Vol.:(0123456789)1 3 https://doi.org/10.1007/s00210-022-02375-4 RESEARCH Novel combination treatment of CDK 4/6 inhibitors with PARP inhibitors in triple negative breast cancer cells Gamze Guney Eskiler1   · Zeynep Ozman2 · Ayten Haciefendi3 · Demet Cansaran‑Duman4 Received: 22 August 2022 / Accepted: 23 December 2022 © The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2023 Abstract Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors provide promising results for treating hormone receptor-positive breast cancer. However, the efficacy of CDK4/6 inhibitors remains uncertain in triple negative breast cancer (TNBC) patients with particularly carrying RB-deficient tumors. Poly-(ADP-ribose) polymerase (PARP) inhibitors offer a therapeutic strategy for the treatment of BRCA​-mutated TNBC patients. However, the acquired drug resistance, changes in the cell cycle regulation, and DNA damage repair have demonstrated the necessity for developing new combination strategies. This preclinical study assessed a combinatory treatment of the CDK4/6 inhibitor abemaciclib with PARP inhibitors talazoparib (TAL) in HCC1937 BRCA​-mutated RB-deficient TNBC cells and TAL-resistant HCC1937-R cells through WST-1 analysis, annexin V, cell cycle, acridine orange/propidium iodide staining, RT-PCR, and apoptosis array. Our findings revealed that abemaciclib and TAL combination synergistically suppressed the growth of TNBC cells and overcame TAL resistance through G0/G1 arrest and the activity of both intrinsic and extrinsic apoptotic pathways. These preliminary results suggest that the combination of abemaciclib and TAL could expand the use of these inhibitors in BRCA​ mutated and RB deficient TNBC patients and potentially overcomes PARP inhibitors resistance. Keywords  Abemaciclib · Talazoparib · Triple-negative breast cancer · Apoptosis · Resistance Introduction Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are potent inhibitors in the treatment of cancer due to suppressing the phosphorylation of retinoblastoma (RB) in G1/S transition. In different types of cancer, the amplification and aber- rant activation of the CDK4/6-cyclin D1-RB-E2F pathway have been observed. Therefore, selective CDK4/6 inhibi- tors (palbociclib, abemaciclib, and ribociclib) have been approved by FDA for the treatment of estrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer in combination with endocrine therapy (Choo and Lee 2018; Murphy 2019). However, recent studies have focused on the therapeutic efficacy of CDK4/6 inhibitors in triple negative breast cancer (TNBC) which is the most aggressive subtype of breast cancer with limited treatment options (Matutino et al 2018; Hu et al 2021; Saleh et al 2021; Wang et al 2021). Actually, the inhibition of CDK4/6 is not potential targeted therapy for the treatment of TNBC patients due to the loss of RB and overexpression of cyclin E (Lanceta et al 2021). However, TNBC cells are sensitive to CDK4/6 inhibitors in preclinical studies (O'Brien et al 2018; Li et al 2019). Addi- tionally, multiple ongoing clinical trials have evaluated the effectiveness of CDK4/6 inhibitors alone or in combination with other targeted therapies in TNBC patients (Lanceta et al 2021). Our previous study reveals that abemaciclib poten- tially inhibits the growth of MDA-MB-231 RB-proficient TNBC cells through apoptosis (Ozman et al 2021). On the other hand, Fassl et al. (2020) state that most of TNBC cells and especially MDA-MB-468 and HCC1937 cells carry- ing inactivating mutations of the RB1 gene are resistant to CDK4/6 inhibitors especially palbociclib through enhanced * Gamze Guney Eskiler gamzeguney@sakarya.edu.tr 1 Department of Medical Biology, Faculty of Medicine, Sakarya University, Korucuk Campus, Sakarya, Turkey 2 Department of Medical Biochemistry, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey 3 Department of Medical Biology, Faculty of Medicine, Bursa Uludag University, Bursa, Turkey 4 Biotechnology Institute, Ankara University, Ankara, Turkey / Published online: 4 January 2023 Naunyn-Schmiedeberg's Archives of Pharmacology (2023) 396:1031–1041 http://crossmark.crossref.org/dialog/?doi=10.1007/s00210-022-02375-4&domain=pdf http://orcid.org/0000-0002-2088-9914 1 3 lysosomal biogenesis. In the study of Lanceta et al. (2021), alterations of lipid metabolism in MDA-MB-231 cells lead to acquired palbociclib resistance. Additionally, Collins et al. (2021) note that CDK4/6 inhibitors may be worse in germline BRCA​ mutant metastatic breast cancer patients than patients with germline BRCA​ wild type and unknown germline BRCA​ status. In this context, there is controversial results for the effectiveness of CDK4/6 inhibitors in TNBC cells. Therefore, identifying new drug–drug interactions and combining treatment strategies will be crucial for the further improvement of targeted treatment modality and the use of CDK4/6 inhibitors in TNBC. Poly (ADP-ribose) polymerase (PARP) inhibitors have been the most promising therapeutic strategy leading to impaired DNA damage repair in BRCA1/2 mutant cancer types and are approved by the FDA for the treatment of breast and ovarian cancer patients with mutations of the BRCA1/2 genes (Cortesi et al 2021; Vanacker et al 2021). Among them, olaparib and talazoparib (TAL) as targeted therapy have been successfully used to treat patients with BRCA​ mutant advanced breast cancer patients (Guney Eskiler et al 2018; McCann and Hurvitz 2021; Singh et al 2021). However, resistance to PARP inhibitors through the restoration of homologous recombination repair, DNA end resection, reversion mutations, protection of DNA replica- tion fork, RAD51-single strand DNA filament and D-loop formation, epigenetic modification, and pharmacologi- cal alteration restricts the success of therapy response (Li et al 2020a, b; Singh et al 2021). Thus, new combination treatment strategies are needed to broaden the use of PARP inhibitors to overcome resistance in the treatment of TNBC. In the literature, some recent studies have demonstrated the synergistic effects of CDK4/6 and PARP inhibitors on RB-proficient and deficient breast cancer as well as blad- der cancer cells (Li et al 2020a, b; Klein et al 2021; Zhu et al 2021). In general, CDK4/6 inhibitors are ineffective in RB-deficient cancer cells (Fassl et al 2020; Zhu et al 2021). However, the combination of PARP inhibitors with CDK4/6 inhibitors exerts potential therapeutic effects on RB-deficient cells and could play a crucial role in CDK4/6 responsive- ness in breast cancer cells (Li et al 2020a, b; Zhu et al 2021). Especially, the combination of CDK4/6 inhibitors (palboci- clib) and PARP inhibitors (niraparib) in RB-deficient TNBC cells increases DNA damage through ROS ((Li et al 2020a, b). Thus, this combination could expand the use of CDK4/6 inhibitors and PARP inhibitors for the treatment of BRCA​ mutated and RB deficient TNBC patients. In this context, we, for the first time, assessed the com- bination of abemaciclib as CDK4/6 inhibitor and TAL as PARP inhibitor in BRCA1 mutated RB deficient HCC1937 TNBC cells to reveal the therapeutic potential of CDK4/6 inhibitors in the sensitivity of PARP inhibitors. Further- more, we evaluated the reversal of TAL resistance upon administration of abemaciclib in TAL resistant HCC1937 TNBC cells. Materials and methods Cell lines HCC1937 and HCC1937-R cells were cultured from early passages in RPMI (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% FBS (Gibco), 1% Pen/Strep (Gibco), at 5% CO2 in a humidified incuba- tor. Stock solutions of TAL (Selleck Chemicals LLC, Hou- ston, TX, USA) and abemaciclib (Biovision, San Francisco, CA, USA) were prepared in DMSO and diluted with fresh medium. HCC1937-R (TAL resistant) cells were generated by 0.1 nM TAL exposure for 12 months in our previous study (Guney Eskiler et al 2021) and the IC50 value of HCC1937-R cells was nearly eight times increased that of the parent cells. Cell viability A total of 5000–20,000 cells per well were seeded in 96-well plates and treated with abemaciclib for 24 and 48 h or TAL for 7 days. The concentrations of abemaciclib or TAL were determined in our previous studies (Guney Eskiler et al 2021; Guney Eskiler and Ozturk 2022). To assess the com- bination treatment, the cells were firstly pre-treated with TAL for 7 days and then incubated abemaciclib and TAL for further 24 and 48 h. After the defined incubation, 100 μl of WST-1 dye (Biovision, San Francisco, CA, USA) was added to the cells and incubated for 45 min in the dark. Finally, the optical density at 450 nm was measured with a microplate reader (Allsheng). The most effective abemaciclib and TAL combination concentrations were selected for further experi- ments and compared with abemaciclib alone treatment. Apoptosis analysis Cells (1–3 × 105/well) were seeded in 6-well plates and treated with abemaciclib (1 µM) alone or pretreated with TAL (0.1 and 1 nM) alone for 7 days and then combined with abemaciclib (1 µM) for further 24 h. Following treat- ment with the defined duration, the cells were washed and stained with Annexin V & Dead Cell Assay kit (Luminex Corporation, Austin, TX, USA) in the dark and the percent- age of apoptotic cells was measured using Muse Cell Ana- lyzer (Millipore, Germany). Cell cycle analysis The cells were seeded in 6-well plates and treated with abe- maciclib alone or combination with TAL. After treatment 1032 Naunyn-Schmiedeberg's Archives of Pharmacology (2023) 396:1031–1041 1 3 with the defined incubation time, the cells were harvested and fixed in pre-chilled 70% ethanol. The fixed cells were then collected and stained with Muse™ Cell Cycle Kit (Luminex Corporation) in the dark for 30 min and subjected to Muse Cell Analyzer (Millipore, Germany). Acridine orange (AO)/propidium iodide (PI) staining A total of 1–5 × 105/well cells were seeded in 6-well plates and incubated with abemaciclib alone or combination with TAL at the indicated times. Afterward, the cells were fixed with 4% paraformaldehyde (PFA) for 30 min and stained with AO (100  mg/mL) and PI solution (1  mg/mL) for 30 min. After washing with PBS, the cells were captured with EVOS FL Cell Imaging System (Thermo Fisher Sci- entific Inc.). Acidic vesicular organelles (AVO) staining Cells (1–5 × 105/well) were seeded in 6-well plates and treated with abemaciclib (1 µM) alone for 12 and 24 h or pretreated with TAL (0.1 and 1 nM) alone for 7 days and then combined with abemaciclib (1 µM) for further 12 and 24 h. Following incubation, the cells were stained with AO (1 μg/ml) for 15 min at 37 °C. Afterward, the cells were washed with PBS and the formation of AVO was observed using EVOS FL Cell Imaging System (Thermo Fisher Sci- entific Inc.). RT‑PCR Total RNA was obtained from cells using E.Z.N.A. Total RNA Kit (Omega Bio-Tek, Norcross, GA). The quantified RNA concentration by Qubit 4.0, cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). CCDN1 (cyclin D1) and RB1 expression levels were analyzed in triplicate on a Step One Plus Real- Time PCR (Applied Biosystems, Foster City, CA). ACTB was used as an internal control. The 2−ΔΔCT method was used for normalization to ACTB levels. Apoptosis array To simultaneously assess the expression of multiple pro- teins associated with apoptosis, Human Apoptosis Array (43 targets) (RayBiotech Life) was performed according to the manufacturer protocol. After treatment with the most effective abemaciclib and TAL combination, protein isola- tion was conducted and an equal amount of total protein was loaded per array. Finally, membranes were incubated with detection buffers C and D (kit components) and visualized by ECL detection kit (Biovision) in a chemiluminescent sys- tem (Syngene, USA). Statistical analysis Data were analyzed by GraphPad Prism 8 software and p < 0.05 was accepted as statistical significance. Data were presented as the mean ± SD. The significance of differ- ences between groups was assessed by one-way ANOVA post-Tukey analysis. The Chou–Talalay method was con- ducted to determine the combination index (Chou 2006). Results The synergistic effects of abemaciclib and talazoparib combination on TNBC cells To assess the potential anti-cancer activity of abemaci- clib and talazoparib combination, WST-1 analysis was conducted. As shown in Fig. 1, abemaciclib alone sig- nificantly inhibited HCC1937 cells viability at concentra- tions ≥ 1.5 µM for 24 h (1 µM: 70.8 ± 1.2% and 1.5 µM: 51.9 ± 1.7%) (p < 0.05). Furthermore, abemaciclib and TAL combination synergistically suppressed both HCC1937 and HCC1937-R viabilities with the greatest effect at a concentration of 1 µM abemaciclib and 1 nM TAL for 24 h. The synergism between abemaciclib and TAL was summarized in Tables 1 and 2. According to CI values, 1 µM abemaciclib + 0.1 nM TAL and 1 µM abe- maciclib + 1 nM TAL combinations for 24 h were accepted as the most effective combinations in both TAL sensitive and resistant TNBC cells. In our previous results, 0.1 and 1 nM TAL alone reduced the viability of HCC1937 cells to 92.2 ± 3.0% and 76.5 ± 2.0%, respectively for 7 days (Guney Eskiler et  al. 2021).The combination of 1 µM abemaciclib + 0.1 nM TAL and 1 µM abemaciclib + 1 nM TAL considerably decreased the viability of HCC1937 cells to 55.5 ± 0.7% and 45.9 ± 1.7%, respectively com- pared with abemaciclib alone (70.5 ± 1.1%) (p < 0.01). Furthermore, 26.5 ± 2.5% and 33.7 ± 3.1% reduction were observed at 1 µM abemaciclib + 0.1 nM TAL and 1 µM abemaciclib + 1 nM TAL combination, respectively in the growth of HCC1937-R cell. On the other hand, abe- maciclib treatment did not decrease the viability of TAL resistant HCC1937-R cells for 24 h (1 µM: 100.4 ± 2.9% and 1.5 µM: 100.6 ± 2.2%). The cytotoxic effects of TAL alone on these cells did not further analyzed in this study due to our previous findings. TAL alone did not induce any toxicity in TAL resistant cells due to acquired resistance (Guney Eskiler et al. 2021). Therefore, abemaciclib and TAL combination more significantly suppressed the pro- liferation of HCC1937 cells (p < 0.01) than abemaciclib or TAL alone treatment and could overcome TAL resistance at a certain degree. 1033Naunyn-Schmiedeberg's Archives of Pharmacology (2023) 396:1031–1041 1 3 The apoptotic effects of abemaciclib and TAL combination in TNBC cells To assess abemaciclib and TAL combination mediated cell death, Annexin V and AO/PI staining were per- formed. As shown in Fig. 2A, 1 µM abemaciclib and 1 nM TAL combination considerably caused apoptosis in both cells. Compared with abemaciclib alone (17.9 ± 0.5% and 6.5 ± 0.3%), this combination significantly increased total apoptotic rate to 59.2 ± 0.8% and 21.2 ± 1.2% in HCC1937 and HCC1937-R cells, respectively (p < 0.01). Furthermore, co-treatment of abemaciclib and TAL led to the nuclear blebbing formation and chromatin conden- sation in these cells compared with abemaciclib alone (Fig. 2B). However, abemaciclib induced lysosomal bio- mass in these cells and this effect was less observed in HCC1937 cells than HCC1937-R cells following com- bination with TAL. To verify the association between autophagy and abemaciclib treatment, AVO staining by AO was performed in two different incubation time. Our results demonstrated that abemaciclib did not induce autophagy related cell death in both cells due to green fluorescence dots (Fig. 3). Fig. 1   Synergistic effects of abemaciclib and TAL on the growth of TNBC cells were determined by WST-1 analysis. The HCC1937 and HCC1937 cells were pre-treated with TAL for 7  days and fur- ther incubated with simulatenously abemaciclib and TAL for further 24 and 48 h compared with abemaciclib alone (p < 0.05*, p < 0.01**) Table 1   CI values of abemaciclib (1 and 1.5 µM) and TAL (0.1 and 1 nM) combination in TNBC cells CI, combination index; CI < 1, synergism; CI = 1, additive effect; CI > 1, antagonism HCC1937 HCC1937-R TAL 0.1 nM 1 nM 0.1 nM 1 nM CI at 1 µM abemaciclib 0.76 0.75 0.44 0.42 CI at 1.5 µM abemaciclib 0.92 1.56 0.61 0.70 1034 Naunyn-Schmiedeberg's Archives of Pharmacology (2023) 396:1031–1041 1 3 The effects of abemaciclib and TAL combination on cell cycle arrest in TNBC cells Cell cycle analysis and the mRNA levels of cyclin D1 and RB1 were conducted for the identification of the cell cycle distribution in these cells (Fig. 4). Compared with abemaci- clib alone treatment, co-treatment of abemaciclib and TAL significantly induced G0/G1 phase arrest in both TNBC cells (p < 0.05). The combination of abemaciclib and 1 nM TAL caused a significant increase in the accumulation of cells in G0/G1 phase (78.4 ± 0.4% and 63.9 ± 0.5%) in HCC1937 and HCC1937-R cells, respectively unlike abemaciclib alone (58.3 ± 0.5% and 57.6 ± 0.5%, respectively) a (p < 0.01, Fig. 4A, B). Additionally, abemaciclib and TAL signifi- cantly inhibited the expression levels of cyclin D1 and RB1 in HCC1937 cells due to RB-deficiency (p < 0.01, Fig. 4C). However, this combination increased the mRNA level of RB1 with the downregulation of cyclin D1 in HCC1937-R cells. Therefore, the effectiveness of abemaciclib and TAL combination differentially affected cell cycle regulation in terms of TAL sensitivity or resistance. Additionally, abe- maciclib and TAL combination were more induced apop- tosis than abemaciclib alone in HCC1937 and HCC1937-R cells. The molecular mechanism of abemaciclib and TAL combination mediated apoptosis in TNBC cells at protein level We further analyzed the levels of multiple proteins in both intrinsic and extrinsic apoptosis pathways. Our findings showed that apoptosis-associated protein expression levels were different between HCC1937 and HCC1937-R cells upon administration of 1 µM abemaciclib and 1 nM TAL combination as summarized in Fig. 5. In HCC1937 cells, pro-apoptotic proteins Bax (1.8-fold), Bid (1.5-fold), Bim (2.0-fold), Cytochrome c (1.2-fold), Caspase-3 (5.3-fold), HTRA2 (1.3-fold) as well as p21 (1.9-fold), p27 (1.3-fold), and p53 (1.6-fold) were upregulated. On the other hand, anti- apoptotic Bcl-2 (1.3-fold), Bcl-w (2.0-fold), HSP27 (1.3- fold), HSP60 (1.2-fold), HSP70 (1.8-fold), and Livin (1.2- fold) were increased after abemaciclib and TAL combination treatment despite of the downregulation of cIAP-2 (0.9-fold), Survivin (0.9-fold), XIAP (0.6-fold), and TNFα/β. Further- more, the expression levels of pro-apoptotic Bad (2.7-fold), Bax (2.4-fold), Bid (2.1-fold), Bim (1.6-fold), Caspase-3 (1.7-fold), Caspase-8, (1.7-fold), Cytochrome c (2.8-fold), and HTRA2 (2.1-fold) proteins were also more upregulated in HCC1937-R cells. However, abemaciclib and TAL com- bination induced the overexpression of anti-apoptotic Bcl-2 (2.1-fold), Bcl-w (2.3-fold), cIAP-2 (1.5-fold), XIAP (2.1- fold), Livin (2.8-fold), Survivin (1.3-fold), HSP27 (2.0-fold), HSP60 (1.8-fold), HSP70 (1.8-fold), and TNFα/β proteins upon combination treatment despite of increased p21 (4.0- fold), p27 (2.5-fold), and p53 (2.0-fold) protein levels. Taken together, abemaciclib and TAL combination induced cell apoptosis through both intrinsic and extrinsic apoptotic pathways in both TNBC cells. However, we detected an increased level of anti-apoptotic proteins in HCC1937-R cells due to TAL resistance. Discussion Herein, we first provided evidence that the combination of abemaciclib with TAL exerted a synergistic anti-cancer effect on HCC1937 BRCA1-mutated RB deficient TNBC cells and induced apoptotic cell death through activating mitochondrial apoptotic pathway. Notably, abemaciblib and TAL combination could potentially overcome TAL resist- ance at a certain degree. In this context, CDK4/6 and PARP inhibitors combination could expand the patient population that can not benefit from PARP or CDK4/6 inhibitors alone in patients carrying BRCA and RB defects. Recent studies have drawn significant attention for identifying molecular predictive factors to determine the sensitivity of TNBC to CDK4/6 inhibitors. Androgen receptor expression, the activation of Myc, MEK and Akt signaling pathway, RB expression status, TP53 mutations, CCNE1 amplification, and CDK2 and other cyclin depend- ent kinases activation play a crucial role in the response of CDK4/6 inhibitors in preclinical and clinical studies (Wang et al 2021). Therefore, combining CDK4/6 inhibi- tors with different targeted drugs could provide a promis- ing treatment strategy for the treatment of TNBC (Saleh et al 2021). In Yamamoto et al. (2019) study, palbociclib Table 2   DRI values of abemaciclib (1 and 1.5 µM) and TAL (0.1 and 1 nM) combination in TNBC cells DRI, dose reduction index; DRI: < 1, not favorable dose reduction; DRI = 1, no-dose reduction; DRI > 1, favorable dose reduction HCC1937 HCC1937-R TAL 0.1 nM 1 nM 0.1 nM 1 nM ABE TAL ABE TAL ABE TAL ABE TAL 1 µM ABE 1.35 49.12 1.57 8.68 2.29 1253.8 2.43 215.4 1.5 µM ABE 1.09 102.37 0.80 3.18 1.66 2520.2 1.45 82.8 1035Naunyn-Schmiedeberg's Archives of Pharmacology (2023) 396:1031–1041 1 3 and MLN0128 mTOR kinase inhibitor exert synergistic anti-cancer activity in both pRb + TNBC cell lines and PDX model (Yamamoto et al. 2019). Huang et al. (2020) state that pre-treatment palbociclib could enhance the efficacy of cisplatin on MDA-MB-231 TNBC in vitro and in vivo (Huang et al 2020) supported by Cretella et al. (2019) study. In this study, the efficacy of paclitaxel could be improved by a pre-treatment with palbociclib in TNBC Fig. 2   The combined apoptotic effects of abemaciclib and TAL on TNBC cells were measured by (A) Annexin V assay and (B) AO/PI staining. (a) Control, (b) 1 µM abemaciclib, (c) 1 µM abemaciclib + 0.1 nM TAL, and (d) 1 µM abemaciclib + 1 nM TAL 1036 Naunyn-Schmiedeberg's Archives of Pharmacology (2023) 396:1031–1041 1 3 cells (Cretella et al 2019). Furthermore, niraparib or olapa- rib and palbociclib combination show potentially syner- gistic effects on RB-deficient TNBC cells (MDA-MB-468, MDA-MB436, BT549, and HCC1937) in a ROS-depend- ent manner (Li et al. 2020a, b). These findings are sup- ported by Zhu et al. (2021) study. In this study, olaparib and palbociclib combination exhibit the synergistic effects on BRCA​ mutant TNBCs cells (olaparib-sensitive (MDA- MB-436) and olaparib-resistant BRCA​mut/TNBC cell lines (HCC1937and SUM149)) (Zhu et al 2021). In this context, we, for the first time, demonstrated that the com- bination of abemaciclib and TAL exhibited a strong syn- ergism in both BRCA​ mutant and RB deficient HCC1937 TNBC cells and improved TAL sensitivity. Fig. 3   The combined effects of abemaciclib and TAL on the formation of AVO by AO staining in TNBC cells for 12 and 24 h. a Control, b 1 µM abemaciclib, c 1 µM abemaciclib + 0.1 nM TAL, and d 1 µM abemaciclib + 1 nM TAL 1037Naunyn-Schmiedeberg's Archives of Pharmacology (2023) 396:1031–1041 1 3 1038 Naunyn-Schmiedeberg's Archives of Pharmacology (2023) 396:1031–1041 1 3 The dysfunction of RB is observed in nearly 30% of TNBC cases and the loss of RB occurs in 7–20% of TNBC patients and leads to CDK4/6 inhibitors resistance (Wang et al 2021). In the study of Patel et al. (2020), 51% of 180 TNBC tissues are Rb positive expression and there is an association between AR expression, lower histologic grade, a lack of a germline BRCA​ mutation, and bone metasta- ses and RB expression. In the literature, the combination of olaparib or niraparib and palbociclib exerts synergis- tic effects on RB deficient TNBC cells (Li et al 2020a, b; Zhu et al 2021). However, MDA-MB-468 and HCC1937 cells with inactivating mutations in the RB1 gene show a lack of palbociclib response in Fassl et al. (2020). In our study, higher concentrations (1, 1.5, and 2 µM) of abe- maciclib significantly suppressed the growth of HCC1937 cells, unlike HCC1937-R cells. Additionally, co-treatment with abemaciclib and talazoparib enhanced the efficacy of abemaciclib and TAL alone and reversed TAL resistance. Furthermore, increased mRNA level of RB1 was detected in particularly HCC1937-R cells. Therefore, higher mRNA level of RB1 could correlate with abemaciclib and TAL response rate. However, further studies are required to identify the molecular mechanism underlying the over- expression of RB1 in HCC1937-R cells. Additionally, post- transcriptional and/or translational mechanisms define pro- tein abundance (Greenbaum et al 2003). Therefore, further molecular experiments including western blot and proteom- ics analysis should be performed for the identification of RB and associated signaling pathway in HCC1937-R cells. In the literature, abemaciclib treatment causes atypi- cal cell death based on swollen lysosomes in the A549 lung cancer cell line (Hino et al 2020). Additionally, Fassl et al. (2020) state that increased lysosomal mass leads to the intrinsic resistance of TNBC and a subset of hormone receptor positive breast cancer tumors. In the current study, co-treatment with abemaciclib and TAL treatment did not induce autophagy due to non-acidic vesicles as in our pre- vious study (Ozman et al 2021). However, the number of lysosomal biomass or swollen lysosomes mentioned in the previous studies (Fassl et al 2020; Hino et al 2020) was more pronounced in HCC1937-R cells. Therefore, the underly- ing molecular mechanisms of abemaciclib and lysosomal biomass and their associations with drug resistance need further investigation. Fig. 4   The synergistic effects of abemaciclib and TAL combina- tion on the regulation of cell cycle. (A) Cell cycle distribution of TNBC cells. (a) Control, (b) 1 µM abemaciclib, (c) 1 µM abemaci- clib + 0.1  nM TAL, and (d) 1  µM abemaciclib + 1  nM TAL. (B) Statistical analysis of the percentage of total apoptosis and G0/G1, S, and G2/M phase in TNBC cells. (C) The mRNA levels of Cyc- lin D1 and RB1 were measured by RT-PCR analysis in TNBC cells. (p < 0.05*, p < 0.01**) ◂ Fig. 5   The combinatory effects of abemaciclib and TAL on the expression of multiple proteins associated with apoptosis. A Apop- tosis array of HCC1937 and HCC1937-R cells following the admin- istration of abemaciclib and TAL combination treatment. B The expression levels of pro-apoptotic and anit-apoptotic proteins in co- treated TNBC cells compared with control 1039Naunyn-Schmiedeberg's Archives of Pharmacology (2023) 396:1031–1041 1 3 Furthermore, abemaciclib and TAL combination sig- nificantly induced G0/G1 arrest in TNBC cells. Changes in DNA damage and homologous recombination (HR) repair affect the cell cycle distribution. Typically, the inhibition of PARP treatment leads to G2/M accumulation in especially TNBC cells due to repair of DNA damage by HR (Chuang et al 2012; Eskiler et al 2018). We speculate that increased DNA damage leads to G0/G1 phase accumulation due to decreased cyclin D1 level and pre-treatment of TAL may cause DNA damage. Thus, the molecular mechanism of each cell cycle phase will be further investigated upon abemaci- clib and TAL combination treatment. Finally, we revealed changes in the pro-apoptotic and anti-apoptotic protein levels by apoptosis array. Our find- ings showed that abemaciclib and TAL combination treat- ment caused over-expression of pro-apoptotic protein and these effects were more pronounced in HCC1937-R cells than HCC1937 parental cells. However, the expression of some anti-apoptotic proteins including Bcl-2, Bcl-w, Livin, and heat shock proteins was increased in both TNBC cells. Bcl-2 and Bcl-w are members of anti-apoptotic Bcl-2 pro- teins, and upregulation of these proteins leads to survival and chemotherapy resistance in different types of cancer (Inao et al 2018). Additionally, higher expression or activity of HSP27, HSP60, and HSP70 is associated with increased tumorigenicity, metastatic potential, and resistance to chemotherapy, resulting in poor prognosis (Albakova et al 2021). Furthermore, over-expressed XIAP and Livin levels are associated with lymph node metastasis, tumor size, and TNM stage in patients with TNBC (Han et al 2017; Hussain et al 2017). Despite apoptosis through intrinsic and extrinsic pathways induced by abemaciclib and TAL combination, the overexpression of anti-apoptotic proteins and increased pro- inflammatory cytokines (TNFα and TNFβ) (Pileczki et al 2013) may cause aggressiveness and apoptotic resistance in HCC1937-R cells. Therefore, co-treatment of abemaciclib with TAL could overcome TAL resistance at certain degree. In this context, further sequentially or simultaneously com- bination strategies should be improved for targeting multi- ple pathways and overcoming TAL resistance in vitro and in vivo. Conclusion Collectively, our preliminary findings provide a preclinical rationale for identifying the synergistic therapeutic effects of abemaciclib and TAL on BRCA​ mutant RB deficient TNBCs cells and the reversal of TAL resistance. However, further studies will focus on validating the efficacy of CDK4/6 inhibitors and PARP inhibitors combination and broadening the utility of these inhibitors for the prevention and/or treat- ment of drug-resistant TNBC patients. Additionally, in vivo experiments should be performed to validate the combined anti-cancer effects of CDK4/6 inhibitors and PARP inhibi- tors on TNBC treatment. Author contribution  GGE and ZO conceived and designed research. ZO and AH conducted experiments. GGE and DCD analyzed data and wrote the manuscript. All authors read and approved the manuscript. Data availability  All data generated or analyzed during this study are included in this published article. Declarations  Ethics approval  Not applicable. Consent to participate  Not applicable. Consent for publication  Not applicable. Competing interests  The authors declare no competing interests. References Albakova Z, Siam MKS, Sacitharan PK, Ziganshin RH, Ryazantsev DY, Sapozhnikov AM (2021) Extracellular heat shock proteins and cancer: new perspectives. Transl Oncol 14(2):100995 Choo JRE, Lee SC (2018) CDK4–6 inhibitors in breast cancer: current status and future development. Expert Opin Drug Metab Toxicol 14(11):1123–1138 Chou TC (2006) Theoretical basis, experimental design, and computer- ized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 58(3):621–681 Chuang HC, Kapuriya N, Kulp SK, Chen CS, Shapiro CL (2012) Dif- ferential anti-proliferative activities of poly (ADP-ribose) poly- merase (PARP) inhibitors in triple-negative breast cancer cells. Breast Cancer Res Treat 134(2):649–659 Collins JM, Nordstrom BL, McLaurin KK, Dalvi TB, McCutcheon SC, Bennett JC, Murphy BR, Singhal PK, McCrea C, Shinde R, Bri- ceno JM (2021) A real-world evidence study of CDK4/6 inhibitor treatment patterns and outcomes in metastatic breast cancer by germline BRCA mutation status. Oncol Ther 9(2):575–589 Cortesi L, Rugo HS, Jackisch C (2021) An overview of PARP inhibitors for the treatment of breast cancer. Targeted oncology 16(3):255–282. https://​doi.​org/​10.​1007/​s11523-​021-​00796-4 Cretella D, Fumarola C, Bonelli M, Alfieri R, La Monica S, Digiacomo G, Cavazzoni A, Galetti M, Generali D, Petronini PG (2019) Pre- treatment with the CDK4/6 inhibitor palbociclib improves the efficacy of paclitaxel in TNBC cells. Sci Rep 9(1):1–11 Eskiler GG, Cecener G, Egeli U, Tunca B (2018) Synthetically lethal BMN 673 (talazoparib) loaded solid lipid nanoparticles for BRCA1 mutant triple negative breast cancer. Pharm Res 35(11):1–20 Fassl A, Brain C, Abu-Remaileh M, Stukan I, Butter D, Stepien P, ... and Sicinski P (2020) Increased lysosomal biomass is responsi- ble for the resistance of triple-negative breast cancers to CDK4/6 inhibition. Sci Adv 6(25):eabb2210 Greenbaum D, Colangelo C, Williams K, Gerstein M (2003) Compar- ing protein abundance and mRNA expression levels on a genomic scale. Genome Biol 4(9):117 Guney Eskiler G, Ozturk M (2022) Therapeutic potential of the PI3K inhibitor LY294002 and PARP inhibitor talazoparib combination 1040 Naunyn-Schmiedeberg's Archives of Pharmacology (2023) 396:1031–1041 https://doi.org/10.1007/s11523-021-00796-4 1 3 in BRCA-deficient triple negative breast cancer cells. Cell Signal 91:110229 Guney Eskiler G, Cecener G, Egeli U, Tunca B (2018) Triple nega- tive breast cancer: new therapeutic approaches and BRCA status. APMIS 126(5):371–379 Guney Eskiler G, Yanar S, Akpinar G, Kasap M (2021) Proteomic analysis of talazoparib resistance in triple-negative breast cancer cells. J Biochem Mol Toxicol 35(3):e22678 Han Y, Zhang L, Wang W, Li J, Song M (2017) Livin promotes the progression and metastasis of breast cancer through the regulation of epithelial-mesenchymal transition via the p38/GSK3β pathway. Oncol Rep 38(6):3574–3582 Hino H, Iriyama N, Kokuba H, Kazama H, Moriya S, Takano N, Hira- moto M, Aizawa S, Miyazawa K (2020) Abemaciclib induces atypical cell death in cancer cells characterized by formation of cytoplasmic vacuoles derived from lysosomes. Cancer Sci 111(6):2132–2145 Hu Y, Gao J, Wang M, Li M (2021) Potential prospect of CDK4/6 inhibitors in triple-negative breast cancer. Cancer Manag Res 13:5223 Huang Y, Wu H, Li X (2020) Novel sequential treatment with pal- bociclib enhances the effect of cisplatin in RB-proficient triple- negative breast cancer. Cancer Cell Int 20(1):1–14 Hussain AR, Siraj AK, Ahmed M, Bu R, Pratheeshkumar P, Alrashed AM, Qadri Z, Ajarim D, Al-Dayel F, Beg S, Al-Kuraya KS (2017) XIAP over-expression is an independent poor prognostic marker in Middle Eastern breast cancer and can be targeted to induce efficient apoptosis. BMC Cancer 17(1):640 Inao T, Iida Y, Moritani T, Okimoto T, Tanino R, Kotani H, Harada M (2018) Bcl-2 inhibition sensitizes triple-negative human breast cancer cells to doxorubicin. Oncotarget 9(39):25545 Klein FG, Granier C, Zhao Y, Pan Q, Tong Z, Gschwend JE, Holm PS, Nawroth R (2021) Combination of talazoparib and palboci- clib as a potent treatment strategy in bladder cancer. J Pers Med 11(5):340 Lanceta L, Lypova N, O’Neill C, Li X, Rouchka E, Chesney J, Imbert- Fernandez Y (2021) Differential gene expression analysis of pal- bociclib-resistant TNBC via RNA-seq. Breast Cancer Res Treat 186(3):677–686 Li T, Xiong Y, Wang Q, Chen F, Zeng Y, Yu X, Wang Y, Zhou F, Zhou Y (2019) Ribociclib (LEE011) suppresses cell proliferation and induces apoptosis of MDA-MB-231 by inhibiting CDK4/6- cyclin D-Rb-E2F pathway. Artif Cells Nanomed Biotechnol 47(1):4001–4011 Li H, Liu ZY, Wu N, Chen YC, Cheng Q, Wang J (2020a) PARP inhibi- tor resistance: the underlying mechanisms and clinical implica- tions. Mol Cancer 19(1):1–16 Li S, Zhang Y, Wang N, Guo R, Liu Q, Lv C, Wang L, Yang QK (2020b) Pan-cancer analysis reveals synergistic effects of CDK4/6i and PARPi combination treatment in RB-proficient and RB-deficient breast cancer cells. Cell Death Dis 11(4):1–16 Matutino A, Amaro C, Verma S (2018) CDK4/6 inhibitors in breast cancer: beyond hormone receptor-positive HER2-negative dis- ease. Ther Adv Med Oncol 10:1758835918818346 McCann KE, Hurvitz SA (2021) Innovations in targeted therapies for triple negative breast cancer. Curr Opin Obstet Gynecol 33(1):34–47 Murphy CG (2019) The role of CDK4/6 inhibitors in breast cancer. Curr Treat Options Oncol 20(6):1–13 O’Brien N, Conklin D, Beckmann R, Luo T, Chau K, Thomas J, Nulty AM, Marchal C, Kalous O, Euw E, Hurvitz S, Mockbee C, Sla- mon DJ (2018) Preclinical activity of abemaciclib alone or in combination with antimitotic and targeted therapies in breast can- cer. Mol Cancer Ther 17(5):897–907 Ozman Z, Guney Eskiler G, Sekeroglu MR (2021) In vitro therapeu- tic effects of abemaciclib on triple-negative breast cancer cells. J Biochem Mol Toxicol 35(9):e22858 Patel JM, Goss A, Garber JE, Torous V, Richardson ET, Haviland MJ, Tung N (2020) Retinoblastoma protein expression and its predic- tors in triple-negative breast cancer. NPJ Breast Cancer 6(1):1–6 Pileczki V, Braicu C, Gherman CD, Berindan-Neagoe I (2013) TNF-α gene knockout in triple negative breast cancer cell line induces apoptosis. Int J Mol Sci 14(1):411–420 Saleh L, Wilson C, Holen I (2021) CDK4/6 inhibitors: A potential therapeutic approach for triple negative breast cancer. Med Comm 2(4):514–530. https://​doi.​org/​10.​1002/​mco2.​97 Singh DD, Parveen A, Yadav DK (2021) Role of PARP in TNBC: mechanism of inhibition, clinical applications and resistance. Biomedicines 9:1512 Vanacker H, Harter P, Labidi-Galy SI, Banerjee S, Oaknin A, Lorusso D, Ray-Coquard I (2021) PARP-inhibitors in epithelial ovarian cancer: actual positioning and future expectations. Cancer Treat Rev 99:102255 Wang R, Xu K, Gao F, Huang J (1876) Guan X (2021) Clinical con- siderations of CDK4/6 inhibitors in triple-negative breast cancer. Biochim Biophys Acta (BBA)-Rev Cancer 2:188590 Yamamoto T, Kanaya N, Somlo G, Chen S (2019) Synergistic anti- cancer activity of CDK4/6 inhibitor palbociclib and dual mTOR kinase inhibitor MLN0128 in pRb-expressing ER-negative breast cancer. Breast Cancer Res Treat 174(3):615–625 Zhu X, Chen L, Huang B, Li X, Yang L, Hu X, Jiang Y, Shao Z, Wang Z (2021) Efficacy and mechanism of the combination of PARP and CDK4/6 inhibitors in the treatment of triple-negative breast cancer. J Exp Clin Cancer Res 40(1):122 Publisher's note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. 1041Naunyn-Schmiedeberg's Archives of Pharmacology (2023) 396:1031–1041 https://doi.org/10.1002/mco2.97 Novel combination treatment of CDK 46 inhibitors with PARP inhibitors in triple negative breast cancer cells Abstract Introduction Materials and methods Cell lines Cell viability Apoptosis analysis Cell cycle analysis Acridine orange (AO)propidium iodide (PI) staining Acidic vesicular organelles (AVO) staining RT-PCR Apoptosis array Statistical analysis Results The synergistic effects of abemaciclib and talazoparib combination on TNBC cells The apoptotic effects of abemaciclib and TAL combination in TNBC cells The effects of abemaciclib and TAL combination on cell cycle arrest in TNBC cells The molecular mechanism of abemaciclib and TAL combination mediated apoptosis in TNBC cells at protein level Discussion Conclusion References