in ratio of \u003cem\u003elacZ\u003c/em\u003e, \u003cem\u003eRB1\u003c/em\u003eKO or \u003cem\u003eBRCA2\u003c/em\u003e KO vs. wild-type MCF7 cells were measured on day 4 and day 7 using flow cytometry, as \u003cem\u003eRB1\u003c/em\u003e KO and \u003cem\u003elacZ\u003c/em\u003e are fluorescent (Supplementary Materials and Methods). Data represent mean ± SD. Right panel: statistical analysis. Data represent mean ± SD, Ordinary 2-way ANOVA, Šídák’s multiple comparisons test. \u003cstrong\u003ed\u003c/strong\u003e: Same as e, but cells were treated with 250nM palbociclib on Day 1.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7253927/v1/55221c544f8c29f3d1a20efd.png"},{"id":88948782,"identity":"46375d8c-6ae4-4da0-aebf-4c3592aea2a0","added_by":"auto","created_at":"2025-08-13 05:36:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":126636,"visible":true,"origin":"","legend":"\u003cp\u003eGeneration and proliferation analysis of \u003cem\u003eRB1\u003c/em\u003e/\u003cem\u003eBRCA2\u003c/em\u003edouble-KO \u003cem\u003ein vitro\u003c/em\u003e models.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e: RB1 protein expression in MCF7 single cell clones (SCC). SCC4 and SCC6 show complete loss of RB1. \u003cstrong\u003eb\u003c/strong\u003e: BRCA2 protein expression in MCF7 single cell clones (SCC). SCC1 and SCC3 show complete loss of BRCA2. \u003cstrong\u003ec\u003c/strong\u003e: \u003cem\u003eRB1\u003c/em\u003e KO SCC4 and SCC6 were transduced with lentivirus for acute KO of \u003cem\u003eBRCA2\u003c/em\u003e via addition of Doxycycline (DOX). \u003cstrong\u003ed\u003c/strong\u003e: \u003cem\u003eBRCA2\u003c/em\u003eKO SCC1 and SCC3 were transduced with lentivirus for acute KO of \u003cem\u003eRB1\u003c/em\u003e via addition of DOX.\u003cstrong\u003e e\u003c/strong\u003e: Cell counting experiment (Supplementary Materials and Methods) of MCF7 \u003cem\u003elacZ\u003c/em\u003e, \u003cem\u003eRB1\u003c/em\u003e KO SCC4/6 (pooled) and \u003cem\u003eBRCA2\u003c/em\u003eKO SCC1/3 (pooled) after 72h of gRNA expression. Cell count of induced population was normalized to uninduced. Data represent mean ± SD, n=3 for \u003cem\u003elacZ\u003c/em\u003e, n=6 for \u003cem\u003eRB1\u003c/em\u003e KO and \u003cem\u003eBRCA2\u003c/em\u003e KO (pooled SCC), Ordinary 2-way ANOVA, Šídák’s multiple comparisons test. \u003cstrong\u003ef\u003c/strong\u003e: Immunoblot of gH2AX (Ser139) in MCF7 cells.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7253927/v1/f98d537559ec6c214526f289.png"},{"id":88948788,"identity":"f80bd187-21b8-4377-900d-5566716fae81","added_by":"auto","created_at":"2025-08-13 05:36:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":623467,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eRB1\u003c/em\u003e/\u003cem\u003eBRCA2\u003c/em\u003e single- and double-KO on CDK4/6i response.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e: Cell cycle analysis of MCF7 cells harboring \u003cem\u003elacZ\u003c/em\u003e, \u003cem\u003eRB1\u003c/em\u003e KO and \u003cem\u003eBRCA2\u003c/em\u003e KO mutation in the presence and absence of the CDK4/6i palbociclib (250nM, 72h). Data represent mean ± SD, n=3. \u003cstrong\u003eb\u003c/strong\u003e: Analysis of changes in percentage of MCF7 cells in S-phase of (a). Data represent mean ± SD, Ordinary 2-way ANOVA, Šídák’s multiple comparisons test. \u003cstrong\u003ec\u003c/strong\u003e: Cell cycle analysis of MCF7 \u003cem\u003elacZ\u003c/em\u003e and \u003cem\u003eRB1\u003c/em\u003e KO SCC 4/6 + \u003cem\u003eBRCA2\u003c/em\u003e KO in the presence and absence of the CDK4/6i palbociclib (250nM, 72h). Data represent mean ± SD, n=3 for \u003cem\u003elacZ\u003c/em\u003e, n=6 for others (pooled SCC4 and SCC6 with induced \u003cem\u003eBRCA2\u003c/em\u003e KO). \u003cstrong\u003ed\u003c/strong\u003e: Analysis of changes in percentage of MCF7 cells in S-phase of (c). Data represent mean ± SD, Ordinary 2-way ANOVA, Šídák’s multiple comparisons test. \u0026nbsp;\u003cstrong\u003ee\u003c/strong\u003e: Cell cycle analysis of MCF7 \u003cem\u003elacZ\u003c/em\u003e, \u003cem\u003eRB1\u003c/em\u003e KO SCC4/6, \u003cem\u003eBRCA2\u003c/em\u003e KO SCC1/3 and \u003cem\u003eRB1\u003c/em\u003e KO SCC4/6 + \u003cem\u003eBRCA2\u003c/em\u003eKO cells in the presence and absence of the CDK4/6i abemaciclib (250nM, 72h). Data represent mean ± SD, n=3 for \u003cem\u003elacZ\u003c/em\u003e, n=6 for others (pooled SCC) \u003cstrong\u003ef\u003c/strong\u003e: Analysis of changes in percentage of MCF7 cells in S-phase of (e). Data represent mean ± SD, Ordinary 2-way ANOVA, Šídák’s multiple comparisons test.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7253927/v1/399e4d7c7e14a31d38c34ca6.png"},{"id":88950089,"identity":"80c72a32-b79d-4476-8659-7f891ab4715b","added_by":"auto","created_at":"2025-08-13 05:44:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":503247,"visible":true,"origin":"","legend":"\u003cp\u003eOlaparib is effective in \u003cem\u003eRB1\u003c/em\u003e-deficient CDK4/6i resistant cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e: Cell cycle analysis of MCF7 \u003cem\u003elacZ\u003c/em\u003e, \u003cem\u003eRB1 \u003c/em\u003eKO SCC4/6\u003cem\u003e \u003c/em\u003e+ \u003cem\u003eBRCA2\u003c/em\u003eKOs and \u003cem\u003eBRCA2 \u003c/em\u003eKO SCC1/3+ \u003cem\u003eRB1\u003c/em\u003e KO cells after treatment with olaparib (10µM, 96h). Data represent mean ± SD, n=3 for \u003cem\u003elacZ\u003c/em\u003e, n=6 for others (pooled SCC). \u003cstrong\u003eb\u003c/strong\u003e: Percentage of cells in G2/M-phase of (a). Data represent mean ± SD, Ordinary 2-way ANOVA, Šídák’s multiple comparisons test. \u003cstrong\u003ec\u003c/strong\u003e: Cell counting (Supplementary Materials and Methods) experiment of MCF7 \u003cem\u003elacZ\u003c/em\u003e, \u003cem\u003eRB1 \u003c/em\u003eKO SCC4/6\u003cem\u003e, BRCA2 \u003c/em\u003eKO SCC1/3\u003cem\u003e \u003c/em\u003eand\u003cem\u003eRB1 \u003c/em\u003eKO SCC4/6\u003cem\u003e + BRCA2 \u003c/em\u003eKO cells after 96h of olaparib treatment (10µM). Data represent mean ± SD, n=3 for \u003cem\u003elacZ\u003c/em\u003e, n=6 for others (pooled SCC), Ordinary 2-way ANOVA, Šídák’s multiple comparisons test. \u003cstrong\u003ed\u003c/strong\u003e: Percentage of live MCF7 \u003cem\u003elacZ\u003c/em\u003e, \u003cem\u003eRB1 \u003c/em\u003eKO SCC4/6\u003cem\u003e, BRCA2 \u003c/em\u003eKO SCC1/3\u003cem\u003e \u003c/em\u003eand\u003cem\u003e RB1 \u003c/em\u003eKO SCC4/6\u003cem\u003e+ BRCA2 \u003c/em\u003eKO cells after 96h of olaparib treatment (10µM). Data represent mean ± SD, n=3 for \u003cem\u003elacZ\u003c/em\u003e, n=6 for others (pooled SCC), Ordinary 2-way ANOVA, Šídák’s multiple comparisons test.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7253927/v1/5c63439a0d068e4ac6aa65f4.png"},{"id":88948790,"identity":"932a1bad-2778-4627-a688-e06101898288","added_by":"auto","created_at":"2025-08-13 05:36:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":310538,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRB1\u003c/em\u003em is associated with mutations in HRD genes and shorter time to treatment discontinuation (TTD) after first-line CDK4/6i + ET.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e: Assessment of the percentage of \u003cem\u003eRB1\u003c/em\u003emutations (\u003cem\u003eRB1\u003c/em\u003em) in \u003cem\u003eBRCA1/2 \u003c/em\u003ewild-type (\u003cem\u003eBRCA\u003c/em\u003e_wt), \u003cem\u003eBRCA1\u003c/em\u003emutated (\u003cem\u003eBRCA1\u003c/em\u003em),\u003cem\u003e BRCA2\u003c/em\u003e mutated (\u003cem\u003eBRCA2\u003c/em\u003em) and \u003cem\u003ePALB2\u003c/em\u003emutated (\u003cem\u003ePALB2\u003c/em\u003em) clinical samples. \u003cstrong\u003eb\u003c/strong\u003e: Analysis of % of \u003cem\u003eRB1\u003c/em\u003em in clinical samples with \u003cem\u003eBRCA\u003c/em\u003e-wt, monoallelic (mono) or biallelic (bi) co mutation in \u003cem\u003eBRCA1\u003c/em\u003e, \u003cem\u003eBRCA2\u003c/em\u003e and \u003cem\u003ePALB2\u003c/em\u003e. \u003cstrong\u003ec\u003c/strong\u003e: Percentage of \u003cem\u003eRB1\u003c/em\u003em stratified by 13q status in clinical samples from all breast cancer subtypes (all breast) and HR+/HER2− breast cancer. \u003cstrong\u003ed\u003c/strong\u003e: Percentage of \u003cem\u003eRB1\u003c/em\u003em stratified by HRDsig in clinical samples from all breast cancer subtypes (all breast) and HR+/HER2− breast cancer. \u003cstrong\u003ea-d:\u003c/strong\u003eData represent mean and 95% confidence interval (CI). \u003cstrong\u003ee-k\u003c/strong\u003e: Time to treatment discontinuation analysis (TTD). Outcomes were stratified by biomarker status based on sequencing results of a solid tumor biopsy taken prior to therapy start in patients who were \u003cstrong\u003ee\u003c/strong\u003e: \u003cem\u003eBRCA1\u003c/em\u003e mutant positive, \u003cstrong\u003ef\u003c/strong\u003e: \u003cem\u003eBRCA2\u003c/em\u003e mutant positive, \u003cstrong\u003eg\u003c/strong\u003e: biallelic \u003cem\u003eBRCA1\u003c/em\u003e mutant positive, \u003cstrong\u003eh\u003c/strong\u003e: biallelic \u003cem\u003eBRCA2\u003c/em\u003e mutant positive, \u003cstrong\u003ei\u003c/strong\u003e: \u003cem\u003eRB1\u003c/em\u003e mutant positive, \u003cstrong\u003ej\u003c/strong\u003e: \u003cem\u003eBRCA2\u003c/em\u003e biallelic and \u003cem\u003eRB1\u003c/em\u003e mutant positive and \u003cstrong\u003ek\u003c/strong\u003e:\u003cstrong\u003e \u003c/strong\u003eHRD positive. \u003cem\u003eBRCA\u003c/em\u003e negative patients were defined as those who did not have a known or likely pathogenic alteration in either the \u003cem\u003eBRCA1\u003c/em\u003e or \u003cem\u003eBRCA2\u003c/em\u003egene.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7253927/v1/dd65033200fe367de51013a0.png"},{"id":89325380,"identity":"30332e9b-d68e-4245-9654-0216c44945fd","added_by":"auto","created_at":"2025-08-18 20:16:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3792070,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7253927/v1/444d8458-e890-4957-a941-ca0276db57fb.pdf"},{"id":88948789,"identity":"e10238a9-c4c4-4d93-8f9a-9658b5a1ffd1","added_by":"auto","created_at":"2025-08-13 05:36:16","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1672838,"visible":true,"origin":"","legend":"","description":"","filename":"HaasetalSupplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-7253927/v1/0fe48941e09035bf8f6e4ef8.docx"}],"financialInterests":"Competing interest reported. E.S.S. and M.M. are Employees at Foundation Medicine and Shareholders in Roche. M.B. received honoraria from AstraZeneca, Daiichi Sankyo, Eli Lilly, Gilead, Menarini, MSD, Novartis, Pierre Fabre, Pfizer, Seagen, Stemline, and research funding from Astra Zeneca, Daiichi Sankyo, Novartis, and Pfizer. C.E.G. received research funding to institutions, travel support to attend research meetings and provide lectures, honoraria for lectures from AstraZeneca and research funding to institutions and travel support to attend research meetings from Daiichi Sankyo, Genentech, Roche and Exact Sciences.","formattedTitle":"\u003cp\u003eImpact of homologous recombination deficiency on CDK4/6 inhibitor sensitivity in HR+/HER2-breast cancer\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWhile advancements in screening methods and innovative therapeutic strategies have significantly improved the overall survival of breast cancer (BC) patients over the past few decades\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, metastatic BC (mBC) continues to be an incurable disease and the leading cause of cancer-related death in women\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The standard of care for advanced hormone-receptor positive/human epidermal growth factor receptor 2 negative (HR+/HER2-) BC involves endocrine therapy (ET) in combination with cyclin-dependent-kinase 4/6 inhibitors (CDK4/6i) such as palbociclib, abemaciclib and ribociclib\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. These treatment combinations significantly increase progression-free survival (PFS)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e and overall survival (OS)\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e in mBC. However, disease progression is frequently observed and acquired therapy resistance constitutes a significant clinical problem in patients with mBC\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMutations impacting cell cycle genes, such as upregulation of CDK2 or FGFR1, have been described leading to CDK4/6i resistance and disease progression\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. However, recent findings show that patients with germline pathogenic variants (PVs) in the \u003cem\u003eBRCA2\u003c/em\u003e gene have a shorter PFS when treated with ET combined with CDK4/6i\u003csup\u003e10\u003c/sup\u003e. Inactivating mutations or loss of heterozygosity (LOH) of \u003cem\u003eBRCA2\u003c/em\u003e have been linked to BC development and are observed in approximately 30\u0026ndash;40% of patients with sporadic disease\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Yet, the precise mechanism by which \u003cem\u003eBRCA2\u003c/em\u003e mutations confer resistance to CDK4/6i remains unclear. A recent report suggests that \u003cem\u003eBRCA2\u003c/em\u003e PVs may not directly cause resistance, but instead promote CDK4/6i resistance due to the close genomic proximity of \u003cem\u003eBRCA2\u003c/em\u003e and retinoblastoma protein 1 (\u003cem\u003eRB1\u003c/em\u003e) on chromosome 13q, which can result in the simultaneous loss of both genes\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. This phenomenon has previously been described in prostate cancer where it is associated with epithelial-mesenchymal transition, higher grade of invasiveness and poor clinical outcome\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Since RB1 is a direct downstream target of CDK4/6, its inactivation or LOH confers significant resistance against CDK4/6 inhibition\u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThus far, \u003cem\u003eBRCA2\u003c/em\u003e alterations are primarily associated with homologous recombination deficiency (HRD)\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. HRD is associated with an elevated BC risk and an elevated mutational burden, resulting not only from \u003cem\u003eBRCA2\u003c/em\u003e loss but also from mutations in other genes involved in homologous recombination repair, such as \u003cem\u003eBRCA1\u003c/em\u003e or partner and localizer of \u003cem\u003eBRCA2 (PALB2)\u003c/em\u003e\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Inactivating mutations in \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003ePALB2\u003c/em\u003e are more frequently observed in patients with triple-negative BC (TNBC)\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, whereas \u003cem\u003eBRCA2\u003c/em\u003e mutations are associated with both TNBC and HR+/HER2- BC\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eBRCA\u003c/em\u003e-mutated (\u003cem\u003eBRCA\u003c/em\u003em) triple-negative breast cancer (TNBC), PARP inhibitors (PARPi) are the preferred treatment, whereas standard care for HR+/HER2- mBC typically consists of ET alone or combined with CDK4/6i, irrespective of HRD status\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, in early-stage BC, both CDK4/6i as well as PARPi have been approved for treatment and are associated with improved disease-free survival (DFS) and OS in independent studies\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Nevertheless, it remains uncertain whether PARPi would be a better option compared to CDK4/6i in combination with ET for HRD-positive HR+/HER2- mBC cases.\u003c/p\u003e\u003cp\u003eUsing CRISPR-engineered HR+/HER2- breast cancer cell lines, we show here that \u003cem\u003eRB1\u003c/em\u003e loss promoted CDK4/6i resistance, while \u003cem\u003eBRCA2\u003c/em\u003e-deficient cells remained CDK4/6i-sensitive. Interestingly, while associated with increased sensitivity to PARPi, dual loss of \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e led to marked resistance to CDK4/6 inhibitors. Genomic profiling revealed that not only \u003cem\u003eBRCA2\u003c/em\u003e loss, but HRD in general is linked to shorter time to treatment discontinuation and \u003cem\u003eRB1\u003c/em\u003e co-loss in real world clinical data. Our findings highlight the need for a more comprehensive understanding of how HRD \u0026ndash; beyond \u003cem\u003eBRCA2\u003c/em\u003e mutations \u0026ndash; contributes to CDK4/6i resistance and the incidence of \u003cem\u003eRB1\u003c/em\u003e alterations. These data also suggest the need for more tailored and optimized treatment strategies for patients with \u003cem\u003eBRCA2\u003c/em\u003e mutations or other forms of HRD.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eCell culture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMCF7 and T47D cells were obtained from the Tissue Culture Core Facility at the Medical University of Graz. All cell lines were authenticated prior to use and routinely checked for mycoplasma contamination. MCF7 cells were cultivated in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) and T47D cells were cultivated in high-glucose DMEM (both from Gibco\u0026trade; by Thermo Fisher Scientific). Media were supplemented with 10% fetal bovine serum (Serana Europe GmbH), 1% Antibiotic-Antimycotic and 2 mM L-glutamine (both from Gibco\u0026trade; by Thermo Fisher Scientific). Both cell lines were grown as a monolayer and maintained at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% humidity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCRISPR/Cas9 mediated genome engineering\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn order to establish \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e knockout cell lines, the protocol published by Andrew J. Kueh and Marco J. Herold, WEHI, Melbourne was followed\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBriefly, guide RNAs targeting \u003cem\u003eRB1\u003c/em\u003e, \u003cem\u003eBRCA2\u003c/em\u003e and \u003cem\u003elacZ\u003c/em\u003e were designed using CRISPick and ordered from Eurofins Genomics. The guide RNAs were cloned into two different inducible lentiviral expression vectors with different fluorescent reporters. For the \u003cem\u003eBRCA2\u003c/em\u003e and \u003cem\u003elacZ\u003c/em\u003e guide RNA, Fgh1tUTG (Addgene plasmid #70183), and for the \u003cem\u003eRB1\u003c/em\u003e guide RNA either Fgh1tUTG (Addgene plasmid #70183) or FgH1tUTCyan (Addgene plasmid #70183) was used. Cas9 protein was expressed constitutively by the vector FUCas9mCherry (Addgene plasmid #70182). The plasmids were amplified by High Efficiency Transformation of NEB\u0026reg; Stable Competent \u003cem\u003eE.coli\u003c/em\u003e (C3040H) and isolated using the Plasmid Miniprep Kit by Monarch. Correct insertion of the sgRNA in the lentiviral vector was confirmed by sequencing using the Mix2Seq Kit (Eurofins). Lentiviruses were generated using packaging vectors pMDL/pRRE (Addgene plasmid #12251), pRSV-rev (Addgene plasmid #12253) and pVSV-G (Addgene plasmid #138479), as well as the respective plasmid for guide RNA or Cas9 in 293FT cells (Tissue Culture Core Facility, Medical University of Graz). MCF7 and T47D cells were infected by adding 1 ml of lentivirus containing Cas9 and guide RNA followed by a 2 hour centrifugation (2200 rpm, 32\u0026deg;C). Cells were sorted by flow cytometry and the expression of the guide RNA was induced by doxycycline (Merck, Darmstadt, Germany). Bulk or single cell clones were used for further experiments.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunoblotting\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were harvested and lysed in cold Pierce\u0026trade; RIPA Buffer containing Protease Inhibitor Cocktail (both from Thermo Fisher Scientific). Protein concentration was measured using the BCA Protein Assay Kit (EMD Millipore Corporation). The samples were diluted in RIPA Buffer to the desired concentration. Laemmli Buffer (Bio-Rad) was added and the samples were heated to 95\u0026deg;C at 650 rpm for 10 minutes. After separating the proteins by SDS-PAGE, the proteins were transferred onto nitrocellulose (Bio-Rad) or Hybond\u0026reg; PVDF (Amersham\u0026trade;) membranes. After the transfer, the membranes were blocked in 5% skim milk for 1h. Then, the membranes were incubated with following antibodies overnight at 4\u0026deg;C: Rb (4H1) Mouse mAb (#9309, Cell Signaling Technology), BRCA2 (D9S6V) Rabbit mAb (#10741, Cell Signaling Technology), Phospho-Histone H2A.X (Ser 139) (20E3) Rabbit mAB (#9718, Cell Signaling Technology), HSP70 (produced at WEHI antibody facility, Melbourne, Australia), Anti-mouse IgG, HRP-linked Antibody (#7076, Cell Signaling Technology) and Anti-rabbit IgG, HRP-linked Antibody (#7074, Cell Signaling Technology). Proteins were detected using Immobilon\u0026reg; Classico Western HRP substrate (Millipore).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell cycle analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were harvested and washed two times before they were fixed by adding cold 70% ethanol dropwise on the cell pellet. After fixation for at least 30 minutes at 4\u0026deg;C, cells were washed two times in PBS followed by adding ribonuclease A (Thermo Fisher Scientific). After adding the propidium iodide solution (Sigma Aldrich), the suspension was incubated for 10 minutes at room temperature (RT) in the dark. The PI signal was measured at 605 nm with the CytoFLEX S (Beckman Coulter GmbH) flow cytometer. Data were analyzed using Modfit LT\u0026trade; 5.0 software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell viability analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo analyze apoptosis by flow cytometry, cells were harvested, washed two times in PBS and resuspended in Annexin A5 binding buffer (0.01 M HEPES, 0.14 M NaCl, 2.5 mM CaCl\u003csub\u003e2\u003c/sub\u003e) containing allophycocyanine (APC) - Annexin V (Biolegend) and 7-Aminoactinomycin D (7-AAD) (Biolegend) per sample. After incubation for 15 minutes at RT in the dark, cells were analyzed by the CytoFLEX S flow cytometer (Beckman Coulter GmbH) at 660 nm. Data were analyzed using CytExpert 2.6.\u003c/p\u003e\u003cp\u003e\u003cb\u003eComprehensive Genomic Profiling\u003c/b\u003e\u003c/p\u003e\u003cp\u003eComprehensive genomic profiling of formalin-fixed, paraffin-embedded (FFPE) tissue sections from breast cancer biopsies (local, metastatic and lymph nodes) was performed using FoundationOne\u0026reg; or FoundationOne\u0026reg;CDx in a Clinical Laboratory Improvement Amendments\u0026ndash;certified, College of American Pathologists\u0026ndash;accredited laboratory (Foundation Medicine Inc.). Hybrid capture was carried out on at least 324 cancer-related genes and selected introns from 34 genes frequently rearranged in cancer, as previously described\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. For the BC cohort, HER2 status was determined from the HER2 (\u003cem\u003eERBB2\u003c/em\u003e) amplification status based on the FoundationOne\u0026reg; or FoundationOne\u0026reg;CDx assay and ER status was derived from accompanying pathology reports, where available. Approval for this study, including a waiver of informed consent and Health Insurance Portability and Accountability Act (HIPAA) waiver of authorization, was obtained from the Western Institutional Review Board. Prevalence and co-occurrence were compared using a Fisher\u0026rsquo;s exact test implemented using the SciPy stats package in Python2.\u003c/p\u003e\u003cp\u003eAn HRD signature status (HRDsig) was calculated from the genomic profiling results. The HRDsig algorithm evaluates genome-wide copy number patterns linked to HRD using a comprehensive set of copy number features\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Scores range from 0 to 1, with a predefined cutoff of 0.7, determined from prior analyses demonstrating 90% sensitivity for identifying biallelic \u003cem\u003eBRCA\u003c/em\u003e alterations in \u003cem\u003eBRCA\u003c/em\u003e-driven tumors.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDefinition of\u003c/b\u003e \u003cb\u003eBRCA1\u003c/b\u003e, \u003cb\u003eBRCA2\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eRB1\u003c/b\u003e \u003cb\u003epositive status\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e positive status were defined based on the presence of a known or likely pathogenic alteration in the \u003cem\u003eBRCA1\u003c/em\u003e or \u003cem\u003eBRCA2\u003c/em\u003e gene. Biallelic \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e positive status was defined as any patient who had at least two known or likely pathogenic short variant alterations, at least one known or likely pathogenic homozygous short variant alteration, a homozygous deletion, or at least two known or likely pathogenic rearrangements in the \u003cem\u003eBRCA1\u003c/em\u003e or the \u003cem\u003eBRCA2\u003c/em\u003e gene. \u003cem\u003eBRCA\u003c/em\u003e negative was defined as any patient without a known or likely pathogenic alteration in either the \u003cem\u003eBRCA1\u003c/em\u003e or the \u003cem\u003eBRCA2\u003c/em\u003e gene. \u003cem\u003eRB1\u003c/em\u003e positive status was defined as any patient with a known or likely pathogenic alteration (short variant, copy deletion, or rearrangement) in the \u003cem\u003eRB1\u003c/em\u003e gene.\u003c/p\u003e\u003cp\u003e\u003cb\u003eClinicogenomic database (CGDB) Methods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eReal-world time to treatment discontinuation (TTD) was assessed utilizing the nationwide (US-based) Flatiron Health-Foundation Medicine clinicogenomic database (FH-FMI CGDB). All data were de-identified and originated from approximately 280 US cancer clinics (~\u0026thinsp;800 sites of care). The CGDB analysis assessed 2,092 unique patients and only one sample per patient was used for the analysis. Clinical data were derived from electronic health records, comprised of patient-level structured and unstructured data, and were linked to genomic data derived from FoundationOne\u0026reg; or FoundationOne\u0026reg;CDx comprehensive genomic profiling results via de-identified, deterministic matching\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Treatment information was abstracted from oncologist-defined, rule-based lines of therapy. IRB approval of the study protocol was obtained prior to study conduct and included a waiver of informed consent.\u003c/p\u003e\u003cp\u003eTTD was calculated from the start of first-line standard of care (receipt of a CDK4/6 inhibitor (abemaciclib, palbociclib, or ribociclib) with endocrine therapy (anastrozole, elacestrant, exemestane, fulvestrant, goserelin, letrozole, leuprolide, megestrol, tamoxifen, or toremifene) in any combination, excluding maintenance therapy) until the time of treatment discontinuation, for any reason, including death. Patients that did not have documentation of death, ending a treatment, or beginning a new treatment were subsequently censored at the date of last clinic visit or structured activity. Survival probability was estimated using the Kaplan-Meier method and survival curves were compared using a log-rank test. Biomarker status was determined from a solid tumor biopsy taken prior to start of therapy and significance was determined by a univariate Cox proportional hazards model. To mitigate bias, at-risk intervals were left-truncated and patients were treated as at risk of death only after the later of their first Foundation Medicine report date and their second visit in the Flatiron Health network, as both are requirements for inclusion in the database.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData analysis and graph generation were performed using GraphPad Prism 10.5.0 and CGDB analyses were performed using R version 4.3.2. The number of biological replicates is indicated by \"n\". Statistical significance for \u003cem\u003ein vitro\u003c/em\u003e studies was assessed using one-way or two-way analysis of variance (ANOVA), with multiple comparison testing, as specified in the figure legends. Adjusted p-values less than 0.05 were considered statistically significant. Significance levels are denoted as follows: * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and **** p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll relevant data are provided within the article and its accompanying Supplementary Data. Because of HIPAA requirements, we are not consented to share individualized patient genomic data, which contains potentially identifying or sensitive patient information. Foundation Medicine is committed to collaborative data analysis, and has well-established and widely utilized mechanisms by which investigators can query the core genomic database of \u0026gt;850,000 de-identified sequenced cancers to obtain aggregated datasets. More information and mechanisms for data access can be obtained by contacting the corresponding authors or the Foundation Medicine Data Governance Council at [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eLoss of\u003c/b\u003e \u003cb\u003eRB1\u003c/b\u003e \u003cb\u003ealters cell proliferation in cancer cell lines\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the impact of \u003cem\u003eRB1\u003c/em\u003e or \u003cem\u003eBRCA2\u003c/em\u003e single loss on cell proliferation and response to CDK4/6i, we used the HR+/HER2- breast cancer cell lines MCF7 and T47D and generated \u003cem\u003eRB1\u003c/em\u003e- and \u003cem\u003eBRCA2\u003c/em\u003e-deficient (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea, b) cells using an inducible CRISPR/Cas9 system\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Single-gene knockout (KO) cells were used to study competitive interactions and investigate selective growth advantages under both untreated conditions and in the presence of the CDK4/6i palbociclib.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs expected, we observed that \u003cem\u003eRB1\u003c/em\u003e-deficient MCF7 cells showed significantly enhanced proliferation and outcompeted wild-type cells when co-cultured over a 7-day period in both untreated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) and palbociclib-treated conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). In contrast, loss of \u003cem\u003eBRCA2\u003c/em\u003e provided no growth advantage, irrespective of CDK4/6i treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d).\u003c/p\u003e\u003cp\u003eA similar trend was observed in the T47D model: while \u003cem\u003eRB1\u003c/em\u003e-deficient cells showed increased proliferation and a competitive advantage, particularly under CDK4/6 inhibition (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec-e), \u003cem\u003eBRCA2\u003c/em\u003e-deficient cells had no advantage under both untreated and palbociclib-treated conditions (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec-e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCo-alteration of\u003c/b\u003e \u003cb\u003eRB1\u003c/b\u003e\u003cb\u003e/\u003c/b\u003e\u003cb\u003eBRCA2\u003c/b\u003e \u003cb\u003ereduces fitness\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo study the interaction between the loss of both \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e in HR+/HER2- breast cancer cells, we established double-KO models for MCF7 and T47D cells. For MCF7, we selected two single-cell clones (SCCs) derived from the \u003cem\u003eRB1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) or \u003cem\u003eBRCA2\u003c/em\u003e KO (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) bulk populations. Then, they were transduced with a doxycycline-inducible lentiviral construct for expression of the additional guide RNA targeting \u003cem\u003eBRCA2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) or \u003cem\u003eRB1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), respectively, resulting in acute induction of a \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e double KO. The inducible single- and double-KO was also introduced in the T47D cell line. Here, a \u003cem\u003eRB1\u003c/em\u003e KO bulk population was subsequently transduced with a lentivirus carrying the \u003cem\u003eBRCA2\u003c/em\u003e-specific guide RNA (Fig. S2a).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNotably, acute double-KO induction of \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e resulted in impaired proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee and Fig. S2b). In MCF7 cells, the acute induction of the \u003cem\u003eBRCA2\u003c/em\u003e knockout in addition to the pre-existing \u003cem\u003eRB1\u003c/em\u003e-deficiency resulted in significantly decreased proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). This effect could also be seen in \u003cem\u003eBRCA2\u003c/em\u003e-deficient MCF7 cells upon induction of the \u003cem\u003eRB1\u003c/em\u003e knockout, although the effect was slightly less pronounced (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). We speculated that this might be due to increased DNA-damage as both genes, either directly or indirectly, play a role in DNA damage repair\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Indeed, double KO cells showed increased gamma-H2A.X (γH2A.X) levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef), indicating elevated DNA double-strand break (DSB) formation or DNA damage response activation.\u003c/p\u003e\u003cp\u003eA similar impact on proliferation was observed upon induction of acute double KO of \u003cem\u003eBRCA2\u003c/em\u003e and \u003cem\u003eRB1\u003c/em\u003e in T47D cells (Fig. S2b). This suggests that the combined loss of these genes provides a growth disadvantage for the cells at steady-state, which is potentially driven, at least in part, by increased DNA damage. However, given that \u003cem\u003eRB1\u003c/em\u003e-deficiency is a major driver of CDK4/6i resistance, co-alteration of \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e may still increase selective advantage under therapeutic pressure.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRB1\u003c/b\u003e\u003cb\u003e-deficiency is the major driver of CDK4/6i resistance in\u003c/b\u003e \u003cb\u003eRB1\u003c/b\u003e\u003cb\u003e/\u003c/b\u003e\u003cb\u003eBRCA2\u003c/b\u003e\u003cb\u003e-co-mutated cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNext, we investigated the impact of \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e co-loss on sensitivity to CDK4/6i. As expected, palbociclib treatment had no significant effect on cell cycle distribution in \u003cem\u003eRB1\u003c/em\u003e-deficient MCF7 cells, whereas \u003cem\u003eBRCA2\u003c/em\u003e-deficient cells showed an increased G1-phase arrest (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) and decreased S-phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) indicating therapy sensitivity. However, in \u003cem\u003eRB1\u003c/em\u003e plus \u003cem\u003eBRCA2\u003c/em\u003e KO cells, palbociclib failed to induce efficient G1-phase arrest (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) and S-phase was significantly less reduced compared to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), indicating resistance to CDK4/6 inhibition. The same effect was observed with the alternative CDK4/6i abemaciclib, where \u003cem\u003eBRCA2\u003c/em\u003e loss alone was not sufficient to induce therapy resistance, but only the presence of \u003cem\u003eRB1\u003c/em\u003e deficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, f).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSimilar results were observed in T47D cells, where G1-phase arrest and reduced S-phase occurred in control and \u003cem\u003eBRCA2\u003c/em\u003e-deficient cells but were absent in \u003cem\u003eRB1\u003c/em\u003e-deficient or double-KO cells (Fig. S3a,b).\u003c/p\u003e\u003cp\u003eTaken together, these results suggest that \u003cem\u003eBRCA2\u003c/em\u003e loss alone does not confer resistance to CDK4/6 inhibition \u003cem\u003ein vitro\u003c/em\u003e and resistance is primarily mediated by \u003cem\u003eRB1\u003c/em\u003e deficiency. Although \u003cem\u003eRB1\u003c/em\u003e/\u003cem\u003eBRCA2\u003c/em\u003e co-loss may reduce fitness in untreated conditions, these cells may gain a selective advantage under therapeutic pressure.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePARPi are effective in\u003c/b\u003e \u003cb\u003eRB1/BRCA2\u003c/b\u003e\u003cb\u003e-deficient cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePARPi, such as olaparib, are used in patients with mBC who progress on ET in combination with CDK4/6i and harbor germline mutations e.g., in \u003cem\u003eBRCA1\u003c/em\u003e or \u003cem\u003eBRCA2\u003c/em\u003e, due to their ability to induce synthetic lethality.\u003c/p\u003e\u003cp\u003eTo assess whether the PARPi olaparib is also effective in \u003cem\u003eRB1\u003c/em\u003e/\u003cem\u003eBRCA2\u003c/em\u003e-deficient cells, we evaluated their response to olaparib treatment. Upon PARPi treatment, MCF7 \u003cem\u003eRB1\u003c/em\u003e/\u003cem\u003eBRCA2\u003c/em\u003e single- and double-knockout cells exhibited significantly increased G2-phase arrest, a hallmark of PARPi sensitivity\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, compared to control cells (\u003cem\u003elacZ\u003c/em\u003e), which primarily exhibited an increase in G1-phase arrest (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). Notably, after PARPi treatment, \u003cem\u003eRB1\u003c/em\u003e/\u003cem\u003eBRCA2\u003c/em\u003e single- and double- knockout cells displayed a strong reduction in cell count (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) and live cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), indicating therapy sensitivity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor the p53-mutated T47D cell line, the results were similar. This cell line is already relatively sensitive to olaparib and double knockout of \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e did not significantly impact the sensitivity of the cells (Fig. S4).\u003c/p\u003e\u003cp\u003eThese findings suggest that PARPi are effective in \u003cem\u003eBRCA2\u003c/em\u003e-deficient tumors, regardless of the \u003cem\u003eRB1\u003c/em\u003e status, and might be an alternative treatment method for patients with CDK4/6i-resistance caused by loss of \u003cem\u003eRB1\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRB1\u003c/b\u003e \u003cb\u003emutations are associated with HRD and loss of 13q\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBased on our \u003cem\u003ein vitro\u003c/em\u003e data and given that patients with \u003cem\u003eBRCA2\u003c/em\u003e mutations have worse prognosis on ET\u0026thinsp;+\u0026thinsp;CDK4/6i treatment, possibly because of the co-loss of \u003cem\u003eRB1\u003c/em\u003e\u003csup\u003e10\u003c/sup\u003e, we investigated the frequency of co-alteration of \u003cem\u003eRB1\u003c/em\u003e and HRD-genes, such as \u003cem\u003eBRCA2.\u003c/em\u003e Therefore, we analyzed a large genomic database of 55,747 mutational profiles from formalin-fixed paraffin-embedded (FFPE) samples. Only one sample per patient was included.\u003c/p\u003e\u003cp\u003eIn the absence of \u003cem\u003eBRCA1/2\u003c/em\u003e mutations, \u003cem\u003eRB1\u003c/em\u003e mutations were observed in 7.46% (n\u0026thinsp;=\u0026thinsp;3930/52664; 95% CI [7.24; 7.69]) of all BC samples. In \u003cem\u003eBRCA1\u003c/em\u003e-mutated (\u003cem\u003eBRCA1\u003c/em\u003em) BC samples, the frequency of concurrent \u003cem\u003eRB1\u003c/em\u003e mutations was higher than in \u003cem\u003eBRCA\u003c/em\u003ewt (13.84%; n\u0026thinsp;=\u0026thinsp;289/2088; 95% CI [12.39; 15.40]; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). For \u003cem\u003eBRCA2\u003c/em\u003e-mutated (\u003cem\u003eBRCA2\u003c/em\u003em) samples, 12.79% (n\u0026thinsp;=\u0026thinsp;341/2666; 95% CI [11.55; 14.12]; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) of all BC samples displayed concurrent \u003cem\u003eRB1\u003c/em\u003em (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The percentage of \u003cem\u003ePALB2\u003c/em\u003e-mutated (\u003cem\u003ePALB2\u003c/em\u003em) BC samples with co-alteration in \u003cem\u003eRB1\u003c/em\u003e was 10.74% (n\u0026thinsp;=\u0026thinsp;78/726; 95% CI [8.59; 13.23]; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 v \u003cem\u003eBRCA\u003c/em\u003ewt) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). These data indicate that mutations in \u003cem\u003eRB1\u003c/em\u003e and HR genes frequently co-occur in BC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAdditionally, samples with biallelic loss of \u003cem\u003eBRCA1\u003c/em\u003e or \u003cem\u003eBRCA2\u003c/em\u003e loss exhibited a higher fraction of co-occurring \u003cem\u003eRB1\u003c/em\u003e mutations compared to tumors with monoallelic loss (\u003cem\u003eBRCA\u003c/em\u003ewt: 7.46%; n\u0026thinsp;=\u0026thinsp;3930/52664; 95% CI [7.24;7.69]; \u003cem\u003eBRCA1\u003c/em\u003e monoallelic: 11.22%; n\u0026thinsp;=\u0026thinsp;23/205; 95% CI [7.25; 16.36]; \u003cem\u003eBRCA1\u003c/em\u003e biallelic: 15.59%; n\u0026thinsp;=\u0026thinsp;222/1424; 95% CI [13.74; 17.58]; \u003cem\u003eBRCA2\u003c/em\u003e monoallelic: 6.71%; n\u0026thinsp;=\u0026thinsp;22/328; 95% CI [4.25; 9.98]; \u003cem\u003eBRCA2\u003c/em\u003e biallelic: 14.76%; n\u0026thinsp;=\u0026thinsp;279/1890; 95% CI [13.19; 16.44]; \u003cem\u003ePALB2\u003c/em\u003e monoallelic: 12.11%; n\u0026thinsp;=\u0026thinsp;27/223; 95% CI [8.13; 17.13]; \u003cem\u003ePALB2\u003c/em\u003e biallelic: 9.45%; n\u0026thinsp;=\u0026thinsp;36/381; 95% CI [6.71; 12.84];) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The most pronounced difference in the frequency of \u003cem\u003eRB1\u003c/em\u003e mutations could be observed between samples with monoallelic and biallelic loss of \u003cem\u003eBRCA2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Additionally, \u003cem\u003eBRCA2\u003c/em\u003em samples showed the highest tendency to gain \u003cem\u003eRB1\u003c/em\u003e mutations over time in patients with multiple biopsies (16.04%; n\u0026thinsp;=\u0026thinsp;17/106; 95% CI [9.63;24.43]; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), when compared to \u003cem\u003eBRCA\u003c/em\u003ewt (6.94%; n\u0026thinsp;=\u0026thinsp;5/72; 95% CI [2.29;15.47]), \u003cem\u003ePALB2\u003c/em\u003em (5.41%; n\u0026thinsp;=\u0026thinsp;2/37; 95% CI [0.66; 18.19]) or \u003cem\u003eBRCA1\u003c/em\u003em (5.03%; n\u0026thinsp;=\u0026thinsp;124/2465; 95% CI [4.20; 5.97]) samples (Fig. S5a). This reflects possible genomic evolution during disease progression and therapy resistance.\u003c/p\u003e\u003cp\u003e\u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e are located in proximity on chromosome 13q. Analyzing samples from all BC subtypes, we observed that 7.45% (n\u0026thinsp;=\u0026thinsp;2054/27566; 95% CI [7.14;7.77]) of samples without 13q loss showed mutations in \u003cem\u003eRB1\u003c/em\u003e whereas this was the case for 10.13% of samples with 13q loss (n\u0026thinsp;=\u0026thinsp;1577/15574; 95% CI [9.66; 10.61]) (loss v no loss: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). In the HR+/HER2- subset, 4.95% (n\u0026thinsp;=\u0026thinsp;59/1193; 95% CI [3.79;6.33]) of samples without 13q loss displayed \u003cem\u003eRB1\u003c/em\u003e mutations versus 8.65% (n\u0026thinsp;=\u0026thinsp;68/786; 95% CI [6.78;10.84]) of samples with loss of 13q (loss v no loss: p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Furthermore, HRD\u0026thinsp;+\u0026thinsp;samples based on genomic scarring (HRDsig+) exhibited a higher frequency of \u003cem\u003eRB1\u003c/em\u003e mutations, regardless of subtype. In all breast samples, we could observe that 6.81% (n\u0026thinsp;=\u0026thinsp;3125/45907; 95% CI [6.58;7.04]) of HRDsig- samples vs. 14.68% of HRDsig\u0026thinsp;+\u0026thinsp;samples harbor mutations in \u003cem\u003eRB1\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1280/8722; 95% CI [13.94;15.44]) (HRD\u0026thinsp;+\u0026thinsp;vs. HRD-: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Similar results were observed in the HR+/HER2- subset (HRDsig-: 5.07%; n\u0026thinsp;=\u0026thinsp;103/2031 95% CI [4.16;6.12]; vs. HRDsig+: 11.71%; n\u0026thinsp;=\u0026thinsp;35/299; 95% CI [8.29;15.90] p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Our analysis further revealed that \u003cem\u003eBRCA2\u003c/em\u003e mutations are associated with a higher percentage of mutations in \u003cem\u003eRB1\u003c/em\u003e, especially in recent years (Fig. S5b). Together, these data indicate that HRD and the resulting genomic instability, especially mediated by loss of \u003cem\u003eBRCA2\u003c/em\u003e, are linked to \u003cem\u003eRB1\u003c/em\u003e alterations.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHRD and\u003c/b\u003e \u003cb\u003eRB1\u003c/b\u003e \u003cb\u003emutations are associated with reduced time to treatment discontinuation (TTD)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe examined the outcomes of patients with HR+/HER2- metastatic breast cancer on 1st line CDK4/6i\u0026thinsp;+\u0026thinsp;ET in a real-world clinco-genomic database. In this setting, \u003cem\u003eBRCA\u003c/em\u003ewt patients exhibited a median time to treatment discontinuation (TTD) of 15.0 months. TTD was significantly shorter for patients with \u003cem\u003eBRCA1\u003c/em\u003em (median TTD of 5.6mo, hazard ratio (HR) relative to \u003cem\u003eBRCA\u003c/em\u003ewt of 2.3; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee) or \u003cem\u003eBRCA2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef) (median TTD of 11.1mo; HR relative to \u003cem\u003eBRCA\u003c/em\u003ewt of 1.6; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) mutations compared to \u003cem\u003eBRCA\u003c/em\u003ewt. This effect was more pronounced when considering only biallelic \u003cem\u003eBRCA1\u003c/em\u003e (median TTD of 4.2mo, HR relative to \u003cem\u003eBRCA\u003c/em\u003ewt of 3.8; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg) or \u003cem\u003eBRCA2\u003c/em\u003e (median TTD of 10.7mo; HR relative to \u003cem\u003eBRCA\u003c/em\u003ewt of 1.8; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh) alterations. As a positive control, we examined outcomes in patients with \u003cem\u003eRB1\u003c/em\u003e mutations, which were associated with significantly shorter TTD compared to \u003cem\u003eRB1\u003c/em\u003ewt (median TTD 4.3mo v 15.1mo; HR\u0026thinsp;=\u0026thinsp;3.2; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei). Analysis of TTD in patients harboring both \u003cem\u003eBRCA2\u003c/em\u003e and \u003cem\u003eRB1\u003c/em\u003e mutations revealed a marked reduction in TTD (median TTD 7.7mo v 15.4mo; HR\u0026thinsp;=\u0026thinsp;3.3; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). When considering a scar-based measure of HRD (HRDsig), patients that were HRDsig\u0026thinsp;+\u0026thinsp;had a worse outcome relative to HRDsig- patients (median TTD of 10.7 vs. 15.1 months; HR\u0026thinsp;=\u0026thinsp;1.6; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek). This suggests that HRD, which induces genome-wide LOH and other scars that can induce a second hit, is associated with poor outcomes on 1st line CDK4/6i\u0026thinsp;+\u0026thinsp;ET.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe addition of CDK4/6i to ET has led to increased PFS and OS in BC patients with advanced HR+/HER2- disease\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e and is now considered standard of care\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, regardless of HRD-status. While patients with TNBC and inactivating mutations in HRD-associated genes are treated with PARPi\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, it remains unclear whether this is an effective treatment option for patients with HR+/HER2- metastatic or early-stage BC.\u003c/p\u003e\u003cp\u003eA recent study demonstrated that patients with HR+/HER2- BC and germline \u003cem\u003eBRCA2\u003c/em\u003e mutations experience shorter PFS and OS when treated with CDK4/6i and ET likely as a result of gained \u003cem\u003eRB1\u003c/em\u003e mutations\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Although further experimental and clinical data on \u003cem\u003eRB1\u003c/em\u003e/\u003cem\u003eBRCA2\u003c/em\u003e co-alterations HR+/HER2- BC remain limited, studies in prostate cancer have shown that co-deletions are strongly correlated\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e and linked to increased invasiveness and shorter relapse-free survival\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In contrast, patients with high-grade serous ovarian cancer tend to have improved survival outcomes when their tumors exhibit loss of \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e\u003csup\u003e40\u003c/sup\u003e, highlighting a complex interaction between these genes.\u003c/p\u003e\u003cp\u003eTherefore, to further investigate the effects of \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e single- and co-alteration in HR+/HER2 BC, we modelled these mutations \u003cem\u003ein vitro\u003c/em\u003e. We found that \u003cem\u003eRB1\u003c/em\u003e loss was strongly correlated with increased proliferation, while \u003cem\u003eBRCA2\u003c/em\u003e KO led to decreased cell growth. Notably, loss of \u003cem\u003eBRCA2\u003c/em\u003e did not increase CDK4/6i resistance, while loss of \u003cem\u003eRB1\u003c/em\u003e, as expected, led to increased palbociclib and abemaciclib resistance. Co-loss of \u003cem\u003eBRCA2\u003c/em\u003e and \u003cem\u003eRB1\u003c/em\u003e resulted in significantly increased resistance to CDK4/6i. Given the importance of RB1 in the CDK4/6-pathway, loss of RB1 is a strong predictor of CDK4/6i resistance\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and was dominant over \u003cem\u003eBRCA2\u003c/em\u003e mutations. Therefore, our data support the recent findings on \u003cem\u003eBRCA2\u003c/em\u003e and \u003cem\u003eRB1\u003c/em\u003e co-alterations in BC leading to increased resistance towards CDK4/6i\u003csup\u003e10\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTo further assess the impact of HRR-associated alterations beyond \u003cem\u003eBRCA2\u003c/em\u003e, such as those in \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003ePALB2\u003c/em\u003e, on response to CDK4/6i in BC, patient data from a large real-world dataset were analyzed. These analyses demonstrated that HRD was correlated with significantly shorter TTD compared to the general cohort of CDK4/6i-treated BC patients. We observed that HRDsig\u0026thinsp;+\u0026thinsp;samples, particularly those harboring \u003cem\u003eBRCA2\u003c/em\u003e mutations, exhibited an increased frequency of \u003cem\u003eRB1\u003c/em\u003e alterations, a phenomenon also seen in prostate\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and ovarian cancer\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eInterestingly, our findings indicated that co-alteration of \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e negatively impacts cell proliferation in absence of treatment pressure. We hypothesize that this might be a result of the impaired ability to repair DNA double-strand breaks. Strikingly, the enrichment of proliferation-related hallmark gene sets, including E2F targets, G2/M checkpoint regulators and mitotic spindle targets is associated with elevated \u003cem\u003eBRCA2\u003c/em\u003e expression\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. This occurrence might be due to the essential role of \u003cem\u003eBRCA2\u003c/em\u003e in homologous recombination repair (HRR)\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Additionally, \u003cem\u003eRB1\u003c/em\u003e has a non-canonical role in the non-homologous end joining pathway\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e and is crucial to maintain chromosomal integrity\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Hence, inactivation of both genes might increase the dependence of the cells on other error-prone repair pathways, such as single-strand annealing or micro-homology-mediated end joining (MMEJ, also known as alternative end joining). Olaparib, in addition to targeting other PARP family members, also inhibits PARP1\u003csup\u003e45\u003c/sup\u003e, a component of the MMEJ repair pathway\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. This pathway is upregulated in \u003cem\u003eRB1\u003c/em\u003e-deficient cells\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, which may explain why \u003cem\u003eRB1\u003c/em\u003e- and \u003cem\u003eBRCA2\u003c/em\u003e-deficient cells \u0026ndash; whether single or co-mutated \u0026ndash; exhibit similar responses to olaparib. The increased efficacy of PARPi in \u003cem\u003eRB1\u003c/em\u003e-deficient cells is also seen in osteosarcoma\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, prostate cancer\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e and lung adenocarcinoma preclinical models\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Although to date this has not been studied thoroughly in BC, our findings suggest that PARPi may have the potential to overcome therapy failure in \u003cem\u003eRB1\u003c/em\u003e-deficient, CDK4/6i-resistant breast tumors.\u003c/p\u003e\u003cp\u003eClinical data analysis suggests that patients with HRD may benefit more from PARPi\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, although it remains uncertain whether PARPi could be a better treatment option than CDK4/6i plus endocrine therapy for patients with HR+/HER2- mBC or early BC. The optimal approach\u0026mdash;whether to employ PARPi instead or sequentially with CDK4/6i \u0026mdash;requires further investigation. Currently, a phase 3 clinical trial in mBC is performed, screening patients for \u003cem\u003eBRCA1/2\u003c/em\u003e and \u003cem\u003ePALB2\u003c/em\u003e alterations and randomizing them to treatment with the PARP1i saruparib and ET versus CDK4/6i combined with ET in the first-line setting (EvoPAR-Br01; ClinicalTrials.gov identifier: NCT06380751), which may help address some of these questions. Additionally, recent studies have demonstrated that \u003cem\u003eRB1\u003c/em\u003e-deficient cells are sensitive to the BCL-XL inhibitor navitoclax, with co-alteration of \u003cem\u003eBRCA2\u003c/em\u003e further enhancing this sensitivity in preclinical models\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. These findings are of high relevance for patients with advanced, and potentially also early-stage HR+/HER2- BC, as CDK4/6i are increasingly used for higher risk disease\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Additionally, the OlympiA phase III trial has demonstrated benefit of adjuvant olaparib in patients with \u003cem\u003eBRCA1\u003c/em\u003e/\u003cem\u003e2\u003c/em\u003e mutations\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. It is important to determine whether patients with HRD benefit less or not at all from the addition of CDK4/6i, and if there is a benefit, to establish the optimal treatment sequence.\u003c/p\u003e\u003cp\u003eOur findings contribute to the growing body of evidence for treatment selection, nevertheless, our study has several limitations that should be considered. First, the functional experiments were conducted in only two cell line models, which may not fully capture the heterogeneity of HR+/HER2- breast cancer. Additionally, we did not include patient-derived organoid (PDO) models or \u003cem\u003ein vivo\u003c/em\u003e studies, which are important for validating findings in more physiologically relevant systems. Finally, the analysis of real-world clinical data is inherently limited by potential biases, incomplete clinical annotations, and variability in treatment and follow-up, which may affect the robustness of the observed associations. Therefore, further research is needed to clarify the mechanisms and timing of RB1 mutations in HRD patients.\u003c/p\u003e\u003cp\u003eIn conclusion, we propose that patients with HR+/HER2- BC should be evaluated for the presence of an HRD-signature prior to initiating treatment with CDK4/6i. Our findings demonstrate that CDK4/6i are effective in \u003cem\u003eBRCA2\u003c/em\u003e-deficient cell line models. However, given the higher prevalence of \u003cem\u003eRB1\u003c/em\u003e co-alterations observed in patients with \u003cem\u003eBRCA2\u003c/em\u003e mutations, PARPi may represent a more effective therapeutic option for this subset of patients. Therefore, while identifying HR+/HER2- patients with these alterations at diagnosis requires extensive screening, collaborative efforts are essential to optimize treatment strategies for this highly selected BC patient population.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Petra Franzmayr, BSc, Sophie Kienreich, MSc and Bettina Flasch, MSc for their technical support. Cell sorting was performed at the Flow Cytometry Core Facility of the Center for Medical Research, Medical University of Graz. D.H. is funded by the Medical University of Graz within the PhD Program \u0026ldquo;Molecular Medicine\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.H.: Conceptualization; acquisition, analysis, and interpretation of data; visualization; writing \u0026ndash; original draft/review \u0026amp; editing. M.A.D. and M.B.: Conceptualization; project supervision; interpretation of data; writing \u0026ndash; original draft/review \u0026amp; editing. E.S.S. and M.M.: Data curation; data analysis and interpretation; writing \u0026ndash; original draft/review \u0026amp; editing. S.W., U.S. and M.K.: Investigation; data acquisition and analysis; writing \u0026ndash; review \u0026amp; editing. S.O., A.V.L., C.E.G, P.J.J., N.D.: Data interpretation; writing \u0026ndash; review \u0026amp; editing. All authors have read and agreed to the version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrant support\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNothing to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE.S.S. and M.M. are Employees at Foundation Medicine and Shareholders in Roche. M.B. received honoraria from AstraZeneca, Daiichi Sankyo, Eli Lilly, Gilead, Menarini, MSD, Novartis, Pierre Fabre, Pfizer, Seagen, Stemline, and research funding from Astra Zeneca, Daiichi Sankyo, Novartis, and Pfizer. C.E.G. received research funding to institutions, travel support to attend research meetings and provide lectures, honoraria for lectures from AstraZeneca and research funding to institutions and travel support to attend research meetings from Daiichi Sankyo, Genentech, Roche and Exact Sciences.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWojtyla, C., Bertuccio, P., Wojtyla, A. \u0026amp; La Vecchia, C. European trends in breast cancer mortality, 1980\u0026ndash;2017 and predictions to 2025. \u003cem\u003eEur. J. Cancer\u003c/em\u003e 152, 4\u0026ndash;17 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBray, F. \u003cem\u003eet al.\u003c/em\u003e Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. \u003cem\u003eCA. Cancer J. 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Oncol.\u003c/em\u003e e2300091 (2023) doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1200/PO.23.00091\u003c/span\u003e\u003cspan address=\"10.1200/PO.23.00091\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVarkaris, A. \u003cem\u003eet al.\u003c/em\u003e BH3 mimetics targeting BCL-XL have efficacy in solid tumors with RB1 loss and replication stress. \u003cem\u003eNat. Commun.\u003c/em\u003e 16, 4931 (2025).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7253927/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7253927/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWhile the combination of endocrine therapy (ET) and CDK4/6 inhibitors (CDK4/6i) improves progression-free survival (PFS) in HR+/HER2- metastatic breast cancer, resistance remains a major challenge. \u003cem\u003eBRCA2\u003c/em\u003e pathogenic variants have been linked to reduced PFS, potentially due to co-deletion of the neighboring \u003cem\u003eRB1\u003c/em\u003e gene on chromosome 13q. As RB1 is a key target of CDK4/6, its loss drives resistance. Using CRISPR/Cas9, we generated cell lines with single and combined \u003cem\u003eBRCA2\u003c/em\u003e and \u003cem\u003eRB1\u003c/em\u003e deletions. Loss of \u003cem\u003eRB1\u003c/em\u003e but not \u003cem\u003eBRCA2\u003c/em\u003e increased proliferation and conferred resistance to the CDK4/6i palbociclib and abemaciclib. Dual loss reduced proliferation but increased resistance to CDK4/6i \u003cem\u003ein vitro\u003c/em\u003e. However, sensitivity to the PARP inhibitor olaparib was maintained. Finally, analysis of real-world clinical data revealed that \u003cem\u003eRB1\u003c/em\u003e mutations were more frequent in tumors exhibiting homologous recombination deficiency signatures and 13q loss. These genomic features were associated with shorter treatment duration on CDK4/6i plus ET. In conclusion, our findings suggest that \u003cem\u003eRB1\u003c/em\u003e loss, alone or with \u003cem\u003eBRCA2\u003c/em\u003e deletion, contributes to CDK4/6 inhibitor resistance and may help explain reduced efficacy in patients with \u003cem\u003eBRCA2\u003c/em\u003e mutations. Importantly, despite this resistance, sensitivity to PARP inhibition is retained, highlighting a potential therapeutic vulnerability in this molecular context.\u003c/p\u003e","manuscriptTitle":"Impact of homologous recombination deficiency on CDK4/6 inhibitor sensitivity in HR+/HER2-breast cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-13 05:36:11","doi":"10.21203/rs.3.rs-7253927/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2600730e-3946-4c67-8295-e91f1a48334b","owner":[],"postedDate":"August 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53050025,"name":"Biological sciences/Cancer"},{"id":53050026,"name":"Biological sciences/Genetics"},{"id":53050027,"name":"Biological sciences/Molecular biology"},{"id":53050028,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2025-08-18T20:08:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-13 05:36:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7253927","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7253927","identity":"rs-7253927","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}