A network modeling approach to elucidate drug resistance mechanisms and predict combinatorial drug treatments in breast cancer

Durable control of invasive solid tumors is thwarted by the lack of knowledge of effective drug combinations and of the acquired and intrinsic resistance mechanisms of drugs. In an effort to tackle this problem, the SU2C-NSF-TVF Drug Combination Convergence Team is using mechanistic models of cancer cell signaling based on therapeutic and cell line data in order to identify elements within cancer cells that might eventually be exploited through therapeutic combinations. Here we present a comprehensive mechanistic network model of signal transduction in ER+ PIK3CA-mutant breast cancer. Focusing on PI3K inhibitors, the model recapitulates known resistance mechanisms and predicts other possibilities for resistance: loss of RB1, FOXO3, P27, or PRAS40. To test these predictions, we analyzed genome-wide CRISPR screens of two breast cell lines in the presence of PI3K inhibitors (BYL719 and GDC0032) and found that the predicted genes (RB1, FOXO3, P27, and PRAS40) were significantly enriched in the screens. Some of these resistance genes (e.g. loss of RB1) were found to be cell-line specific and follow-up experiments in RB1-KO cells confirmed the cell-line-specific nature of PI3K-inhibitor resistance. The model also reveals known and novel combinatorial interventions that are more effective than PI3K inhibition alone. For example, the model predicts that the combination of PI3K inhibitors with inhibitors of anti-apoptotic protein MCL1 would be effective. Follow up experiments in cell lines using cell viability assays, cell death analyses, and dynamic BH3 profiling experiments to determine increases in apoptotic priming upon treatment confirmed that MCL1 inhibitors (S63845) enhance the effect of PI3K inhibitors (BYL719) and that this combinatorial effect is cell-line-specific, similarly to what was found in the resistance genes case. In conclusion, the model predicted drug resistance mechanisms and effective drug combinations, some of which were verified experimentally and found to be cell-line-specific. Next iterations of the model will incorporate the identified discrepancies and newly identified resistance mechanisms to drugs of clinical interest. Citation Format: Jorge Gómez Tejeda Zañudo, Pingping Mao, Joan Montero, Guotai Xu, Kailey J. Kowalski, Gabriela N. Johnson, José Baselga, Maurizio Scaltriti, Anthony G. Letai, Nikhil Wagle, Reka Albert. Network modeling of drug resistance mechanisms and drug combinations in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 675.

The PIK3CA gene is mutated in ∼40% of HR+/HER2- breast cancers. Alpelisib and inavolisib are currently FDA approved for treatment of PIK3CA-mutant breast cancers, although neither is selective for mutant PIK3CA. Inhibition of wild-type PI3K results in multiple adverse events, including hyperglycemia, which limit the dose and duration of treatment. Further, inhibition of PI3K/AKT relieves feedback suppression of receptor tyrosine kinase (RTK) expression and activity, thus limiting complete inhibition of PI3K/AKT signaling in cancer cells and resulting in adaptive drug resistance. Whether PI3K mutant-selective inhibitors can more potently block this compensatory adaptive resistance is unclear. In this study, we interrogated early adaptive resistance mechanisms to the PI3Kα mutant-selective inhibitor RLY-2608 in HR+ breast cancer cells with PIK3CA mutations. Phospho-RTK arrays of MCF7 and T47D cells treated with the IC75 of RLY-2608 for 0-48h revealed feedback activation of the ERBB family of RTKs (EGFR, HER2, HER3, HER4) at 24 h. These results were confirmed by immunoblot analysis with phospho-specific antibodies. After an initial inhibition of MAPK and PI3K/AKT signaling at 6 h, we found rebound activation of pERK1/2, pAKT1, and pS6 at 24 h, which was maintained for 48 h, suggesting a potential mechanism of adaptive resistance. To identify the ERBB RTKs causing this adaptation, we tested the TKIs erlotinib, neratinib, and tucatinib, and the HER3 antibodies patritumab and zenocutuzumab, each in combination with RLY-2608. RLY-2608 combined with the pan-HER TKI neratinib and the HER2-selective TKI tucatinib most effectively reduced pERK1/2, pAKT1 S473, and pS6 at 48 h, suggesting inhibition of HER2 could block this adaptive mechanism. In short and long-term proliferation assays, neratinib and tucatinib also synergized better with RLY-2608 compared to the other inhibitors. GSEA of RNA-seq data from MCF7 and T47D cells treated with RLY-2608 for 48 h revealed upregulation of pathways related to tyrosine kinase signaling, extracellular matrix, and redox signaling. FoxO signaling was one of the top upregulated KEGG and Reactome pathways, suggesting that activation of FoxO transcriptional activity may compensate for PI3K/AKT inhibition. The Hallmark Estrogen signaling pathway was also upregulated following RLY-2608 treatment; this was validated by immunoblot analysis, showing that ERα levels increased in T47D and MCF7 cells treated with RLY-2608 over a time course. Other upregulated Hallmark pathways in both cell lines included Myogenesis, Hypoxia, and KRAS signaling down. These findings suggest that, in addition to RTK activation, adaptive resistance to RLY-2608 may involve metabolic reprogramming, increased oxidative stress, and hypoxia signaling, highlighting the need for combination strategies to overcome resistance and enhance treatment efficacy. Citation Format: Fabiana Napolitano, Chang-Ching Lin, Khushi Ahuja, Dan Ye, María Rosario Chica-Parrado, Yasuaki Uemoto, Pamela Luna, Yuki Matsunaga, Saurabh Mendiratta, Nisha Unni, Jeon Lee, Ariella B. Hanker, Carlos L. Arteaga. Targeting mechanisms of adaptive resistance to the PI3Kαmutant selective inhibitor RLY-2608 in HR+/PIK3CA mutant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3003.

241P APOBEC3-induced PIK3CA mutations predict better clinical outcomes with PI3K inhibitors in patients with PIK3CA-mutated advanced breast cancer

Background: Taselisib (GDC-0032) is a next-generation PI3K inhibitor with increased anti-tumor activity against PIK3CA mutant (MT) cancers. Taselisib is an orally bioavailable, potent, and selective inhibitor of Class I PI3K alpha, delta, and gamma isoforms, with 30-fold less inhibition of the PI3K beta isoform relative to the PI3K alpha isoform. Preclinical data show that taselisib has enhanced activity against PI3K alpha isoform (PIK3CA) MT breast cancer cell lines and enhanced antitumor activity when combined with letrozole. Clinical data with single-agent taselisib also showed increased tumor shrinkage in patients with PIK3CA MT breast cancer as compared to patients with PIK3CA wildtype (WT) breast cancer. Material and Methods: A Phase 1b dose escalation study was conducted with evaluation of taselisib doses ranging from 6-9 mg QD in combination with letrozole 2.5mg QD in a modified 3+3 design. A dose expansion cohort was conducted with taselisib 6 mg QD. Safety and tolerability of GDC-0032 was assessed, as well as pharmacokinetics (PK), pharmacodynamic (PD) assessment by FDG-PET, and anti-tumor activity by RECIST. Results: As of 31 January 2014, 28 patients were enrolled onto this study with the completion of dose escalation and the dose expansion cohort. No dose limiting toxicities (DLTs) were observed at either the 6 mg (n = 20) or 9 mg (n = 8) dose levels. Adverse events (AEs) assessed by the investigator as related to taselisib in ≥10% of patients (any grade) included diarrhea, nausea, stomatitis, fatigue, rash, decreased appetite, hyperglycemia, dysgeusia, mucosal inflammation, vomiting, muscle spasms, asthenia, dry mouth, dry skin, pruritus, and aspartate aminotransferase increased. Grade 3 and 4 adverse events assessed by the investigator as drug-related and occurring in greater than one patient included diarrhea (14%), hyperglycemia (7%), and mucosal inflammation (7%). No apparent PK interactions were observed between taselisib and letrozole. The median number of prior systemic therapies was six, and promising efficacy data has been observed in these heavily pretreated patients. Metabolic partial responses via FDG-PET (≥ 20% decrease in mean SUVmax) were observed in 11 out of 18 patients assessed (61%). Confirmed partial responses by RECIST have been observed at both the 6mg and 9mg taselisib dose levels. For patients with measurable disease at baseline, the overall response rate of 38% was observed in patients with PIK3CA MT breast cancer and 9% in patients with PIK3CA WT breast cancer. Updated data on safety, PD, efficacy, and biomarker correlates will be presented. Conclusions: The combination of taselisib and letrozole is a well-tolerated regimen with promising preliminary efficacy in PIK3CA MT breast cancer patients. This preliminary Ph1b clinical data is consistent with taselisib preclinical and single-agent clinical data showing increased anti-tumor activity for taselisib in PIK3CA MT breast cancer as compared to PIK3CA WT breast cancer. Taselisib is being further investigated in the neoadjuvant setting in combination with letrozole in the LORELEI study in patients with untreated hormone receptor-positive breast cancer. Citation Format: Cristina Saura, Jasgit Sachdev, Manish R Patel, Andres Cervantes, Dejan Juric, Jeffrey R Infante, Donald Richards, Sandra Sanabria, Xuyang Lu, Joseph Ware, Timothy R Wilson, Hema Parmar, Jerry Y Hsu, Mafalda Oliveira, Eric P Winer, Daniel D Von Hoff, Jose Baselga, Ian E Krop. Ph1b study of the PI3K inhibitor taselisib (GDC-0032) in combination with letrozole in patients with hormone receptor-positive advanced breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD5-2.

Durable control of invasive solid tumors necessitates identifying therapeutic resistance mechanisms and effective drug combinations. In this work, we used a network-based mathematical model to identify sensitivity regulators and drug combinations for the PI3Kα inhibitor alpelisib in estrogen receptor positive (ER+) PIK3CA-mutant breast cancer. The model-predicted efficacious combination of alpelisib and BH3 mimetics, for example, MCL1 inhibitors, was experimentally validated in ER+ breast cancer cell lines. Consistent with the model, FOXO3 downregulation reduced sensitivity to alpelisib, revealing a novel potential resistance mechanism. Cell line-specific sensitivity to combinations of alpelisib and BH3 mimetics depended on which BCL2 family members were highly expressed. On the basis of these results, newly developed cell line-specific network models were able to recapitulate the observed differential response to alpelisib and BH3 mimetics. This approach illustrates how network-based mathematical models can contribute to overcoming the challenge of cancer drug resistance. SIGNIFICANCE: Network-based mathematical models of oncogenic signaling and experimental validation of its predictions can identify resistance mechanisms for targeted therapies, as this study demonstrates for PI3Kα-specific inhibitors in breast cancer.

The role of radiation therapy in the management of primary breast cancer

Alpelisib-induced thyroiditis in a patient with metastatic breast cancer: Is routine monitoring of thyroid function required?

The PI3K/Akt/mTOR signaling pathway is involved in the inhibition of tumor cell apoptosis, the promotion of cell survival, cell cycle regulation, tumor angiogenesis, invasion, and metastasis and therefore plays an important role intumorigenesis, tumor growth, patient prognosis, and tumor treatment. Recent studies have identified this signaling pathway in breast cancer, and the PI3K/Akt/mTOR pathway is therefore being considered as a new therapeutic target anda hotspot in breast cancer research. Pre-clinical studies have confirmed that PI3K inhibitors and mTOR inhibitors achieve anticancer effects by targeting different levels of the PI3K/Akt/mTOR signaling pathway. Among the PI3K inhibitors, some molecules target only PI3K, whereas others target both PI3K and mTOR. Currently, researchers tend to focus on molecular targets based on the different PI3K subtypes to achieve more targeted and specific inhibition of the PI3K pathway. However, the clinical efficacy and efficiency of these inhibitors need to be further studied. The mTOR inhibitors target mTORC1 and have become relatively well-developed targeted therapies for the PI3K/AKT/mTOR pathway. Rapamycin derivatives have been studied in Phase II / III clinical trials in breast cancer, and these derivatives achieved positive results in the treatment of metastatic breast cancer when combined with endocrine therapy or HER2-targeted therapies. This review summarizes the activation of the PI3K/AKT/mTOR pathway, its role in breast cancer, and the latest clinical trials of a variety of PI3K and mTOR inhibitors to improve the understanding of the role of these drugs in breast cancer treatment.

Dear Editor, Despite the improved outcome of advanced estrogen receptor-positive (ER+) breast cancer patients treated with endocrine therapy in combination with either a cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) or a phosphoinositide 3-kinase inhibitor (PI3Ki), the disease will eventually progress, and the optimal treatment strategy upon progression remains undefined [1-4]. To address this, we developed MCF-7- and T47D-derived PIK3CA-mutated breast cancer cell lines [5] resistant to combined CDK4/6i palbociclib and fulvestrant (MPF-R and TPF-R) or combined PI3Ki alpelisib and fulvestrant (MAF-R and TAF-R), respectively (Supplementary Materials and Methods). Drug-sensitive isogenic cells (M-S and T-S) grown in parallel with MPF-R and TPF-R cells and the original MCF-7/S0.5 and T47D cells were analyzed for comparison. To characterize the cell lines, we initially performed gene expression microarray analysis. The Gene Set Enrichment Analysis (GSEA) showed that alterations in the regulators of various pathways, including the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, apoptosis, cholesterol homeostasis, and interferon alpha response were significantly enriched gene datasets in MPF-R cells compared to M-S cells (Supplementary Table S1, Supplementary Figure S1A). Notably, alterations in the regulators of the cell cycle and the PI3K/AKT/mammalian target of the rapamycin (mTOR) signaling pathway were identified as significantly enriched in MAF-R cells compared to MCF-7/S0.5 cells (Supplementary Table S2, Supplementary Figure S1B). Conversely, the estrogen-dependent gene expression gene set was found to be significantly enriched in M-S and MCF-7/S0.5 cells compared to MPF-R and MAF-R cells, respectively (Supplementary Figure S1C-D), which suggests that the resistant cells are less dependent on the ER pathway than the parental cell lines. Although most of the genomic alterations identified, such as PIK3CA and ERBB2 mutations, MYC amplification, and ESR1 fusion, were common to both sensitive and resistant cells, a unique RB1 mutation (c.2125T>G, p.Tyr709Asp) was found in MPF-R cells, which likely is causally linked to the CDK4/6i-resistant phenotype in these cells (Supplementary Tables S3-S5). Based on these data, we firstly evaluated whether addition of an inhibitor of the PI3K/AKT/mTOR or the cell cycle pathway would overcome resistance to combined fulvestrant and palbociclib or alpelisib, respectively. The combination of alpelisib and fulvestrant was recently approved by the U.S. Food and Drug Administration for the treatment of ER+ PIK3CA-mutated metastatic breast cancer following progression on endocrine therapy [6]. However, whether this drug combination would suppress the growth of tumors resistant to combined CDK4/6i and endocrine therapy remains to be determined, although the clinical trial BYLieve showed some response in patients who progressed on combined CDK4/6i and endocrine therapy [2]. In addition, it is unknown whether the clinical combination of CDK4/6i and fulvestrant therapy is efficacious following progression on combined PI3Ki and fulvestrant. Therefore, we tested the effects of the PI3Ki alpelisib, CDK4/6i palbociclib, and ER degrader fulvestrant as single drugs and in various dual and triple combinations on growth and viability of breast cancer cells resistant to the combination of palbociclib or alpelisib and fulvestrant. We found that all the combinations were quite effective in the sensitive cell lines, whereas only the triple combination significantly reduced cell growth and viability of all resistant cells compared to the standard dual combinations (Supplementary Figures S2-S3). Next, we assessed the long-term effect of the different treatments and confirmed that all combinations were comparably effective in the sensitive cell lines (Figure 1A-C). In resistant MPF-R and TPF-R cells, combined alpelisib and fulvestrant was more effective than combined palbociclib and fulvestrant, but failed to maintain growth inhibition over the 8 weeks of treatment (Figure 1D-E). In contrast, the triple combination was highly effective in both resistant cell lines, preventing the outgrowth of resistant colonies in the 8-week period in MPF-R, while few resistant colonies of TPF-R cells were observed at weeks 7 and 8 (Figure 1D-E). Similarly, only the triple combination completely arrested the growth of the resistant MAF-R cells over the entire treatment period, while combined fulvestrant and palbociclib or alpelisib failed to maintain growth inhibition as cell confluency was observed as early as week 2 (Figure 1F). Triple combination with alpelisib, palbociclib, and fulvestrant prevents or significantly delays the emergence of resistance and significantly inhibits the growth of cell line-based xenograft and PDX tumors resistant to combined fulvestrant and palbociclib or alpelisib. (A-F) MCF-7- and T47D-derived cells resistant to combined palbociclib and fulvestrant (MPF-R and TPF-R) or to combined alpelisib and fulvestrant (MAF-R) and corresponding sensitive cell lines (M-S, T-S, MCF-7/S0.5) were treated weekly with vehicle, fulvestrant alone (Fulv, 100 nmol/L), palbociclib alone (Palbo, 200 nmol/L) and alpelisib alone (Alp, 1 μmol/L for M-S/MPF-R, 500 nmol/L for T-S/TPF-R, and 5 μmol/L for MCF7/S0.5/MAF-R) or different combinations of these for 8 weeks. Medium with the treatments was changed once a week. The percentage of wells (n = 48) exhibiting 50 % or greater confluence (defined as positive) was assessed weekly. The experiment was performed in three biological replicates and data are presented as mean ± SEMs. The triple combination therapy durably impairs growth of resistant breast cancer cells and prevents the emergence of resistance. (G-H) MPF-R and MAF-R cells were transplanted into the mammary fat pad of NOG CIEA mice. Mice were then treated with fulvestrant (Fulv, 100 mg/kg, subcutaneously, weekly) combined with either palbociclib (Palbo, 25 mg/kg, oral gavage, 5 days per week, n = 8-10) or alpelisib (Alp, 25 mg/kg, oral gavage, 5 days per week, n = 8-9) or a triple combination of alpelisib, palbociclib, and fulvestrant (n = 8). Tumor size was measured weekly for 4-5 weeks. For each subfigure, tumor growth curves are shown to the left and endpoint bar graphs of the same values are shown to the right. Data are presented as mean ± SEMs. Significant differences are calculated by one-way ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001, **** P < 0.0001; ns, non-significant) at endpoint (week 4 or 5). Triple combination of alpelisib, palbociclib and fulvestrant significantly inhibited growth of MPF-R and MFA-R tumor xenografts. (I) Left panel: Average tumor growth curves from mice bearing the Gar15-13-FPR patient-derived xenograft (PDX) model resistant to combined palbociclib and fulvestrant treated with palbociclib (25 mg/kg, 5 days per week, oral gavage) and fulvestrant (2 mg/body, subcutaneously weekly, n = 5), or a triple combination of alpelisib (50 mg/kg, 5 days per week, oral gavage), palbociclib, and fulvestrant (n = 5). The addition of alpelisib resulted in a clear inhibition of tumor growth. Growth rate of palbociclib- and fulvestrant-resistant PDX was similar to that of the untreated parental PDX (Supplementary Figure S6). Right panel: Tumor volumes at endpoint (3 weeks for palbociclib and fulvestrant; and 5 weeks for triple combination). (J) Quantification of Ki67 expression in PDX-Gar15-13FPR tumor sections treated with double or triple drug combinations, performed by ImageJ. (K) Representative images of Ki67 expression in 6 PDX-Gar15-13-FPR tumors treated with double or triple combinations (Scale bar 100 μm). (L-M) Kaplan-Meier curves evaluating PFS according to the levels of PDK-1 and PTEN in metastatic lesions (n = 80) from patients with advanced ER+ breast cancer treated with combined CDK4/6i (palbociclib or ribociclib) and endocrine therapy (fulvestrant or letrozole). A two-sided P-value was calculated using log-rank testing. (N-Q) Representative images of breast cancer metastasis sections showing low PDK-1 expression (intensity < 2, N), High PDK-1 expression (intensity ≥ 2, O), low nuclear and cytoplasmic PTEN expression (intensity < 2, P) and high nuclear and cytoplasmic PTEN expression (intensity ≥ 2, Q) (Scale bar 50 μm). Abbreviations: Alp, alpelisib; Fulv, fulvestrant; Palbo, palbociclib; PDX, patient-derived xenografts; PDK-1, phosphoinositide-dependent kinase-1; PTEN, phosphatase and tensin homolog; SEM, standard error of the mean. By studying the underlying mechanisms of the inhibitory effect caused by the triple combination, we observed a marked increase in apoptosis and cleaved poly(ADP-ribose) polymerase (PARP) levels in both MPF-R and M-S cells treated with the triple combination compared to the palbociclib and fulvestrant combination (Supplementary Figure S4A-B), but these changes were not observed in TPF-R and T-S cells (Supplementary Figure S4C). A slight increase in apoptosis was also observed in the MAF-R cells, but not in TAF-R cells (Supplementary Figure S4D-E). Furthermore, triple combination efficiently inhibited cell proliferation (Supplementary Figure S4F-I), and reduced the expression of key proteins of the ER, PI3K/AKT/mTOR and cyclin D-CDK4/6-Rb pathways in both MPF-R and TPR-R cells (Supplementary Figure S5A-D), and induced cell cycle arrest in MPF-R cells (Supplementary Figure S5E-F). Next, we evaluated the antitumor activity of the triple combination and the clinically used dual combinations in MPF-R and MAF-R cell line xenografts. The triple combination significantly inhibited growth of both MPF-R and MAF-R tumors compared to the clinically used dual combinations (Figure 1G-H). In addition, we evaluated the efficacy of the triple combination in a palbociclib- and fulvestrant-resistant patient-derived xenograft (PDX) model (Gar15-13-FPR) and found that the triple combination significantly reduced tumor growth compared with combined palbociclib and fulvestrant (P < 0.0001) (Figure 1I, Supplementary Figure S6). The triple combination-treated tumors demonstrated a greater decrease in Ki67 expression compared to combined palbociclib and fulvestrant-treated tumors (Figure 1J-K). To investigate the clinical relevance of our findings, we next assessed whether p-S6 and p-PRAS40 levels, found to be upregulated in the combined palbociclib- and fulvestrant-resistant cell lines compared to sensitive cells (Supplementary Figure S5A-B), correlated with the clinical outcome in ER+ advanced breast cancer patients treated with combined CDK4/6i and endocrine therapy. Survival analysis did not show a significant correlation between the expression level of either p-S6 or p-PRAS40 and progression-free survival (PFS, Supplementary Figure S7). High p-AKT levels have been previously found in the combined palbociclib- and fulvestrant-resistant cells and were correlated with shorter PFS in the advanced ER+ breast cancer patients treated with combined CDK4/6i and fulvestrant [7]. Therefore, we next evaluated the clinical relevance of other markers of the PI3K/AKT/mTOR pathway, including 3-phosphoinositide dependent protein kinase-1 (PDK-1) and phosphatase and tensin homolog (PTEN), which are key regulators of the pathway and have previously been shown to play a key role in the resistance mechanisms to CDK4/6i [8, 9]. Moreover, PDK-1 was found to be among the genes significantly enriched in the palbociclib- and fulvestrant-resistant cell line in the GSEA analysis (Supplementary Table S1). Although PDK-1 and p-PDK-1 protein expression were not increased in cells resistant to combined CDK4/6i and endocrine therapy compared to the sensitive cells, we observed induction of phosphorylated and non-phosphorylated PDK-1 expression upon treatment with combined fulvestrant and CDK4/6i (Supplementary Figure S8), which suggests involvement of this regulator in the response to the combined therapy. The expression levels of PDK-1 and PTEN were evaluated in a cohort of advanced ER+ patients treated with combined CDK4/6i and endocrine therapy (n = 80). The survival analysis indicated strong associations between PDK-1 and PTEN levels and PFS. High levels (intensity ≥ 2) of PDK-1 were significantly associated with shorter PFS compared to low levels of PDK-1 (P = 0.019) (Figure 1L). PTEN showed, as expected, the opposite association, as tumors exhibiting low (intensity < 2) nuclear and cytoplasmic PTEN expression were associated with poor outcome compared to tumors with high nuclear and cytoplasmic PTEN expression (Figure 1M). Univariate Cox regression analysis showed that PDK-1 status (P = 0.022), PTEN status (P = 0.030), endocrine status (P = 0.007), and line of therapy (P = 0.006) were all significantly associated with PFS for patients treated with the combination of the endocrine therapy and CDK4/6i (Supplementary Table S6). Multivariate Cox regression analysis of these parameters revealed that both PTEN (P = 0.006) and PDK-1 expression (P = 0.031) were independent prognostic factors for PFS (Supplementary Table S6). Description of patient clinicopathological characteristics in the early and advanced settings are shown in Supplementary Tables S7-S8, respectively. Although χ2 test identified a significant difference in line of therapy for combined CDK4/6 inhibitor and endocrine therapy between low and high PDK-1 groups (Supplementary Table S8), multivariate analysis showed that PDK-1, but not line of therapy, was an independent prognostic factor for PFS in this patient cohort (Supplementary Table S6). No significant association between PDK-1 or PTEN expression and other clinical parameters at the metastatic disease was identified (Supplementary Table S8). Collectively, we found that the triple combination with fulvestrant, palbociclib and alpelisib is superior in abolishing the growth of breast cancer resistant to combined fulvestrant and palbociclib or alpelisib. Moreover, our data suggested that the benefit of treating tumors resistant to combined palbociclib and fulvestrant with the alternative approved combination of alpelisib and fulvestrant, or vice versa, was very limited. Our data supported the clinical development of triple combination therapy targeting PI3K, CDK4/6, and ER in advanced ER+ breast cancer following progression on the combined CDK4/6i or PI3Ki and endocrine therapy. The immunohistochemical study was approved by the Ethics committee of the Region of Southern Denmark (approval no S-2008-0115) and the Danish Data Protection Agency. All patient samples were collected in compliance with informed consent policy and coded to maintain patient confidentiality. Procedures and endpoints involving laboratory animals were approved by the Experimental Animal Committee of The Danish Ministry of Justice (2021-15-0201-0084) and the Garvan Institute of Medical Research Animal Ethics Committee (protocol 15/25, 18/20, and 18/26). Not applicable. The gene expression data generated during the study are publicly available in the gene expression omnibus (GEO) database under the accession number GSE210400. Survival analyses and immunohistochemistry data are not publicly available to protect patient privacy but will be made available to authorized researchers who have an approved Institutional Review Board application and have obtained approval from the Regional Committees on Health Research Ethics for Southern Denmark. Please contact the corresponding author with data access requests. All other datasets generated during the study will be made available upon reasonable request to the corresponding author. Uncropped Western blots are part of the supplementary information. Conceptualization: Leena Karimi, Carla L. Alves, and Henrik J. Ditzel. Methodology: Leena Karimi, Carla L. Alves, Mikkel G. Terp, Martina Tuttolomondo, Neil Portman, Sidse Ehmse, Lene E. Johansen, Martin Bak, Elgene Lim, and Henrik J. Ditzel. Investigation: Leena Karimi, Carla L. Alves, Mikkel G. Terp, Martina Tuttolomondo, Neil Portman, Sidse Ehmsen, Lene E. Johansen, Martina Bak, Elgene Lim, and Henrik J. Ditzel. Writing the original draft: Leena Karimi, Carla L. Alves, Henrik J. Ditzel. Writing and Review & Editing: all authors. Funding acquisition, Leena Karimi, Carla L. Alves, and Henrik J. Ditzel. Resources: Henrik J. Ditzel. Supervision: Carla L. Alves and Henrik J. Ditzel. The authors declare that they have no competing interests. This research was funded in part by grants from the Danish Cancer Society (to Henrik J. Ditzel), Health Insurance "Denmark" (to Henrik J. Ditzel), Academy of Geriatric Cancer Research (AgeCare) (to Henrik J. Ditzel), and Pink Tribute (to Henrik J. Ditzel). We thank Lone Christiansen and Helle Wohlleben at the Department of Pathology, Odense University Hospital, for excellent technical assistance with the immunohistochemistry, the Animal Core Facility at University of Southern Denmark for animal care, and Kat Occhipinti for editorial assistance. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

HER2 amplification is found in up to 20% of breast cancers and PIK3CA mutations occur in up to 30%. These cancers are susceptible to targeted therapies directed against oncogenic HER2 and PI3K. Emerging evidence from clinical trials has revealed a surprising heterogeneity in response to targeted therapies, for example within HER2 positive breast cancer patients, the efficacy of HER2 inhibitors has varied. We have previously shown in breast cancer cell lines and a small cohort of HER2 positive breast cancer patients that low levels of the pro-apoptotic BIM can confer intrinsic resistance to HER2 inhibitors, by repressing the apoptotic response following treatment. Here in, we investigated the ability of a BCL-2/Bcl-XL inhibitor to enhance the apoptotic response specifically in low BIM HER2 amplified and PIK3CA mutant breast cancers with intrinsic resistance to targeted therapies, by freeing up BIM from BCL-2:BIM and BCL-xL:BIM complexes. We found that in these cancers, the addition of BCL-2/BCL-xL inhibitors synergized with HER2 inhibitors and PI3K inhibitors to induce apoptosis, and to similar levels induced by targeted therapy alone in the high BIM expressing breast cancers. Biochemical analysis revealed that PI3K inhibition downregulated MCL-1 in PIK3CA mutants, which leads to a further increase in the amount of free BIM, further “maximizing” the otherwise low levels of cellular BIM in these cells. These results translated to tumor regressions in mouse xenografts treated with the combination, similar to what was seen with single-agent PI3K inhibitor in a high BIM expressing PIK3CA mutant breast cancer xenograft model. Thus, combining a BCL-2/BCL-xL inhibitor with the appropriate targeted therapy may overcome intrinsic resistance caused by low BIM expression in HER2 amplified and PIK3CA mutant breast cancer. Citation Format: Anthony C. Faber, Sadhna Vora, Jeffrey A. Engelman. Sensitizing low BIM expressing breast cancers to targeted therapies. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A062.

PurposeThe therascreen PIK3CA mutation assay and the alpha-specific PI3K inhibitor alpelisib are FDA-approved for identifying and treating patients with advanced PIK3CA-mutated (PIK3CAmut) breast cancer (BC). However, it is currently unknown to what extend this assay detects most PIK3CA mutations in BC. This information is critical as patients and clinicians are using this and other genomic assays to indicate alpelisib.MethodsData from 6338 patients with BC was explored across 10 publicly available studies. The primary objective was to evaluate the proportion and distribution of PIK3CA mutations in BC. Secondary objectives were (1) to evaluate in silico the spectrum of PIK3CA mutations in BC that would be captured by the therascreen panel; (2) to evaluate the proportion and distribution of PIK3CA mutations in hormone receptor-positive/HER2-negative (HR+/HER2−), HER2+, and triple-negative BC (TNBC); and (3) to explore the identification of PIK3CA mutations in a cohort of 48 HR+/HER2− advanced BC patients by the Guardant B360 circulating tumor DNA (ctDNA) assay.ResultsPatients with PIK3CAmut tumors represented 35.7% (2261/6338). Five PIK3CA mutations comprised 73% of all PIK3CA mutations: H1047R (35%), E545K (17%), E542K (11%), N345K (6%), and H1047L (4%). Therascreen gene list would capture 72% of all PIK3CA mutations and 80% of patients with a known PIK3CAmut BC. Among patients with double PIK3CAmut tumors (12% of all PIK3CAmut), the therascreen panel would capture 78% as harboring 1 single PIK3CA mutation, 17% as PIK3CAmut undetected, and 5% as PIK3CA double-mut. PIK3CA mutation rates were lower in TNBC (16%) compared to HR+/HER2 (42%) and HER2+ (31%) BC; however, the distribution of the 4 main PIK3CA mutations across subtypes was similar. Finally, 28% of PIK3CA mutations identified in ctDNA in 48 patients with advanced HR+/HER2− BC were not part of the therascreen panel.ConclusionPIK3CA mutations in BC are heterogenous and ~ 20% of patients with a known PIK3CA mutation, and 95% with a known double PIK3CAmut tumor, would not be captured by the therascreen panel. Finally, the clinical utility of PIK3CA mutations not present in the therascreen companion diagnostic assay or identified by other sequencing-based assays needs further investigation.

HER2 amplification and activating mutations in PIK3CA, the gene encoding the p110α subunit of PI3K, often co-occur in breast cancer. We generated a transgenic mouse model of HER2-overexpressing (HER2+), PIK3CAH1047R-mutant breast cancer. In these mice, PIK3CAH1047R accelerates HER2-mediated tumor formation and promotes resistance to HER2 inhibitors (Hanker et al. PNAS 2013). HER2+/PIK3CA tumor growth was inhibited by treatment with the HER2 antibodies trastuzumab and pertuzumab in combination with the pan-PI3K inhibitor BKM120 (TPB). We sought to discover mechanisms of acquired resistance to the triple therapy by long-term treatment of established HER2+/PIK3CA tumors. Tumor transplants derived from a transgenic HER2+/PIK3CA tumor were initially growth inhibited by TPB. After several weeks, a subset of transplants (3/11) resumed growth in the presence of continuous TPB therapy. TPB-resistant tumors were cross-resistant to the combination of T + P + BYL719 (a p110α-specific inhibitor). Whole exome sequencing did not identify acquired somatic alterations in TPB-resistant tumors, including in HER2. However, RNA-seq revealed significant transcriptional upregulation of extracellular matrix (ECM) genes and genes involved in cell adhesion, including collagens, tenascins, and thrombospondins. Likewise, trichrome staining revealed a significant increase in collagen fibers and IHC analysis confirmed increased Tenascin expression in the TPB-resistant tumor stroma. In addition, western blot analysis revealed increased expression of an activated form of integrin β1, a substrate for ECM ligands such as collagen, as well as P-SrcY416 (activated by integrins/focal adhesion). We also found that transcription of many of these genes is induced by short-term TPB treatment in human breast cancer cell lines by qRT-PCR. Interestingly, primary tumor cells derived from TPB-resistant tumors no longer displayed resistance when grown in vitro. These cells regained TPB resistance when re-introduced into mice. Plating primary tumor cells on growth factor-reduced Matrigel or on Collagen I-coated plates restored resistance, suggesting that the ECM directly promotes TPB resistance. We are currently investigating whether inhibition of Integrin/Src signaling reverses TPB resistance. We are also exploring whether components of the ECM are altered in residual disease specimens from HER2+ breast cancer patients treated with neoadjuvant anti-HER2 therapies. Our data suggest that upregulation of ECM/integrin/Src signaling contributes to resistance to combinations of HER2 and PI3K inhibitors, and strongly support the growing body of literature indicating that components of the tumor microenvironment promote resistance to targeted therapies. Citation Format: Ariella B. Hanker, Monica Valeria Estrada, Junfei Zhao, Feixiong Cheng, Preston D. Moore, Darren Tyson, Violeta Sanchez, Brent N. Rexer, Melinda Sanders, Zhongming Zhao, Thomas P. Stricker, Carlos L. Arteaga. ECM/Integrin signaling promotes resistance to the combination of HER2 and PI3K inhibitors in HER2+, PIK3CA-mutant breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 302.

Breast Cancer in the Asia–Pacific Region

Introduction: Metastatic HER2-positive breast cancer is treated with regimens such as taxane with trastuzumab and pertuzumab followed by trastuzumab deruxtecan (T-DXd). The third line options are ado-trastuzumab emtansine (T-DM1), tucatinib with capecitabine, and trastuzumab. Mutations in the PIK3CA gene encoding PI3Kα have been reported in 12%-39% of HER2-positive breast cancers and are associated with worse prognosis. Alpelisib is an oral, α-specific PI3K inhibitor that is approved in combination with fulvestrant in hormone receptor positive, HER2-negative, PIK3CA-mutated advanced breast cancer following progression on or after endocrine therapy. Activating mutations in the PIK3CA gene are associated with resistance to anti-HER2 therapies. Combined inhibition of HER2 and PI3K overcomes this mechanism preclinically. We present a patient with estrogen and progesterone receptor (ER/PR) negative, HER2-positive, PIK3CA-mutated multidrug resistant refractory breast cancer who had a complete response to alpelisib and trastuzumab. Case Presentation: A 63-year-old female patient underwent left mastectomy for a stage I (T1aN0M0) ER/PR-negative, HER2-positive breast cancer in 2014. Four years later, she developed a HER2-positive right axillary breast cancer which was treated with neoadjuvant chemotherapy followed by axillary nodal dissection and regional radiation therapy. Since 2018, right axillary nodal and chest wall metastases progressed on several lines of systemic therapy including lapatinib and capecitabine, trastuzumab and paclitaxel, trastuzumab and vinorelbine, fam-trastuzumab-deruxtecan-nhki (Enhertu), T-DM1, margetuximab with eribulin, and finally trastuzumab with capecitabine. A right chest wall biopsy demonstrated a high grade invasive ductal carcinoma that was ER/PR-negative and HER2-positive. Next generation sequencing revealed a PIK3CA mutation (p.H1047R). Treatment was changed to alpelisib and trastuzumab. After four months of treatment, she developed a complete response of cutaneous metastases with adverse effect of grade 2 mucositis. She continues to be under treatment and is doing well. Conclusions: Alpelisib and trastuzumab can be a viable treatment option for patients with HER2-positive and PIK3CA-mutated metastatic breast cancer. Our case demonstrates the importance of obtaining next generation sequencing in patients with refractory breast cancer. Integrating precision medicine can elucidate mechanisms of resistance and allow treatment with combination and targeted therapies that can improve patient outcomes, similar to our patient presented in this vignette. Citation Format: Farah Shah, Nejina Rijal, Erin H. Lin, Sayeh M. Lavasani, Rita S. Mehta, Ritesh Parajuli. Complete response with Alpelisib and Trastuzumab in a patient with ER/PR-negative, HER2-positive refractory metastatic breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P5-05-19.

Cyclin-dependent kinase 4/6 (CDK4/6) and PI3K inhibitors synergize in PIK3CA-mutant ER-positive HER2-negative breast cancer models. We conducted a phase Ib trial investigating the safety and efficacy of doublet CDK4/6 inhibitor palbociclib plus selective PI3K inhibitor taselisib in advanced solid tumors, and triplet palbociclib plus taselisib plus fulvestrant in 25 patients with PIK3CA-mutant, ER-positive HER2-negative advanced breast cancer. The triplet therapy response rate in PIK3CA-mutant, ER-positive HER2-negative cancer was 37.5% [95% confidence interval (CI), 18.8-59.4]. Durable disease control was observed in PIK3CA-mutant ER-negative breast cancer and other solid tumors with doublet therapy. Both combinations were well tolerated at pharmacodynamically active doses. In the triplet group, high baseline cyclin E1 expression associated with shorter progression-free survival (PFS; HR = 4.2; 95% CI, 1.3-13.1; P = 0.02). Early circulating tumor DNA (ctDNA) dynamics demonstrated high on-treatment ctDNA association with shorter PFS (HR = 5.2; 95% CI, 1.4-19.4; P = 0.04). Longitudinal plasma ctDNA sequencing provided genomic evolution evidence during triplet therapy. SIGNIFICANCE: The triplet of palbociclib, taselisib, and fulvestrant has promising efficacy in patients with heavily pretreated PIK3CA-mutant ER-positive HER2-negative advanced breast cancer. A subset of patients with PIK3CA-mutant triple-negative breast cancer derived clinical benefit from palbociclib and taselisib doublet, suggesting a potential nonchemotherapy targeted approach for this population.This article is highlighted in the In This Issue feature, p. 1.