Luigi Formisano

A Phase Ib Study of Alpelisib (BYL719), a PI3Kα-Specific Inhibitor, with Letrozole in ER+/HER2− Metastatic Breast Cancer

Purpose: Alpelisib, a selective oral inhibitor of the class I PI3K catalytic subunit p110α, has shown synergistic antitumor activity with endocrine therapy against ER+/PIK3CA-mutated breast cancer cells. This phase Ib study evaluated alpelisib plus letrozoles safety, tolerability, and preliminary activity in patients with metastatic ER+ breast cancer refractory to endocrine therapy. Experimental Design: Twenty-six patients received letrozole and alpelisib daily. Outcomes were assessed by standard solid-tumor phase I methods. Tumor blocks were collected for DNA extraction and next-generation sequencing. Results: Alpelisibs maximum-tolerated dose (MTD) in combination with letrozole was 300 mg/d. Common drug-related adverse events included hyperglycemia, nausea, fatigue, diarrhea, and rash with dose-limiting toxicity occurring at 350 mg/d of alpelisib. The clinical benefit rate (lack of progression ≥6 months) was 35% (44% in patients with PIK3CA-mutated and 20% in PIK3CA wild-type tumors; 95% CI, 17%–56%), including five objective responses. Of eight patients remaining on treatment ≥12 months, six had tumors with a PIK3CA mutation. Among evaluable tumors, those with FGFR1/2 amplification and KRAS and TP53 mutations did not derive clinical benefit. Overexpression of FGFR1 in ER+/PIK3CA mutant breast cancer cells attenuated the response to alpelisib in vitro. Conclusions: The combination of letrozole and alpelisib was safe, with reversible toxicities. Clinical activity was observed independently of PIK3CA mutation status, although clinical benefit was seen in a higher proportion of patients with PIK3CA-mutated tumors. Phase II and III trials of alpelisib and endocrine therapy in patients with ER+ breast cancer are ongoing. Clin Cancer Res; 23(1); 26–34. ©2016 AACR.

Treatment of Triple-Negative Breast Cancer with TORC1/2 Inhibitors Sustains a Drug-Resistant and Notch-Dependent Cancer Stem Cell Population.

Approximately 30% of triple-negative breast cancers (TNBC) harbor molecular alterations in PI3K/mTOR signaling, but therapeutic inhibition of this pathway has not been effective. We hypothesized that intrinsic resistance to TORC1/2 inhibition is driven by cancer stem cell (CSC)-like populations that could be targeted to enhance the antitumor action of these drugs. Therefore, we investigated the molecular mechanisms by which PI3K/mTOR inhibitors affect the stem-like properties of TNBC cells. Treatment of established TNBC cell lines with a PI3K/mTOR inhibitor or a TORC1/2 inhibitor increased the expression of CSC markers and mammosphere formation. A CSC-specific PCR array revealed that inhibition of TORC1/2 increased FGF1 and Notch1 expression. Notch1 activity was also induced in TNBC cells treated with TORC1/2 inhibitors and associated with increased mitochondrial metabolism and FGFR1 signaling. Notably, genetic and pharmacologic blockade of Notch1 abrogated the increase in CSC markers, mammosphere formation, and in vivo tumor-initiating capacity induced by TORC1/2 inhibition. These results suggest that targeting the FGFR-mitochondrial metabolism-Notch1 axis prevents resistance to TORC1/2 inhibitors by eradicating drug-resistant CSCs in TNBC, and may thus represent an attractive therapeutic strategy to improve drug responsiveness and efficacy.

Kinome-wide RNA interference screen reveals a role for PDK1 in acquired resistance to CDK4/6 inhibition in ER-positive breast cancer

Acquired resistance to cyclin-dependent kinases 4 and 6 (CDK4/6) small-molecule inhibitors in breast cancer arises through mechanisms that are yet uncharacterized. In this study, we used a kinome-wide siRNA screen to identify kinases that, when downregulated, yield sensitivity to the CDK4/6 inhibitor ribociclib. In this manner, we identified 3-phosphoinositide-dependent protein kinase 1 (PDK1) as a key modifier of ribociclib sensitivity in estrogen receptor-positive MCF-7 breast cancer cells. Pharmacologic inhibition of PDK1 with GSK2334470 in combination with ribociclib or palbociclib, another CDK4/6 inhibitor, synergistically inhibited proliferation and increased apoptosis in a panel of ER-positive breast cancer cell lines. Ribociclib-resistant breast cancer cells selected by chronic drug exposure displayed a relative increase in the levels of PDK1 and activation of the AKT pathway. Analysis of these cells revealed that CDK4/6 inhibition failed to induce cell-cycle arrest or senescence. Mechanistic investigations showed that resistant cells coordinately upregulated expression of cyclins A, E, and D1, activated phospho-CDK2, and phospho-S477/T479 AKT. Treatment with GSK2334470 or the CDK2 inhibitor dinaciclib was sufficient to reverse these events and to restore the sensitivity of ribociclib-resistant cells to CDK4/6 inhibitors. Ribociclib, in combination with GSK2334470 or the PI3Kα inhibitor alpelisib, decreased xenograft tumor growth more potently than each drug alone. Taken together, our results highlight a role for the PI3K-PDK1 signaling pathway in mediating acquired resistance to CDK4/6 inhibitors. Cancer Res; 77(9); 2488-99. ©2017 AACR.

Activating PIK3CA Mutations Induce an Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-regulated Kinase (ERK) Paracrine Signaling Axis in Basal-like Breast Cancer

Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR.

Genomic profiling of ER+ breast cancers after short-term estrogen suppression reveals alterations associated with endocrine resistance

Genomic profiling of ER+/HER2− breast tumors after short-term estrogen deprivation revealed alterations associated with intrinsic resistance and provided mechanistic insights. A patient look at cancer resistance A variety of drugs that inhibit estrogen signaling are in use for breast cancer, but patients often develop resistance to these treatments. To understand how this resistance develops, Giltnane et al. evaluated 143 patients who were receiving the aromatase inhibitor letrozole to block estrogen signaling before undergoing surgery for breast cancer. By performing genomic analysis on these patients’ tumors, the authors were able to identify not only changes in gene expression and estrogen receptor gene fusions that correlated with resistance to therapy but also potential leads for future treatments that could help overcome this resistance. Inhibition of proliferation in estrogen receptor–positive (ER+) breast cancers after short-term antiestrogen therapy correlates with long-term patient outcome. We profiled 155 ER+/human epidermal growth factor receptor 2–negative (HER2−) early breast cancers from 143 patients treated with the aromatase inhibitor letrozole for 10 to 21 days before surgery. Twenty-one percent of tumors remained highly proliferative, suggesting that these tumors harbor alterations associated with intrinsic endocrine therapy resistance. Whole-exome sequencing revealed a correlation between 8p11-12 and 11q13 gene amplifications, including FGFR1 and CCND1, respectively, and high Ki67. We corroborated these findings in a separate cohort of serial pretreatment, postneoadjuvant chemotherapy, and recurrent ER+ tumors. Combined inhibition of FGFR1 and CDK4/6 reversed antiestrogen resistance in ER+ FGFR1/CCND1 coamplified CAMA1 breast cancer cells. RNA sequencing of letrozole-treated tumors revealed the existence of intrachromosomal ESR1 fusion transcripts and increased expression of gene signatures indicative of enhanced E2F-mediated transcription and cell cycle processes in cancers with high Ki67. These data suggest that short-term preoperative estrogen deprivation followed by genomic profiling can be used to identify druggable alterations that may cause intrinsic endocrine therapy resistance.

An ERBB1-3 Neutralizing Antibody Mixture With High Activity Against Drug-Resistant HER2+ Breast Cancers With ERBB Ligand Overexpression

Background Plasticity of the ERBB receptor network has been suggested to cause acquired resistance to anti-human epidermal growth factor receptor 2 (HER2) therapies. Thus, we studied whether a novel approach using an ERBB1-3-neutralizing antibody mixture can block these compensatory mechanisms of resistance. Methods HER2+ cell lines and xenografts (n ≥ 6 mice per group) were treated with the ERBB1-3 antibody mixture Pan-HER, trastuzumab/lapatinib (TL), trastuzumab/pertuzumab (TP), or T-DM1. Downregulation of ERBB receptors was assessed by immunoblot analysis and immunohistochemistry. Paired pre- and post-T-DM1 tumor biopsies from patients (n = 11) with HER2-amplified breast cancer were evaluated for HER2 and P-HER3 expression by immunohistochemistry and/or fluorescence in situ hybridization. ERBB ligands were measured by quantitative reverse transcription polymerase chain reaction. Drug-resistant cells were generated by chronic treatment with T-DM1. All statistical tests were two-sided. Results Treatment with Pan-HER inhibited growth and promoted degradation of ERBB1-3 receptors in a panel of HER2+ breast cancer cells. Compared with TL, TP, and T-DM1, Pan-HER induced a similar antitumor effect against established BT474 and HCC1954 tumors, but was superior to TL against MDA-361 xenografts (TL mean = 2026 mm 3 , SD = 924 mm 3 , vs Pan-HER mean = 565 mm 3 , SD = 499 mm 3 , P = .04). Pan-HER-treated BT474 xenografts did not recur after treatment discontinuation, whereas tumors treated with TL, TP, and T-DM1 did. Post-TP and post-T-DM1 recurrent tumors expressed higher levels of neuregulin-1 (NRG1), HER3 and P-HER3 (all P < .05). Higher levels of P-HER3 protein and NRG1 mRNA were also observed in HER2+ breast cancers progressing after T-DM1 and trastuzumab (NRG1 transcript fold change ± SD; pretreatment = 2, SD = 1.9, vs post-treatment = 11.4, SD = 10.3, P = .04). The HER3-neutralizing antibody LJM716 resensitized the drug-resistant cells to T-DM1, suggesting a causal association between the NRG1-HER3 axis and drug resistance. Finally, Pan-HER treatment inhibited growth of HR6 trastuzumab- and T-DM1-resistant xenografts. Conclusions These data suggest that upregulation of a NRG1-HER3 axis can mediate escape from anti-HER2 therapies. Further, multitargeted antibody mixtures, such as Pan-HER, can simultaneously remove and/or block targeted ERBB receptor and ligands, thus representing an effective approach against drug-sensitive and -resistant HER2+ cancers.

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.

Melanoma response to anti-PD-L1 immunotherapy requires JAK1 signaling, but not JAK2

ABSTRACT Immunotherapies targeting programmed cell death protein 1 (PD-1) or its ligand, programmed cell death ligand 1 (PD-L1), dramatically improve the survival of melanoma patients. However, only ∼40% of treated patients demonstrate a clinical response to single-agent anti-PD-1 therapy. An intact tumor response to type-II interferon (i.e. IFN-γ) correlates with response to anti-PD-1, and patients with de novo or acquired resistance may harbor loss-of-function alterations in the JAK/STAT pathway, which lies downstream of the interferon gamma receptor (IFNGR1/2). In this study, we dissected the specific roles of individual JAK/STAT pathway members on the IFN-γ response, and identified JAK1 as the primary mediator of JAK/STAT signaling associated with IFN-γ-induced expression of antigen-presenting molecules MHC-I and MHC-II, as well as PD-L1 and the cytostatic response to IFN-γ. In contrast to the crucial role of JAK1, JAK2 was largely dispensable in mediating most IFN-γ effects. In a mouse melanoma model, inhibition of JAK1/2 in combination with anti-PD-L1 therapy partially blocked anti-tumor immunologic responses, while selective JAK2 inhibition appeared to augment therapy. Amplification of JAK/STAT signaling in tumor cells through genetic inhibition of the negative regulator PTPN2 potentiated IFN-γ response in vitro and in vivo, and may be a target to enhance immunotherapy efficacy.

Abstract P2-10-01: Genomic profiling of residual ER+ breast cancers treated with prolonged neoadjuvant letrozole reveals novel alterations in clinically resistant tumors

Background: Approximately 20% of patients with early ER+ breast cancer (BC) treated with adjuvant antiestrogen therapy eventually relapse with endocrine-resistant metastatic disease. We hypothesized that profiling newly diagnosed ER+ BC that persist following prolonged estradiol deprivation with letrozole would identify genomic alterations associated with endocrine resistance. Methods: We treated 57 postmenopausal women (median 77 years; range 60-86) with ER+/HER2– BC with neoadjuvant letrozole (median 7.5 months; range 3-36) followed by surgery and adjuvant endocrine therapy. Patients were followed with serial ultrasounds and defined as non-responders if they developed recurrent locally or metastatic disease, or had a preoperative endocrine prognostic index (PEPI) ≥4 (composite score of post-treatment ER, Ki67, T and N status). Post-treatment specimens were profiled by RNA-seq and targeted capture NGS of >300 cancer-related genes. We screened for variants with a high probability of disrupting protein function (GERP score >4) and excluded likely germline variants by filtering out every alteration not present in COSMIC, if the variant had an allele frequency >0.1% as per the ExAC dataset. Results: Ten patients (17.5%) had a PEPI 0 score, 31 (54%) were PEPI 1-3, and 16 (28%) were PEPI ≥4. After a median follow-up of 50 months (12-100), 9 patients (15.7%) had recurred with metastatic disease (4 with PEPI 1-3, 5 with PEPI ≥4). We identified 294 variants with a median coverage >250x (206 nonsynonymous, 21 nonsense, 58 indels, 8 splice site). Recurrent mutations included PIK3CA (38%), KMT2C (28%), CDH1 (15%), NF1 (12 %), TP53 (10%), MAP3K1 (7%), ERBB2 (7%) and ESR1 (5%). Recurrent amplifications were identified in MCL1 (31%), GNAS (19%), CCND1 (16%), FYN (14%), AURKA (12%), and ERBB2 (10%), while recurrent deletions were found in DUSP4 (12%), NCOR1 (8%) and NF1 (6%). Compared to alterations reported in untreated ER+ breast cancers in TCGA, we observed a significant increase in KMT2C , NF1 , MCL1 and FYN alterations (FDR Conclusions: Genomic profiling of residual ER+ breast cancers treated with prolonged neoadjuvant letrozole revealed a different mutational landscape than primary untreated ER+ BC. These alterations may be associated with poor response to estrogen deprivation in early breast cancer and deserve further study. Citation Format: Guerrero AL, Stricker T, Hutchinson KE, Formisano L, Giltnane J, Fidalgo A, Schwarz LJ, Gavila J, Guillen V, Lluch A, Ruiz A, Arteaga CL. Genomic profiling of residual ER+ breast cancers treated with prolonged neoadjuvant letrozole reveals novel alterations in clinically resistant tumors [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 P2-10-01.