XLSX file - 14K, Table S1: Calculated IC50s for the PI3K/mTOR pathway inhibitors against the breast cancer cell line panel. ER: Estrogen Receptor. N/A: IC50 not achieved, N/D: IC50 not determined

PDF file - 45K, Summary of adverse events suspected to be study-drug related by grade (safety set).

PDF file - 42K, Summary of deaths and adverse events (safety set).

PDF file - 40K, Figure S2: Biomarkers of in vivo response to inhibitors of PI3K/mTOR signaling. Reverse Phase Protein Analysis (RPPA) of KPL-1 tumor samples taken after 7 days treatment with the molecules indicated. Tumors taken 24 hours post final dose. A, pAKT Ser473. B, pS6 Ser235/236.

PDF file - 67K, Bayesian logistic regression model: dose-response curve and model inference results at the time of determination of the recommended Phase II dose.

PDF file - 48K, Exposure to buparlisib and trastuzumab (safety set).

PDF file - 772K, Figure S1: Targeting the PI3K/mTOR pathway in HER2-amplified breast cancer cell lines. Effect of A, RAD001, B, BEZ235, C, BKM120 or D, BYL719 treatment on PI3K/mTOR signaling in two representative cell lines; SKBR3 and MDA453 (PIK3CA mutant H1047R). All lysates taken 24 hours post-treatment.

PDF file - 38K, Patient disposition (full analysis set).

PDF file - 65K, Criteria for defining dose-limiting toxicities.

PDF file - 62K, Summary of buparlisib pharmacokinetic parameters.

PDF file - 36K, Figure S3. Xenograft response data for 7-day tissue acquisition studies. A-C, SUM190, UACC812 and KPL-1 xenografts (6 mice per arm) were treated with BKM120 at 35 mg/kg, BYL719 at 50 mg/kg, RAD001 at 10 mg/kg and BEZ235 at 30 mg/kg, all daily PO. Trastuzumab was given at given at 10 mg/kg IP twice weekly. For SUM190 (A), tumors were collected 2.5 hours post final dose, for UACC812 (B) and KPL-1 (C), samples were collected 24 hours post final dose.

XLSX file - 213K, Table S2. Summary of in vivo activity of BKM120, BYL719, RAD001 and BEZ235 in breast cancer tumor models. T/C: Ratio of mean tumor volume of treated (T) to control (C) at final day of treatment. Final day was determined as the final day of treatment for the vehicle control arm for each study in Figure 5. Comparisons between groups were all made by Repeated Measures ANOVA (RMANOVA) followed by Dunnett test for multiple comparisons. BW: body weight; TR: treatment related; NTR: non-treatment related.

Additional file 5: Table S2. Pharmacodynamic changes in protein levels in acquired resistant cells versus parental cells. RPPA data (norm_Log2 values) restricted to the proteins with a > 0.25 or

Abstract Background Combined targeting of CDK4/6 and ER is now the standard of care for patients with advanced ER+/HER2− breast cancer. However, acquired resistance to these therapies frequently leads to disease progression. As such, it is critical to identify the mechanisms by which resistance to CDK4/6-based therapies is acquired and also identify therapeutic strategies to overcome resistance. Methods In this study, we developed and characterized multiple in vitro and in vivo models of acquired resistance to CDK4/6-based therapies. Resistant models were screened by reverse phase protein array (RPPA) for cell signaling changes that are activated in resistance. Results We show that either a direct loss of Rb or loss of dependence on Rb signaling confers cross-resistance to inhibitors of CDK4/6, while PI3K/mTOR signaling remains activated. Treatment with the p110α-selective PI3K inhibitor, alpelisib (BYL719), completely blocked the progression of acquired CDK4/6 inhibitor-resistant xenografts in the absence of continued CDK4/6 inhibitor treatment in models of both PIK3CA mutant and wild-type ER+/HER2− breast cancer. Triple combination therapy against PI3K:CDK4/6:ER prevented and/or delayed the onset of resistance in treatment-naive ER+/HER2− breast cancer models. Conclusions These data support the clinical investigation of p110α-selective inhibitors of PI3K, such as alpelisib, in patients with ER+/HER2− breast cancer who have progressed on CDK4/6:ER-based therapies. Our data also support the investigation of PI3K:CDK4/6:ER triple combination therapy to prevent the onset of resistance to the combination of endocrine therapy plus CDK4/6 inhibition.

PIK3CA mutations occur in approximately 40% of patients with hormone receptor-positive breast cancer. A PI3K inhibitor, alpelisib, combined with fulvestrant led to a median progression-free survival of 11 months, as compared with 5.7 months with placebo plus fulvestrant. Hyperglycemia, rash, and diarrhea were more common with alpelisib. Background PIK3CA mutations occur in approximately 40% of patients with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancer. The PI3K alpha-specific inhibitor alpelisib has shown antitumor activity in early studies. Methods In a randomized, phase 3 trial, we compared alpelisib (at a dose of 300 mg per day) plus fulvestrant (at a dose of 500 mg every 28 days and once on day 15) with placebo plus fulvestrant in patients with HR-positive, HER2-negative advanced breast cancer who had received endocrine therapy previously. Patients were enrolled into two cohorts on the basis of tumor-tissue PIK3CA mutation status. The primary end point was progression-free survival, as assessed by the investigator, in the cohort with PIK3CA-mutated cancer; progression-free survival was also analyzed in the cohort without PIK3CA-mutated cancer. Secondary end points included overall response and safety. Results A total of 572 patients underwent randomization, including 341 patients with confirmed tumor-tissue PIK3CA mutations. In the cohort of patients with PIK3CA-mutated cancer, progression-free survival at a median follow-up of 20 months was 11.0 months (95% confidence interval [CI], 7.5 to 14.5) in the alpelisib-fulvestrant group, as compared with 5.7 months (95% CI, 3.7 to 7.4) in the placebo-fulvestrant group (hazard ratio for progression or death, 0.65; 95% CI, 0.50 to 0.85; P<0.001); in the cohort without PIK3CA-mutated cancer, the hazard ratio was 0.85 (95% CI, 0.58 to 1.25; posterior probability of hazard ratio <1.00, 79.4%). Overall response among all the patients in the cohort without PIK3CA-mutated cancer was greater with alpelisib-fulvestrant than with placebo-fulvestrant (26.6% vs. 12.8%); among patients with measurable disease in this cohort, the percentages were 35.7% and 16.2%, respectively. In the overall population, the most frequent adverse events of grade 3 or 4 were hyperglycemia (36.6% in the alpelisib-fulvestrant group vs. 0.7% in the placebo-fulvestrant group) and rash (9.9% vs. 0.3%). Diarrhea of grade 3 occurred in 6.7% of patients in the alpelisib-fulvestrant group, as compared with 0.3% of those in the placebo-fulvestrant group; no diarrhea of grade 4 was reported. The percentages of patients who discontinued alpelisib and placebo owing to adverse events were 25.0% and 4.2%, respectively. Conclusions Treatment with alpelisib-fulvestrant prolonged progression-free survival among patients with PIK3CA-mutated, HR-positive, HER2-negative advanced breast cancer who had received endocrine therapy previously.

Addition of CDK4/6 inhibitors such as palbociclib, ribociclib or abemaciclib to endocrine-based therapies significantly improves progression-free and in some subgroups, overall survival in patients with advanced estrogen receptor-positive (ER+) breast cancer. However, acquired resistance to CDK4/6 inhibitors remains a significant unmet clinical need. It is critical to ascertain the mechanisms by which tumor cells evade CDK4/6 based therapy. In this study we screen multiple in vitro and in vivo models of acquired resistance to CDK4/6 inhibitors to identify potential resistance/escape pathways and targets for pharmaceutical intervention to overcome resistance. ER+ breast cancer cell lines of diverse molecular backgrounds were conditioned to acquire resistance to CDK4/6 inhibitors through either long-term culture in the presence of clinically relevant concentrations of inhibitors or as cell line xenografts treated through progression on a CDK4/6 inhibitor plus fulvestrant. Baseline and pharmacodynamic changes in cell signaling were measured using reverse phase protein array (RPPA) and RNAseq analysis. Acquired resistance to CDK4/6 inhibitors was associated with decreases in phosphorylated-Rb (pRb) and ER-alpha protein and increases in pAKT and pS6 relative to isogenic controls. The p110α-selective PI3K-inhibitor, alpelisib, in combination with fulvestrant or ribociclib/fulvestrant blocked pAKT signaling in xenografts progressing on palbociclib/fulvestrant and induced significant tumor regressions. Apelisib plus fulvestrant also produced robust anti-tumor responses in xenografts progressing on ribociclib/fulvestrant. Triple combination treatment with ribociclib/alpelisib/fulvestrant induced significant regressions in resistant tumors and in treatment naïve models, where complete tumor regressions occurred regardless of PIK3CA mutation status. Regressions were maintained for >9 weeks post withdrawal of treatment, indicating that therapeutic resistance may be prevented by this triple combination approach. RPPA analysis of responding tumors identified sustained inhibition of both PI3K- and CDK4/6:Rb-pathway signaling accompanied by activation of pro-apoptotic proteins. These data support clinical investigation of targeting PI3K with alpelisib in breast cancers progressing on CDK4/6 based therapies and investigation of upfront triple combination therapy prior to acquisition of resistance to CDK4/6. Citation Format: Neil A. O'Brien, Martina SJ McDermott, Dylan F. Conklin, Alex Gaither, Tong Luo, Raul Ayala, Suruchi Salgar, Emmanuelle DiTomaso, Naveen Babbar, Faye Su, Sara A. Hurvitz, Ronald Linnartz, Kristine Rose, Samit Hirawat, Dennis J. Slamon. Targeting activated PI3K/mTOR signaling overcomes resistance to CDK4/6-based therapies in preclinical ER+ breast cancer models [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 3825.

Background In MONALEESA-2, ribociclib plus letrozole showed improved progression-free survival compared with letrozole alone as first-line treatment for postmenopausal patients with hormone receptor (HR)-positive, HER2-negative, advanced breast cancer. MONALEESA-7 aimed to assess the efficacy and safety of ribociclib plus endocrine therapy in premenopausal women with advanced, HR-positive breast cancer. Methods This phase 3, randomised, double-blind, placebo-controlled trial was done at 188 centres in 30 countries. Eligible patients were premenopausal women aged 18-59 years who had histologically or cytologically confirmed HR-positive, HER2-negative, advanced breast cancer; an Eastern Cooperative Oncology Group performance status of 0 or 1; measurable disease as per Response Evaluation Criteria in Solid Tumors version 1.1 criteria, or at least one predominantly lytic bone lesion; and had not received previous treatment with cyclin-dependent kinases 4 and 6 inhibitors. Endocrine therapy and chemotherapy in the adjuvant or neoadjuvant setting was permitted, as was up to one line of chemotherapy for advanced disease. Patients were randomly assigned (1: 1) via interactive response technology to receive oral ribociclib (600 mg/day on a 3-weeks-on, 1-week-off schedule) or matching placebo with either oral tamoxifen (20 mg daily) or a non-steroidal aromatase inhibitor (letrozole 2.5 mg or anastrozole 1 mg, both oral, daily), all with goserelin (3.6 mg administered subcutaneously on day 1 of every 28-day cycle). Patients and investigators were masked to treatment assignment. Efficacy analyses were by intention to treat, and safety was assessed in all patients who received at least one dose of any study treatment. The primary endpoint was investigator-assessed progression-free survival. MONALEESA-7 is registered with ClinicalTrials.gov, NCT02278120 and is ongoing, but no longer enrolling patients. Findings Between Dec 17, 2014, and Aug 1, 2016, 672 patients were randomly assigned: 335 to the ribociclib group and 337 to the placebo group. Per investigator's assessment, median progression-free survival was 23.8 months (95% CI 19.2-not reached) in the ribociclib group compared with 13.0 months (11.0-16.4) in the placebo group (hazard ratio 0.55, 95% CI 0.44-0.69; p<0.0001). Grade 3 or 4 adverse events reported in more than 10% of patients in either group were neutropenia (203 [61%] of 335 patients in the ribociclib group and 12 [4%] of 337 in the placebo group) and leucopenia (48 [14%] and four [1%]). Serious adverse events occurred in 60 (18%) of 335 patients in the ribociclib group and 39 (12%) of 337 in the placebo group, of which 15 (4%) and six (2%), respectively, were attributed to the study regimen. 12 (4%) of 335 patients in the ribociclib group and ten (3%) of 337 in the placebo group discontinued treatment because of adverse events. No treatment-related deaths occurred. 11 deaths occurred (five [1%] in the ribociclib group and six [2%] in the placebo group) during or within 30 days after treatment, most of which were due to progression of the underlying breast cancer (three [1%] and six [2%]). The remaining two deaths in the ribociclib group were due to an intracranial haemorrhage in an anticoagulated patient, and a pre-existing wound haemorrhage in another patient. Interpretation Ribociclib plus endocrine therapy improved progression-free survival compared with placebo plus endocrine therapy, and had a manageable safety profile in patients with premenopausal, HR-positive, HER2-negative, advanced breast cancer. The combination could represent a new first-line treatment option for these patients. Copyright (c) 2018 Elsevier Ltd. All rights reserved.

Background: Hyperactivation of the phosphatidylinositol-3-kinase (PI3K) pathway can occur due to PIK3CA mutations, present in ∼40% of patients (pts) with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2–) ABC. The Phase 3 randomized, double-blind SOLAR1 trial (NCT02437318) investigated the efficacy and safety of ALP (α-specific PI3K inhibitor) + FUL in pts with HR+, HER2– ABC. Methods: Men/postmenopausal women with HR+, HER2– ABC and 1 prior line of endocrine therapy were randomized (1:1) to ALP (300 mg/day) + FUL (500 mg every 28 days + Cycle 1 Day 15) or placebo (PBO) + FUL. Primary endpoint was locally assessed progression-free survival (PFS) in the PIK3CA-mutant (mut) cohort; PFS was analyzed in the non-mut cohort as a proof of concept (PoC). Safety was assessed in the total population. Other analyses were tumor response and PFS by important prognostic subgroups, including PIK3CA mutation exon/domain and subtype. Results: 572 pts enrolled; 341 had PIK3CA-mut ABC by tissue. Primary endpoint was met; PFS in the mut cohort was significantly longer with ALP+FUL vs PBO+FUL (HR 0.65; 95% CI 0.50–0.85; P = 0.00065; median 11.0 vs 5.7 months [mo]); median follow-up was 20.0 mo. Secondary endpoint of locally assessed PFS in the non-mut cohort did not meet predefined PoC criteria (HR 0.85; 95% CI 0.58–1.25; median 7.4 vs 5.6 mo). In pts with measurable, PIK3CA-mut ABC (n = 262), overall response rate was 36% for ALP+FUL vs 16% for PBO+FUL (p = 0.0002). Overall, most frequent all-grade (G) adverse events (AEs; single preferred term; ALP+FUL vs PBO+FUL) were hyperglycemia (64% vs 10%), diarrhea (58% vs 16%), nausea (45% vs 22%), decreased appetite (36% vs 10%) and rash (36% vs 6%). G 3/4 hyperglycemia (fasting plasma glucose >250 mg/dL) was observed in 37% of patients for ALP+FUL vs < 1% for PBO+FUL; G 3/4 rash in 10% vs < 1%. Discontinuations of ALP+FUL/PBO+FUL due to AEs were 5% vs 1%. Conclusions: ALP+FUL met the primary endpoint by significantly extending PFS vs PBO+FUL and demonstrated a manageable tolerability profile. This is the first study to show statistically significant, clinically meaningful PFS treatment improvement with an α-specific PI3K inhibitor in PIK3CA-mut HR+, HER2– ABC. Clinical trial identification: NCT02437318 (May 7, 2015). Editorial acknowledgement: Editorial assistance was provided by John Munro of ArticulateScience Ltd. Legal entity responsible for the study: Novartis Pharmaceutical Corporation. Funding: Novartis Pharmaceutical Corporation. Disclosure: F. André: Grants: Novartis during the conduct of the study; Grants: AstraZeneca, Pfizer, Eli Lilly, and Roche, outside of the submitted work. G. Rubovszky: Fees paid to institution: Novartis during the conduct of the study; Fees for advisory boards: Novartis outside of the submitted work. M. Campone: Consulting fees and fees for non-CME services related directly from commercial interest or their agents: Novartis, Pfizer, Astra Zeneca, Eli Lilly. S. Loibl: Grants to institution for research funding: Abbvie, Amgen, AstraZeneca, Celgene, Novartis, Pfizer, Roche, Teva, Vifor, outside of the submitted work. H.S. Rugo: Grants to institution: Pfizer, Novartis, Eli Lilly, Genentech, Macrogenics, Plexxikon, Merck, OBI, Eisai; Travel support: Eli Lilly, Pfizer, Mylan, Amgen, Merck Puma, all outside of the submitted work. H. Iwata: Grants and personal fees: Daiichi Sankyo, during the study; Grants and personal fees: Chugai, AstraZeneca, Pfizer; Personal fees: Eisai; Grants: MSD, Kyowahakou Kirin, GSK, Lilly, Novartis, Bayer, outside the work. P. Conte: Speaker's bureau: Roche/Genentech, Novartis, AstraZeneca; Research funding to institution: Roche, Novartis; Merck Serono; Travel & accommodation: Novartis; Celgene; AstraZeneca. I.A. Mayer: Consulting/advisory relationship: Novartis, Genentech; Research funding: Novartis, Pfizer. B. Kaufman: Advisory boards: Novartis, outside of the submitted work. T. Yamashita: Grants and honoraria: Chugai; Honoraria: Eisai, Novartis, Taiho, Sanofi, AstraZeneca; Grants and honoraria: Kyowa Kirin; Honoraria from Pfizer Japan, outside the submitted work. K. Inoue: Grants to institution: Novartis, Pfizer, Chugai, DaiichiSankyo, Parexel / Puma Biotechnology, MSD, Bayer, Eli Lilly, Esai, during the conduct of the study. A-S. Longin: Employment: Novartis. D. Mills, C. Wilke, S. Hirawat: Employment, ownership of stocks: Novartis. D. Juric: Fees from advisory boards: Novartis, Genentech, Eisai, Ipsen, EMD Serono, during the conduct of the study. All other authors have declared no conflicts of interest.