Cl Arteaga

A Phase Ib Trial of RAD001, an mTOR Inhibitor, with Weekly Cisplatin and Paclitaxel in Patients with HER2-Negative Metastatic Breast Cancer.

Background: Pre-clinical data suggests that in basal-like breast cancers (triple-negative and some luminal B), drugs that either negatively modulate p63 and/or activate p73, such as cisplatin and paclitaxel, promote p73-dependent apoptosis. This is enhanced upon concomitant inhibition of mTOR with RAD001 (Everolimus). We conducted a phase Ib trial in patients with HER2-negative metastatic breast cancer to explore the safety, tolerability and anti-tumor activity of the combination of paclitaxel, cisplatin and RAD001.Materials and Methods: This was an open-label, phase Ib dose-escalation study. RAD001 was started at 20 mg/week on a 28-day treatment cycle, and was escalated over 3 dose levels (20, 25 and 30 mg/week). Cisplatin (25 mg/m2) and paclitaxel (80 mg/m2) doses were fixed, and were administered once a week for 3 weeks on a 28-day treatment cycle. Treatment was continued until dose-limiting toxicity (DLT) was observed or until progression of disease. All toxicities were documented using the NCI CTC v.3. Disease assessment was done every 2 months after initiation of therapy.Results: A total of 16 patients were enrolled. Median age was 55 years and the median number of previous chemotherapy regimens in the metastatic setting was 3. Seventy percent of the patients had triple-negative disease, all patients had metastatic visceral disease, and 25% of those had concomitant bone metastasis. Three patients are still on study, but have not had their first assessment of disease. Of the 13 patients that are currently evaluable for best response, 1 had a complete response (CR), 2 had partial response (PR), 7 had stable disease (SD), and 3 had disease progression (PD) at their first disease assessment. All patients with CR or PR received the higher dose level (RAD001 30 mg/week). Twelve patients have discontinued study drugs at this time; 7 due to disease progression, 3 due to toxicity and 2 due to maximum benefit. Overall median time to progression (TTP) was 5 months. The most common toxicities were alopecia (100%), neutropenia (28%), anemia (10%) and fatigue (5%). Grade 3 and 4 toxicities were overall uncommon (8.5%) and consisted of neutropenia which lasted for less than 5 days. No DLTs were seen at any of the dose levels.Discussion: The combination of weekly RAD001, cisplatin and paclitaxel was overall very well tolerated, despite neutropenia (non-dose limiting and without serious sequelae). Significant antitumor activity in this heavily pre-treated patient population was seen at all dose levels and appeared to be higher in the group treated with RAD001 30 mg/week. The recommended phase II doses for this combination are cisplatin 25 mg/m2, paclitaxel 80 mg/m2 given weekly for 3 weeks, and RAD 30 mg/week, on a 28-day treatment cycle. A phase Ib-II study of cisplatin, paclitaxel, and daily doses of RAD001 is ongoing. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3093.

Abstract PD1-6: A randomized phase II neoadjuvant study of cisplatin, paclitaxel with or without everolimus (an mTOR inhibitor) in patients with stage II/III triple-negative breast cancer (TNBC)

Background: mTOR inhibitors can activate p73, a pro-apoptotic member of the p53 family, and enhance the sensitivity of breast cancer cells to cisplatin and paclitaxel. Thus, we hypothesized that combined use of the mTOR inhibitor everolimus, cisplatin, and paclitaxel would have synergistic anti-tumor effects in TNBC. Methods: Patients with clinical stage II/III TNBC were assigned (2:1) to weekly cisplatin 25 mg/m2 + paclitaxel 80 mg/m2 ± daily everolimus 5 mg for 12 weeks, until definitive surgery. Biopsy specimens were obtained in 100% of patients at baseline, at day 5 of cycle 1 and at surgery. Primary endpoint was pathological complete response (pCR). The study design provided 90% power to detect a difference in pCR rate of 35% vs. 20% with a two-sided significance level equal to 0.1 (type I error) for each arm. Results: A total of 145 patients were accrued between 2009 and 2013. To date, 14 patients have not yet completed surgery, and 11 patients were not evaluable (study discontinuation due to disease progression, toxicity or withdrawal). Baseline characteristics between arms were similar and well balanced: median age was 52 (28 – 81), median breast tumor size was 2 cm (0.1 – 7.6), 72% of tumors were histologic grade III, and 70% of patients had clinical stage III disease. Clinical outcomes are summarized in Table 1. View this table: Clinical outcomes Despite similar rates of pCR and clinical response in both arms, the combination of cisplatin/paclitaxel provided comparable pCR rates to anthracycline/cyclophosphamide/taxane containing regimens administered for longer periods of time. Most common adverse events are summarized in Table 2. View this table: Adverse events TNBC subtyping (Lehmann et al. JCI 2009), DNA mutations and alterations, as well as markers of proliferation, apoptosis, PI3K/mTOR and DNA damage response signaling will be presented. Preliminary analysis of Ki67 in a subset of tumors suggest that a reduction in Ki67 (day 5 biopsy) is associated with increased pCR rate. Tumors with androgen receptor expression exhibited a very low pCR rate. Conclusion: To our knowledge, this is the largest randomized neoadjuvant study in TNBC with a PI3K/mTOR pathway inhibitor. Results suggest that the paclitaxel/cisplatin combination is well tolerated and active in TNBC. The addition of Everolimus was associated with more adverse events and did not improve pCR or clinical response rates. A molecular signature or biomarker predictive of benefit from the paclitaxel/cisplatin combination is currently under investigation, and will be presented at the time of the meeting. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD1-6.

PD09-06: Phase II Trial of RAD001 (Everolimus), an mTOR Inhibitor, with Weekly Cisplatin and Paclitaxel in Patients with HER2−Negative Metastatic Breast Cancer (MBC).

Background: In basal-like breast cancers, drugs that either negatively modulate p63 and/or activate p73, such as cisplatin and paclitaxel, promote p73-dependent apoptosis. Further, the serine threonine kinase mTOR negatively regulates p73. Inhibition of mTOR with RAD001 upregulates p73, and results in p73-mediated apoptosis. Therefore, we conducted a phase II trial in patients with HER2−negative MBC to explore safety, tolerability and anti-tumor activity of the combination of paclitaxel, cisplatin and RAD001. Materials and Methods: We initiated an open-label, phase II multi-institutional study of weekly cisplatin (25 mg/m2), paclitaxel (80 mg/m2) and daily RAD001 (5 mg), given on a 28-day cycle. Treatment was continued until unacceptable toxicity or progression of disease. All toxicities were documented using the NCICTCv.4. Disease was assessed every 2 months. Results: A total of 55 patients were enrolled. Median age was 55 years; 62% of patients had prior chemotherapy with a median of 3 previous regimens in the metastatic setting. Sixty-three percent of patients had triple-negative disease, 81% patients had visceral disease, and 35% had bone metastases. Twenty-one patients are still on study. The most common toxicities are summarized in the table below. Neutropenia was the main cause for dose reductions, mainly after cycle 3. Only 1 patient developed febrile neutropenia. Of the 44 patients assessed for best response (RECIST) thus far, 11 had partial response, 21 stable disease, 9 disease progression, and 3 were not evaluable. Thirty eight patients have discontinued treatment so far; 30 due to disease progression, 5 due to toxicity, and 3 withdrew consent. Current median time to progression on the evaluable patients is 6 months. Discussion: The combination of RAD001, cisplatin and paclitaxel was overall very well tolerated despite cumulative pancytopenia. Significant antitumor activity in this heavily pre-treated patient population was seen. All patients’ primary diagnostic and/or metastatic tumor biopsies are currently being analyzed for the presence of PIK3CA and AKT1 mutations, and immunohistochemical expression of p53, p63, p73, and PTEN. Microarrays are being generated to determine whether time to progression is superior in patients with basal-like breast cancers. Microarrays will be mined to identify a pretreatment profile that mirrors a low p63/high p73 gene expression signature. Correlation of mutational analysis and gene signatures with clinical benefit will be presented at the meeting Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD09-06.

A Phase Ib Trial of Erlotinib, an EGFR Inhibitor, and Everolimus (RAD001), an mTOR Inhibitor, in Patients with Metastatic Breast Cancer.

Background: The EGFR and the PI3K pathways have been implicated in breast cancer9s prognosis and mechanisms of resistance to conventional therapies. Preclinical data have shown synergy between EGFR and PI3K/Akt pathway blockade. Therefore, we conducted a phase Ib trial to determine the maximum tolerated dose (MTD) and antitumor activity of Erlotinib, an oral EGFR tyrosine kinase inhibitor, and Everolimus (RAD001), an oral mTOR inhibitor, given daily to patients with metastatic breast cancer.Materials and Methods: This was an open-label, phase I dose-escalation study. Everolimus was started at 2.5 mg/d and Erlotinib was started at 100 mg/d on a 28-day treatment cycle. Doses were escalated over 6 levels (Everolimus 2.5, 5 or 10 mg daily with Erlotinib 100 or 150 mg daily). Treatment was continued until consistent dose-limiting toxicity (DLT) was observed or until progression of disease. All toxicities were documented using the NCI CTC v.3. Disease assessment was done every 2 months after initiation of therapy.Results: A total of 14 patients were enrolled on the first 2 dose levels (Erlotinib 100 mg with Everolimus 2.5 mg [level I] and 5 mg [level II] daily). Eight patients were enrolled on level I and 6 patients were enrolled on level II. The median age of all patients enrolled was 55 years of age, and 92% of them had visceral disease. Half of the patients had ER+ disease, 35% had HER2 + disease, and 15% had triple-negative disease. The median number of previous chemotherapy regimens in the metastatic setting was 4. Two patients on the level II cohort discontinued trial due to grade 3 stomatitis prior to their first disease assessment. Of the remaining 13 patients, 11 progressed at the time of their first disease assessment, and 1 patient on the dose level I cohort had a partial response that lasted for 7 months. The most common toxicities were rash (16%), transaminase elevation (15%), stomatitis (13%), fatigue (7%) and diarrhea (5%). Grade 3 and 4 toxicities were overall uncommon (3.5%), and mostly consisted of stomatitis, transaminase elevation and rash. Stomatitis was the only DLT, and was seen at dose level II. The MTD was determined to be Erlotinib 100 mg and Everolimus 2.5 mg daily.Discussion: In patients with metastatic HER2-negative breast cancer the combination of Erlotinib and Everolimus was overall well tolerated, but clinically ineffective from a tumor activity point of view in this heavily pre-treated patient population. Stomatitis was one of the most common toxicities and also the DLT in the cohort treated with dose level II. In view of the plethora of novel active agents currently in clinical trials in breast cancer, we do not believe the above combination warrants further testing in this disease. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3094.

Exon 9 and exon 20 mutations in PIK3CA confer resistance to HER2 inhibitors in HER2-overexpressing breast cancer cells.

Abstract #4054 The anti-tumor effect of HER2 antagonists in HER2-dependent breast cancer cells has been proposed to rely on inhibition of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Mutations in the p110α catalytic subunit of PI3K occur in up to 40% of breast cancers and activation of this pathway has been implicated in resistance to the HER2 antibody trastuzumab (T). PIK3CA mutations cluster in two regions in the helical (E542K, E545K; exon 9) and catalytic (H1047R; exon 20) domains. We studied the role of these mutants in resistance to HER2 inhibitors in breast cancer cells with HER2 amplification. Two lines with endogenous H1047R p110 (PI3K), SUM190 and HCC1954, and SKBR3, SUM225, and BT474 cells stably transduced with retroviral vectors encoding HA-tagged wild-type, E545K, or H1047R p110, were studied for their response to the HER2 tyrosine kinase inhibitor lapatinib ditosylate (L; GW-572016) and T. In monolayer and 3D cell proliferation assays, partial resistance to L and T was conferred by either mutation compared to WT p110. After prolonged L treatment, Ser473 phosphorylation of Akt recovered in all cells with endogenous or ectopic p110 mutants in spite of continued inhibition of Y1248 P-HER2 and Y1289 P-HER3 by L. Further, BT474 cells with either p110 mutant could be passaged in the continued presence of L. Immunoprecipitation of p85, the regulatory subunit of PI3K, showed that PI3K association with HER3 was abrogated by T and L. RNAi of HER3 in MCF10A/HER2/PI3KE545K and MCF10A/HER2/PI3KH1047R cells markedly but not completely inhibited growth suggesting that the PI3K mutants may still depend on HER3 for full activation. We hypothesized that in cells with mutant PI3K, the mutants coexist in a pool with WT enzyme, and that mutant and WT p110α are bound to p85 in variable proportions. Experiments to measure whether the ratio of mutant to WT p110 bound to p85, assayed by mass spectrometry, will increase in the PI3K-mutant cells with acquired resistance to L are in progress. To test the role of these mutants on resistance to anti-HER2 therapies in vivo, athymic mice bearing BT474 xenografts with ectopic WT, E545K, or H1047R p110 are undergoing treatment with L and T. Finally, we analyzed mutations of PIK3CA in a cohort of 40 patients with locally advanced HER2+ breast cancer treated with weekly single-agent T for 3 weeks, followed by T with docetaxel for a total of 12 weeks before surgery (Mohsin et al. JCO 23:2460, 2005). Sequential core biopsies at weeks 1 and 3 after initiation of T were taken. Eight of 40 tumors (20%) expressed mutant PI3K, 2 in exon 9 and 6 in exon 20. PIK3CA mutations did not correlate with change in Ki67 (p=0.97) or cleaved caspase 3 (p=0.51) in weeks 1 or 3 of treatment nor with pathologic complete response (p=0.21). These data suggest that, if mutant PI3K confers relative resistance to T, a shorter time to recurrence may be a more robust endpoint as the initial cellular or clinical response may not be a good indicator of this resistance. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4054.

Resistance to Endocrine Therapy in Estrogen Receptor-Positive (ER+) Breast Cancer Is Dependent upon Phosphatidylinositol-3 Kinase (PI3K) Signaling.

ER+ breast cancers typically respond to treatment with aromatase inhibitors (AIs), but a significant fraction exhibit de novo or acquired resistance. To model AI resistance, we cultured four ER+, hormone-dependent human breast cancer cell lines under hormone-depleted conditions for several months until hormone-independent populations emerged (termed long-term estrogen-deprived, LTED). LTED cells outgrew parental counterparts under hormone-depleted conditions. While two LTED lines showed increased ER levels and response to estradiol, two LTED lines showed the opposite. Therefore, ER+ breast cancer cells did not consistently overcome hormone deprivation by increasing sensitivity to low estrogen levels.We analyzed protein lysate arrays with 625 antibodies against signaling proteins to discover common mechanisms of hormone-independent growth. All LTED lines showed increased phosphorylation of p70S6K, an mTOR substrate, compared to parental controls. An siRNA library screen targeting 779 kinases revealed that downregulation of kinases linked to the phosphatidylinositol-3 kinase (PI3K) signaling pathway (serum/glucocorticoid-regulated kinase 1; insulin receptor; p110α/PI3K) inhibited the growth of MCF-7/LTED but not parental MCF-7 cells under hormone-depleted conditions. Immunoblot analysis for P-AKT and P-p70S6K showed that PI3K/AKT/mTOR signaling was increased in all LTED lines. Pathway analysis of gene expression microarray data showed significant alteration of genes involved in insulin-like growth factor-I (IGF-I) signaling in 3/4 LTED lines. Receptor tyrosine kinase array analysis revealed increased activation of receptors for IGF-I (IGF-IR) and/or insulin (InsR) in 3/4 LTED lines. These receptors transduce signals to potently activate PI3K. Treatment with the IGF-IR/InsR kinase inhibitor AEW541 decreased P-AKT in 3/4 LTED lines. Treatment with inhibitors of IGF-IR/InsR (AEW541), PI3K/mTOR (BEZ235), or mTOR (RAD001) suppressed the monolayer and anchorage-independent growth of 3/4, 4/4, and 4/4 LTED lines, respectively, and prevented the emergence of hormone-independent cells. Treatment with PI3K and mTOR inhibitors induced apoptosis in 3/4 LTED lines. Finally, we analyzed levels of PI3K pathway markers (P-AKT, P-S6, P-GSK3α/β) in primary tumor lysates from 65 ER+ breast cancer patients using reverse-phase protein arrays. Hierarchical clustering yielded two groups of tumors with high or low PI3K activation. Following adjuvant anastrozole therapy, PI3K-high patients had significantly shorter disease-free survival compared to PI3K-low patients. With a median follow-up of 9.4 months, recurrence rates were 16% and 0%, respectively ( p Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 403.

Transcriptional and Post-Translational Upregulation of HER3 (ErbB3) Counteracts Antitumor Effect of HER2 Tyrosine Kinase Inhibitors.

We hypothesized that sustained inhibition of HER3 and its output to PI3K/Akt is required for the optimal antitumor effect of HER2 inhibitors. Therefore, we examined the temporal effect of the HER2 tyrosine kinase inhibitor (TKI) lapatinib (lap) on feedback upregulation of active HER3 in HER2-overexpressing breast cancer cells. A time course with lap-treated cells showed 3 to 5-fold upregulation of HER3 RNA and protein, beginning at 4 h and increasing through 48 h. P-Tyr immunoblot of HER3 immunoprecipitates revealed recovery of HER3 phosphorylation at and beyond 13 h of treatment. Site-specific antibodies revealed HER3 phosphorylation at Y1197 and Y1289, two of the six p85 binding sites in HER3. Recovery of P-HER3 correlated temporally with recovery of T308 P-Akt. The upregulation of HER3 RNA upon treatment with lap suggested that inhibition of active HER2 and PI3K/Akt derepresses the transcription factor FoxO3a. Putative FoxO3a binding sites were identified within the 59 flanking region upstream of the HER3 transcription start site. Transfection with FoxO3a siRNA reduced basal and lap-induced HER3 RNA levels 2 to 5-fold compared to control cells. Conversely, overexpression of FoxO3a increased HER3 RNA 2.5-fold, which could be further enhanced by lap treatment. In addition to these transcriptional mechanisms, the recovery of P-HER3 upon lap-induced inhibition of HER2 suggested engagement of another tyrosine kinase transactivating HER3 and/or that HER2 had been incompletely inhibited by the TKI. However, IGF-IR, Src, and MET TKIs did not inhibit the recovery of P-HER3. On the other hand, the addition of trastuzumab (tz) to lap-treated cells prevented recovery of P-HER3, suggesting that disruption of a ligand-independent HER2-HER3 interaction was involved in partial maintenance of HER3 phosphorylation.The upregulation of HER3 RNA and partial maintenance of P-HER3 and P-Akt suggested that combined inhibition of HER2 and HER3 will synergistically inhibit tumor cell viability. Transfection with HER3 siRNA sensitized HER2+ breast cancer cells to each lap and tz as assessed by Apo-BrdU (apoptosis) and 3D-Matrigel growth assays. Further, treatment with AMG-888, a HER3 monoclonal antibody (AMGEN-U3), sensitized cells to each lap and tz. Ongoing studies include the treatment of BT474 xenografts in athymic mice with lap ± AMG-888 using [ 18 F]-FDG-PET as a non-invasive imaging biomarker to predict treatment outcome. Finally, we examined HER3 levels by immunohistochemistry in sections from tumor blocks of patients enrolled in a neoadjuvant trial where lap was given alone during the first 6 weeks of therapy. The percent and intensity of tumor cell staining was calculated as a histoscore (Human Pathol. 26:291, 1995). On week 2 of therapy, HER3 levels increased 135% above pre-therapy levels (n=8; p=0.03, Mann-Whitney). These data suggest that upon inhibition of the HER2 tyrosine kinase, HER2+ breast cancers 1) upregulate HER3 by transcriptional mechanisms and partially maintain HER3 function by post-translational mechanisms; 2) this compensatory phosphorylation of HER3 partially maintains PI3K/Akt; and 3) inhibition of HER3 sensitizes HER2-dependent breast cancer cells to HER2 inhibitors. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 63.

Abstract P3-03-05: PI3K/PDK1 mediates resistance to CDK4/6 inhibitors through dysregulation of S-phase cyclins/cyclin dependent kinases (CDKs)

Background: CDK4/6 inhibitors in combination with antiestrogens are approved for the treatment of ER+ advanced breast cancer. However, not all patients benefit from CDK4/6 inhibitors, underscoring the need to develop therapeutic strategies to circumvent de novo and acquired drug resistance. Methods: ER+ breast cancer cells (MCF-7, T47D, HCC1428, and HCC1500) were made resistant to increasing doses to the CDK4/6 inhibitor ribociclib (LEE011; Novartis). LEE011-resistant cells were characterized by 2D/3D growth, cell cycle, and immunoblot analyses. GSK2334470 (PDK1 inhibitor) and dinaciclib (CDK2 inhibitor) were used to modify resistance to ribociclib. PDK1 and pS6 immunohistochemistry (IHC) were performed on primary human tumor explants treated ex vivo with palbociclib. Results: Resistant cell lines (MCF-7/LR, T47D/LR, HCC1428/LR, and HCC1500/LR) exhibited an IC 50 at least 20-fold higher than that of their parental cells. They displayed cross-resistance to the CDK4/6 inhibitors palbociclib and abemaciclib. Immunoblot analysis of ribociclib-resistant cells showed increased levels of 3-phosphoinositide dependent protein kinase 1 (PDK1), S227 pRSK2 (target of PDK1), T308 pAKT (target of PDK1), and pS6 (downstream effector of the PDK1 target p70S6K), compared to parental drug sensitive cells. PDK1 is a master kinase that functions downstream of phosphoinositide 3-kinase (PI3K) and is crucial for the activation of AKT and many other AGC kinases including PKC, S6K, SGK, and RSK. Primary tumor explants treated ex vivo with palbociclib for 96 h also exhibited upregulation of PDK1 and pS6 by IHC. Cell cycle analysis revealed that CDK4/6 inhibition failed to induce G1 arrest, a reduction in S phase, and senescence in MCF-7/LR and T47D/LR compared to parental cells. Progression into S phase in the presence of ribociclib suggested upregulation of S-phase cyclins/CDKs. Indeed, the resistant cells exhibited significantly higher levels of pCDK2, cyclin A, cyclin E and S477/T479 pAKT, a CDK2-dependent phosphorylation of AKT required for full kinase activity and limited to the S-phase of the cell cycle. Pharmacological inhibition of PDK1 (with GSK2334470) or CDK2 (with dinaciclib) re-sensitized the ribociclib-resistant cells to CDK4/6 inhibitors. However, ribociclib/GSK2334470 inhibited MCF-7/LR and T47D/LR cell proliferation better than ribociclib/dinaciclib. Further, ribociclib/GSK2334470 but not ribociclib/dinaciclib completely abrogated pRb, pS6, pRSK2, pCDK2, cyclin A, and cyclin E, suggesting the PI3K/PDK1 pathway mediates acquired resistance to CDK4/6 inhibitors through dysregulation of the cell cycle. Consistent with these data, ribociclib/GSK2334470 inhibited growth of established MCF-7 xenografts in nude mice, significantly more potently than each drug alone. Conclusions: These data support a critical role for PI3K/PDK1 in acquired resistance to CDK4/6 inhibitors in ER+ breast cancer cells. Co-targeting of PI3K/PDK1 and CDK4/6 may overcome resistance to CDK4/6 inhibitors and is worthy of further translational and clinical investigation in patients with ER+ breast cancer. Citation Format: Jansen VM, Formisano L, Witkiewicz A, Estrada MV, Sanchez V, Dugger TC, Knudsen ES, Arteaga CL. PI3K/PDK1 mediates resistance to CDK4/6 inhibitors through dysregulation of S-phase cyclins/cyclin dependent kinases (CDKs) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-05.

Abstract PD2-06: Inhibition of 3-phosphoinositide dependent protein kinase 1 (PDK1) synergizes with CDK4/6 inhibitors against ER-positive breast cancer

Background: Dysregulation in cell cycle checkpoints is common in cancer. Small molecule inhibitors that target the CDK4/6/cyclinD1 pathway of the cell cycle are in clinical development. Recently the combination of the CDK4/6 inhibitor palbociclib and the aromatase inhibitor letrozole was approved for the treatment of post-menopausal women with ER+/HER2- advanced breast cancer. However, not all patients benefit from CDK4/6 inhibitors and a significant fraction of them eventually progress on these agents, underscoring the need to develop potent therapeutic strategies to circumvent drug resistance. Methods: We performed a high-throughput RNA interference (RNAi) kinome screen targeting 720 kinases to identify targetable molecules whose inhibition, in combination with the CDK4/6 inhibitor LEE011 (ribociclib), induced synthetic lethality in MCF7 ER+ breast cancer cells. PDK1 RNAi oligonucleotides and the PDK1 inhibitor GSK2334470 in combination with each of the CDK4/6 inhibitors, palbociclib and LEE011, were tested against ER+ breast cancer cells. In vivo anti-tumor efficacy of LEE011 and GSK2334470 was assessed in ovariectomized athymic nude mice bearing MCF7 xenografts. Results: A siRNA kinome screen identified PDK1 as the top RNA whose downregulation sensitized MCF7 cells to CDK4/6 inhibitors. This was confirmed with independent siRNAs in ER+ MCF7, T47D, HCC1428 and HCC1500 breast cancer cells. Pharmacological inhibition of PDK1 with the ATP-competitive, small molecule inhibitor GSK2334470 in combination with each of the CDK4/6 inhibitors, LEE011 and palbociclib, synergistically inhibited proliferation and increased apoptosis of MCF7 and T47D cells (combination index 0.19-0.89). LEE011-resistant MCF7 and T47D cells were generated by chronic treatment with doses of LEE011 up to 1 µM. Drug-resistant cells displayed increased levels of PDK1, phosphorylated Rb, and phosphorylated S6 ribosomal protein (pS6), an effector of the PDK1 substrate p70S6K, compared to parental drug-sensitive cells. Inhibition of PDK1 with siRNA or GSK2334470 re-sensitized the LEE011-resistant cells to the CDK4/6 inhibitors. Genetic (RNAi) and pharmacological inhibition of PDK1 (with GSK2334470) abrogated pS6 levels whereas inhibition of AKT with the small molecule inhibitor MK2206 did not affect pS6 levels, suggesting PDK1 can induce resistance to CDK4/6 inhibitors via p70S6K/pS6 signaling in an AKT-independent manner. The effects observed in cell lines in culture were recapitulated in vivo using MCF7 xenografts established in ovariectomized nude mice in the absence of estrogen supplementation. Treatment with GSK2334470 and LEE011 induced tumor regressions (8/8 tumors by RECIST criteria) more potently than either drug alone. Conclusions: These data support a critical role of PDK1 in mediating acquired resistance to CDK4/6 inhibitors in ER+ breast cancer cells. Co-targeting of the PDK1 and CDK4/6 pathways may overcome resistance to CDK4/6 inhibitors and is worthy of further translational and clinical investigation in patients with ER+ breast cancer. Citation Format: Jansen VM, Bhola NE, Bauer JA, Formisano L, Moore P, Koch J, Arteaga CL. Inhibition of 3-phosphoinositide dependent protein kinase 1 (PDK1) synergizes with CDK4/6 inhibitors against ER-positive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD2-06.

Abstract P3-07-41: Genomic alterations indicative of a luminal A subtype associate with clinical benefit to buparlisib and letrozole in endocrine-resistant ER+/HER2– metastatic breast cancer

Background: Activation of the phosphoinositide-3-kinase (PI3K) pathway has been associated with resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancer. Recently, we reported a Stand Up 2 Cancer (SU2C) Phase Ib trial of buparlisib, an oral, reversible, pan-PI3K inhibitor in combination with the aromatase inhibitor letrozole in patients with metastatic ER+/HER2– breast cancer (n=51) who had previously progressed on endocrine therapy. In this study, 31% of patients demonstrated clinical benefit (CR, PR and SD ≥6 months; Mayer et al. JCO 2014). Clinical activity was observed in patients with PIK3CA-wild type (PIK3CA-WT) and PIK3CA-mutant (PIK3CA-MUT) tumors. We performed targeted next-generation sequencing (tNGS) to identify somatic alterations associated with clinical benefit to the combination therapy. Methods: tNGS was performed using Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) in DNA extracted from tumors of 33 study patients (22 samples from archived primary/untreated and 11 from metastatic biopsies). Detected alterations were tested for association with clinical benefit (Fisher9s exact test) and progression-free survival (PFS; log-rank test). For PFS analysis, patients were censored if they discontinued buparlisib for toxicity. Results: The most commonly detected alterations occurred in PIK3CA (36%), TP53 (30%), MAP3K1 (27%), GATA3 (24%), CCND1 (24%), CDH1 (21%) and PTEN (21%). Additional alterations of note included FGFR1 amplification (15%), MYC amplification (12%), ESR1 mutations (6%) and ERBB2 mutations (6%). Probable inactivating mutations occurring in MAP3K1 (MAP3K1-MUT) were significantly associated with improved clinical benefit, regardless of other mutations (6/9 patients, 67%, P=0.044). PIK3CA-MUT tumors trended toward greater clinical benefit (7/12, 58%, P=0.067). Patients with coexisting PIK3CA-MUT and MAP3K1-MUT tumors derived the largest clinical benefit (5/7, 70% P=0.07) compared to patients with only PIK3CA-MUT (2/5; 40%, P=1.0) or only MAP3K1-MUT tumors (1/2; 50%, P=1.0). Only 2/19 (11%) patients with PIK3CA-WT/MAP3K1-WT cancers benefitted from treatment. Both MAP3K1 and PIK3CA alterations were each also associated with increased PFS (p=0.03 and p=0.009, respectively). Three of 5 (60%) patients with tumors with FGFR1 amplification achieved clinical benefit (including a MAP3K1-MUT tumor and a PIK3CA-MUT/MAP3K1-MUT tumor), suggesting that FGFR1 may preferentially signal via PI3K and/or FGFR1 amplifications are not associated with resistance to the combination of aromatase inhibitors and PI3K inhibitors. Conclusions: Both MAP3K1 and PIK3CA are mutated at higher frequencies in luminal A breast cancer, suggesting that this alteration pattern (MAP3K1-MUT + PIK3CA-MUT) is a surrogate for low grade ER+ breast cancers with PI3K dependence. It is also possible that MAP3K1 mutations may predispose tumor cells to sensitivity to PI3K inhibition, but this speculation requires further investigation. Finally, patients with ER+/FGFR1-amplified cancers appeared to derive clinical benefit from combined therapy with letrozole and buparlisib. Citation Format: Balko JM, Hicks M, Berger MF, Solit DB, Bouvier N, Sanders ME, Estrada MV, Won H, Cantley LC, Mayer IA, Arteaga CL. Genomic alterations indicative of a luminal A subtype associate with clinical benefit to buparlisib and letrozole in endocrine-resistant ER+/HER2– metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-41.