Evaluation of Copanlisib in Combination with Eribulin in Triple-negative Breast Cancer Patient-derived Xenograft Models.

The PI3K pathway regulates essential cellular functions and promotes chemotherapy resistance. Activation of PI3K pathway signaling is commonly observed in triple-negative breast cancer (TNBC). However previous studies that combined PI3K pathway inhibitors with taxane regimens have yielded inconsistent results. We therefore set out to examine whether the combination of copanlisib, a clinical grade pan-PI3K inhibitor, and eribulin, an antimitotic chemotherapy approved for taxane-resistant metastatic breast cancer, improves the antitumor effect in TNBC. A panel of eight TNBC patient-derived xenograft (PDX) models was tested for tumor growth response to copanlisib and eribulin, alone or in combination. Treatment-induced signaling changes were examined by reverse phase protein array, immunohistochemistry (IHC) and 18F-fluorodeoxyglucose PET (18F-FDG PET). Compared with each drug alone, the combination of eribulin and copanlisib led to enhanced tumor growth inhibition, which was observed in both eribulin-sensitive and -resistant TNBC PDX models, regardless of PI3K pathway alterations or PTEN status. Copanlisib reduced PI3K signaling and enhanced eribulin-induced mitotic arrest. The combination enhanced induction of apoptosis compared with each drug alone. Interestingly, eribulin upregulated PI3K pathway signaling in PDX tumors, as demonstrated by increased tracer uptake by 18F-FDG PET scan and AKT phosphorylation by IHC. These changes were inhibited by the addition of copanlisib. These data support further clinical development for the combination of copanlisib and eribulin and led to a phase I/II trial of copanlisib and eribulin in patients with metastatic TNBC. SIGNIFICANCE: In this research, we demonstrated that the pan-PI3K inhibitor copanlisib enhanced the cytotoxicity of eribulin in a panel of TNBC PDX models. The improved tumor growth inhibition was irrespective of PI3K pathway alteration and was corroborated by the enhanced mitotic arrest and apoptotic induction observed in PDX tumors after combination therapy compared with each drug alone. These data provide the preclinical rationale for the clinical testing in TNBC.

Retraction notice to "Allicin promotes autophagy and ferroptosis in esophageal squamous cell carcinoma by activating AMPK/mTOR signaling" [Heliyon 8 (2022) e11005].

[This retracts the article DOI: 10.1016/j.heliyon.2022.e11005.].

A phase II study of palbociclib plus letrozole plus trastuzumab as neoadjuvant treatment for clinical stages II and III ER+ HER2+ breast cancer (PALTAN).

Patients with ER+/HER2+ breast cancer (BC) are less likely to achieve pathological complete response (pCR) after chemotherapy with dual HER2 blockade than ER-/HER2+ BC. Endocrine therapy plus trastuzumab is effective in advanced ER+/HER2+ BC. Inhibition of CDK4/6 and HER2 results in synergistic cell proliferation reduction. We combined palbociclib, letrozole, and trastuzumab (PLT) as a chemotherapy-sparing regimen. We evaluated neoadjuvant PLT in early ER+/HER2+ BC. Primary endpoint was pCR after 16 weeks. Research biopsies were performed for whole exome and RNA sequencing, PAM50 subtyping, and Ki67 assessment for complete cell cycle arrest (CCCA: Ki67 ≤ 2.7%). After 26 patients, accrual stopped due to futility. pCR (residual cancer burden-RCB 0) was 7.7%, RCB 0/I was 38.5%. Grade (G) 3/4 treatment-emergent adverse events occurred in 19. Among these, G3/4 neutropenia was 50%, hypertension 26.9%, and leucopenia 7.7%. Analysis indicated CCCA in 85% at C1 day 15 (C1D15), compared to 27% at surgery after palbociclib was discontinued. Baseline PAM50 subtyping identified 31.2% HER2-E, 43.8% Luminal B, and 25% Luminal A. 161 genes were differentially expressed comparing C1D15 to baseline. MKI67, TK1, CCNB1, AURKB, and PLK1 were among the genes downregulated, consistent with CCCA at C1D15. Molecular Signatures Database gene-sets analyses demonstrated downregulated processes involved in proliferation, ER and mTORC1 signaling, and DNA damage repair at C1D15, consistent with the study drug's mechanisms of action. Neoadjuvant PLT showed a pCR of 7.7% and an RCB 0/I rate of 38.5%. RNA sequencing and Ki67 data indicated potent anti-proliferative effects of study treatments. ClinicalTrials.gov- NCT02907918.

Proteins in Tumor-Derived Plasma Extracellular Vesicles Indicate Tumor Origin.

Cancer-derived extracellular vesicles (EVs) promote tumorigenesis, premetastatic niche formation, and metastasis via their protein cargo. However, the proteins packaged by patient tumors into EVs cannot be determined in vivo because of the presence of EVs derived from other tissues. We therefore developed a cross-species proteomic method to quantify the human tumor-derived proteome of plasma EVs produced by patient-derived xenografts of four cancer types. Proteomic profiling revealed individualized packaging of novel protein cargo, and machine learning accurately classified the type of the underlying tumor.

Allicin promotes autophagy and ferroptosis in esophageal squamous cell carcinoma by activating AMPK/mTOR signaling.

The antitumor effects of allicin have been demonstrated in various cancers. However, whether allicin improves esophageal squamous cell carcinoma (ESCC) has not yet been explored. The present study aimed to explore the function and underlying mechanism of action of allicin in ESCC treatment. Our data showed that allicin significantly suppressed ESCC cell proliferation in a dose- and time-dependent manner. A green fluorescent protein-light chain 3 (LC3) transfection assay showed that autophagosomes were elevated in ESCC cells treated with allicin compared with control ESCC cells and that 3-methyladenine (an autophagy inhibitor) reversed allicin-induced LC3 puncta. Furthermore, allicin significantly elevated the ratio of LC3II/LC3I but decreased p62 expression in ESCC cells. Allicin also increased adenosine monophosphate-activated protein kinase (AMPK) phosphorylation but decreased that of the mechanistic target of rapamycin kinase (mTOR), which then induced the elevation of autophagy-related 5 and autophagy-related 7 proteins in ESCC cells. Furthermore, allicin treatment increased the expression of nuclear receptor coactivator 4 (a selective cargo receptor) but suppressed the expression of ferritin heavy chain 1 (the major intracellular iron-storage protein) in ESCC cells and elevated malondialdehyde and Fe2+ production levels. In vivo assays showed that allicin significantly decreased tumor weight and volume. In summary, allicin may induce cell death in ESCC cells by activating AMPK/mTOR-mediated autophagy and ferroptosis. Therefore, allicin may have excellent potential for use in the treatment of ESCC.

Exploring the Mechanism of Realgar against Esophageal Cancer Based on Ferroptosis Induced by ROS-ASK1-p38 MAPK Signaling Pathway.

Background: Realgar (REA), a Chinese herbal decoction, has been used to treat various tumors and has produced positive outcomes; however, there is a lack of convincing evidence for the treatment of esophageal cancer. The present study aimed to investigate the effects of REA on esophageal cancer (EC) and explore its mechanism. Methods: EC cells Eca109 and KYSE150 were selected for this study, and different groups of treated cells were set up. We studied the inhibition rate and half inhibition concentration (IC50) by CCK-8 method, the clone formation assay was used to detect the clone formation ability, the scratch assay is used to determine the cell migration ability, the Transwell assay was used to detect the cell invasion ability, the protein expressions of E-cadherin, Slug, N-cadherin, ASK1, p38 MAPK, p-p38 MAPK, and GPX4 were determined using Western blot, the mRNA expressions of ASK1 and p38 MAPK were assessed using qRT-PCR, transmission electron microscopy was used to observe the cellular ultrastructure, Prussian blue staining was used to observe the intracellular iron particle distribution, and biochemical analysis of cellular MDA, SOD, GSH, and GPXS activities, flow cytometric analysis of cellular ROS levels, immunofluorescence staining to detect cellular GPX4 expression, and JC-1 method to detect mitochondrial membrane potential were used. Results: REA inhibited the proliferation of Eca109 and KYSE150 cells in a time- and concentration-dependent manner, and REA significantly inhibited the migration and invasion of Eca109 and KYSE150 cells and activated the cellular ferroptosis and ROS-ASK1-p38 MAPK signaling pathways (P < 0.05). Inhibition of activation of the ROS-ASK1-p38 MAPK signaling pathway promoted the inhibition of proliferation, migration, and invasion of Eca109 and KYSE150 cells and the induction of ferroptosis by REA. Conclusion: REA induced ferroptosis and inhibited the migration of EC cells by activating the ROS-ASK1-p38 MAPK signaling pathway.

Cancer-associated exportin-6 upregulation inhibits the transcriptionally repressive and anticancer effects of nuclear profilin-1.

Aberrant expression of nuclear transporters and deregulated subcellular localization of their cargo proteins are emerging as drivers and therapeutic targets of cancer. Here, we present evidence that the nuclear exporter exportin-6 and its cargo profilin-1 constitute a functionally important and frequently deregulated axis in cancer. Exportin-6 upregulation occurs in numerous cancer types and is associated with poor patient survival. Reducing exportin-6 level in breast cancer cells triggers antitumor effects by accumulating nuclear profilin-1. Mechanistically, nuclear profilin-1 interacts with eleven-nineteen-leukemia protein (ENL) within the super elongation complex (SEC) and inhibits the ability of the SEC to drive transcription of numerous pro-cancer genes including MYC. XPO6 and MYC are positively correlated across diverse cancer types including breast cancer. Therapeutically, exportin-6 loss sensitizes breast cancer cells to the bromodomain and extra-terminal (BET) inhibitor JQ1. Thus, exportin-6 upregulation is a previously unrecognized cancer driver event by spatially inhibiting nuclear profilin-1 as a tumor suppressor.

Proteomic Resistance Biomarkers for PI3K Inhibitor in Triple Negative Breast Cancer Patient-Derived Xenograft Models.

PI3K pathway activation is frequently observed in triple negative breast cancer (TNBC). However, single agent PI3K inhibitors have shown limited anti-tumor activity. To investigate biomarkers of response and resistance mechanisms, we tested 17 TNBC patient-derived xenograft (PDX) models representing diverse genomic backgrounds and varying degrees of PI3K pathway signaling activities for their tumor growth response to the pan-PI3K inhibitor, BKM120. Baseline and post-treatment PDX tumors were subjected to reverse phase protein array (RPPA) to identify protein markers associated with tumor growth response. While BKM120 consistently reduced PI3K pathway activity, as demonstrated by reduced levels of phosphorylated AKT, percentage tumor growth inhibition (%TGI) ranged from 35% in the least sensitive to 84% in the most sensitive model. Several biomarkers showed significant association with resistance, including elevated baseline levels of growth factor receptors (EGFR, pHER3 Y1197), PI3Kp85 regulatory subunit, anti-apoptotic protein BclXL, EMT (Vimentin, MMP9, IntegrinaV), NFKB pathway (IkappaB, RANKL), and intracellular signaling molecules including Caveolin, CBP, and KLF4, as well as treatment-induced increases in the levels of phosphorylated forms of Aurora kinases. Interestingly, increased AKT phosphorylation or PTEN loss at baseline were not significantly correlated to %TGI. These results provide important insights into biomarker development for PI3K inhibitors in TNBC.

PI3K inhibition enhances the anti-tumor effect of eribulin in triple negative breast cancer.

Loss of the tumor suppressor phosphatase and tensin homolog (PTEN) is commonly observed in triple negative breast cancer (TNBC), leading to activation of the phosphoinositide 3-kinase (PI3K) signaling to promote tumor cell growth and chemotherapy resistance. In this study, we investigated whether adding a pan-PI3K inhibitor could improve the cytotoxic effect of eribulin, a non-taxane microtubule inhibitor, in TNBC patient-derived xenograft models (PDX) with loss of PTEN, and the underlying molecular mechanisms. Three TNBC-PDX models (WHIM6, WHIM12 and WHIM21), all with loss of PTEN expression, were tested for their response to BKM120 and eribulin, alone or in combination in vivo. In addition, the effect of drug treatment on cell proliferation and cell cycle progression were also performed in vitro using a panel of TNBC cell lines, including 2 derived from PDX models. The combination of eribulin and BKM120 led to additive or synergistic anti-tumor effect in 2 of the 3 PDX models, accompanied by an enhanced mitotic arrest and apoptosis in sensitive PDX models. In addition, the combination was synergistic in reducing mammosphere formation, and markers for epithelial-mesenchymal transition (EMT). In conclusion, PI3K inhibition induces synergistic anti-tumor effect when combined with eribulin, by enhancing mitotic arrest and apoptosis, as well as, reducing the cancer stem cell population. This study provides a preclinical rationale to investigate the therapeutic potential for the combination of PI3K inhibition and eribulin in the difficult to treat TNBC. Further studies are needed to identify the biomarkers of response for target patient selection.

Mass Spectrometry-Based Proteomics Reveals Potential Roles of NEK9 and MAP2K4 in Resistance to PI3K Inhibition in Triple-Negative Breast Cancers.

Activation of PI3K signaling is frequently observed in triple-negative breast cancer (TNBC), yet PI3K inhibitors have shown limited clinical activity. To investigate intrinsic and adaptive mechanisms of resistance, we analyzed a panel of patient-derived xenograft models of TNBC with varying responsiveness to buparlisib, a pan-PI3K inhibitor. In a subset of patient-derived xenografts, resistance was associated with incomplete inhibition of PI3K signaling and upregulated MAPK/MEK signaling in response to buparlisib. Outlier phosphoproteome and kinome analyses identified novel candidates functionally important to buparlisib resistance, including NEK9 and MAP2K4. Knockdown of NEK9 or MAP2K4 reduced both baseline and feedback MAPK/MEK signaling and showed synthetic lethality with buparlisib in vitro A complex in/del frameshift in PIK3CA decreased sensitivity to buparlisib via NEK9/MAP2K4-dependent mechanisms. In summary, our study supports a role for NEK9 and MAP2K4 in mediating buparlisib resistance and demonstrates the value of unbiased omic analyses in uncovering resistance mechanisms to targeted therapy.Significance: Integrative phosphoproteogenomic analysis is used to determine intrinsic resistance mechanisms of triple-negative breast tumors to PI3K inhibition. Cancer Res; 78(10); 2732-46. ©2018 AACR.

Serum thymidine kinase 1 activity as a pharmacodynamic marker of cyclin-dependent kinase 4/6 inhibition in patients with early-stage breast cancer receiving neoadjuvant palbociclib.

BACKGROUND: Thymidine kinase 1 (TK1) is a cell cycle-regulated enzyme with peak expression in the S phase during DNA synthesis, and it is an attractive biomarker of cell proliferation. Serum TK1 activity has demonstrated prognostic value in patients with early-stage breast cancer. Because cyclin-dependent kinase 4/6 (CDK4/6) inhibitors prevent G1/S transition, we hypothesized that serum TK1 could be a biomarker for CDK4/6 inhibitors. We examined the drug-induced change in serum TK1 as well as its correlation with change in tumor Ki-67 levels in patients enrolled in the NeoPalAna trial (ClinicalTrials.gov identifier NCT01723774). METHODS: Patients with clinical stage II/III estrogen receptor-positive (ER+)/HER2-negative breast cancer enrolled in the NeoPalAna trial received an initial 4 weeks of anastrozole, followed by palbociclib on cycle 1, day 1 (C1D1) for four 28-day cycles, unless C1D15 tumor Ki-67 was > 10%, in which case patients went off study owing to inadequate response. Surgery occurred following 3-5 weeks of washout from the last dose of palbociclib, except in eight patients who received palbociclib (cycle 5) continuously until surgery. Serum TK1 activity was determined at baseline, C1D1, C1D15, and time of surgery, and we found that it was correlated with tumor Ki-67 and TK1 messenger RNA (mRNA) levels. RESULTS: Despite a significant drop in tumor Ki-67 with anastrozole monotherapy, there was no statistically significant change in TK1 activity. However, a striking reduction in TK1 activity was observed 2 weeks after initiation of palbociclib (C1D15), which then rose significantly with palbociclib washout. At C1D15, TK1 activity was below the detection limit (<20 DiviTum units per liter Du/L) in 92% of patients, indicating a profound effect of palbociclib. There was high concordance, at 89.8% (95% CI: 79.2% - 96.2%), between changes in serum TK1 and tumor Ki-67 in the same direction from C1D1 to C1D15 and from C1D15 to surgery time points. The sensitivity and specificity for the tumor Ki-67-based response by palbociclib-induced decrease in serum TK1 were 94.1% (95% CI 86.2% - 100%) and 84% (95% CI 69.6% -98.4%), respectively. The κ-statistic was 0.76 (p < 0.001) between TK1 and Ki-67, indicating substantial agreement. CONCLUSIONS: Serum TK1 activity is a promising pharmacodynamic marker of palbociclib in ER+ breast cancer, and its value in predicting response to CDK4/6 inhibitors warrants further investigation. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01723774. Registered on 6 November 2012.

A Phase II Trial of Neoadjuvant MK-2206, an AKT Inhibitor, with Anastrozole in Clinical Stage II or III PIK3CA-Mutant ER-Positive and HER2-Negative Breast Cancer.

Purpose: Hyperactivation of AKT is common and associated with endocrine resistance in estrogen receptor-positive (ER+) breast cancer. The allosteric pan-AKT inhibitor MK-2206 induced apoptosis in PIK3CA-mutant ER+ breast cancer under estrogen-deprived condition in preclinical studies. This neoadjuvant phase II trial was therefore conducted to test the hypothesis that adding MK-2206 to anastrozole induces pathologic complete response (pCR) in PIK3CA mutant ER+ breast cancer.Experimental Design: Potential eligible patients with clinical stage II/III ER+/HER2- breast cancer were preregistered and received anastrozole (goserelin if premenopausal) for 28 days in cycle 0 pending tumor PIK3CA sequencing. Patients positive for PIK3CA mutation in the tumor were eligible to start MK-2206 (150 mg orally weekly, with prophylactic prednisone) on cycle 1 day 2 (C1D2) and to receive a maximum of four 28-day cycles of combination therapy before surgery. Serial biopsies were collected at preregistration, C1D1 and C1D17.Results: Fifty-one patients preregistered and 16 of 22 with PIK3CA-mutant tumors received study drug. Three patients went off study due to C1D17 Ki67 >10% (n = 2) and toxicity (n = 1). Thirteen patients completed neoadjuvant therapy followed by surgery. No pCRs were observed. Rash was common. MK-2206 did not further suppress cell proliferation and did not induce apoptosis on C1D17 biopsies. Although AKT phosphorylation was reduced, PRAS40 phosphorylation at C1D17 after MK-2206 persisted. One patient acquired an ESR1 mutation at surgery.Conclusions: MK-2206 is unlikely to add to the efficacy of anastrozole alone in PIK3CA-mutant ER+ breast cancer and should not be studied further in the target patient population. Clin Cancer Res; 23(22); 6823-32. ©2017 AACR.

NeoPalAna: Neoadjuvant Palbociclib, a Cyclin-Dependent Kinase 4/6 Inhibitor, and Anastrozole for Clinical Stage 2 or 3 Estrogen Receptor-Positive Breast Cancer.

Purpose: Cyclin-dependent kinase (CDK) 4/6 drives cell proliferation in estrogen receptor-positive (ER+) breast cancer. This single-arm phase II neoadjuvant trial (NeoPalAna) assessed the antiproliferative activity of the CDK4/6 inhibitor palbociclib in primary breast cancer as a prelude to adjuvant studies.Experimental Design: Eligible patients with clinical stage II/III ER+/HER2- breast cancer received anastrozole 1 mg daily for 4 weeks (cycle 0; with goserelin if premenopausal), followed by adding palbociclib (125 mg daily on days 1-21) on cycle 1 day 1 (C1D1) for four 28-day cycles unless C1D15 Ki67 > 10%, in which case patients went off study due to inadequate response. Anastrozole was continued until surgery, which occurred 3 to 5 weeks after palbociclib exposure. Later patients received additional 10 to 12 days of palbociclib (Cycle 5) immediately before surgery. Serial biopsies at baseline, C1D1, C1D15, and surgery were analyzed for Ki67, gene expression, and mutation profiles. The primary endpoint was complete cell cycle arrest (CCCA: central Ki67 ≤ 2.7%).Results: Fifty patients enrolled. The CCCA rate was significantly higher after adding palbociclib to anastrozole (C1D15 87% vs. C1D1 26%, P < 0.001). Palbociclib enhanced cell-cycle control over anastrozole monotherapy regardless of luminal subtype (A vs. B) and PIK3CA status with activity observed across a broad range of clinicopathologic and mutation profiles. Ki67 recovery at surgery following palbociclib washout was suppressed by cycle 5 palbociclib. Resistance was associated with nonluminal subtypes and persistent E2F-target gene expression.Conclusions: Palbociclib is an active antiproliferative agent for early-stage breast cancer resistant to anastrozole; however, prolonged administration may be necessary to maintain its effect. Clin Cancer Res; 23(15); 4055-65. ©2017 AACR.

Proteogenomic integration reveals therapeutic targets in breast cancer xenografts.

Recent advances in mass spectrometry (MS) have enabled extensive analysis of cancer proteomes. Here, we employed quantitative proteomics to profile protein expression across 24 breast cancer patient-derived xenograft (PDX) models. Integrated proteogenomic analysis shows positive correlation between expression measurements from transcriptomic and proteomic analyses; further, gene expression-based intrinsic subtypes are largely re-capitulated using non-stromal protein markers. Proteogenomic analysis also validates a number of predicted genomic targets in multiple receptor tyrosine kinases. However, several protein/phosphoprotein events such as overexpression of AKT proteins and ARAF, BRAF, HSP90AB1 phosphosites are not readily explainable by genomic analysis, suggesting that druggable translational and/or post-translational regulatory events may be uniquely diagnosed by MS. Drug treatment experiments targeting HER2 and components of the PI3K pathway supported proteogenomic response predictions in seven xenograft models. Our study demonstrates that MS-based proteomics can identify therapeutic targets and highlights the potential of PDX drug response evaluation to annotate MS-based pathway activities.

Inhibition of cyclin dependent kinase 9 by dinaciclib suppresses cyclin B1 expression and tumor growth in triple negative breast cancer.

Cyclin-dependent kinases (CDKs) are potential cancer therapeutic targets because of their critical role in promoting cell growth. Dinaciclib is a novel CDK inhibitor currently under clinical evaluation for the treatment of advanced malignancies. In this study, we demonstrated the anti-tumor activity of dinaciclib in triple negative breast cancer (TNBC) patient derived xenograft (PDX) and cell lines in vitro and in vivo. Treatment with dinaciclib induced cell cycle arrest at G2/M phase and marked apoptosis. These changes were accompanied by reduced phosphorylation of CDK1 and retinoblastoma (Rb) protein and decreased protein levels of cyclin B1, cMYC and survivin. We further demonstrated that siRNA knockdown of CDK9, the kinase subunit of positive transcription elongation factor b (P-TEFb), instead of CDK1 or CDK2, reduced the levels of cyclin B1 and MYC in TNBC cell lines. These data support the importance of CDK9, in addition to CDK1, in mediating the growth inhibitory effect of dinaciclib in TNBC. Further investigation of CDK9 as a therapeutic target in TNBC is needed.

A Phase I Study of the AKT Inhibitor MK-2206 in Combination with Hormonal Therapy in Postmenopausal Women with Estrogen Receptor-Positive Metastatic Breast Cancer.

PURPOSE: PI3K/AKT pathway activation is an important endocrine resistance mechanism in estrogen receptor-positive (ER(+)) breast cancer. After promising preclinical modeling of MK-2206, an allosteric pan-AKT inhibitor, with either estrogen deprivation or fulvestrant, we conducted a phase I trial in patients with metastatic ER(+)HER2(-) breast cancer to determine the recommended phase II treatment dose (RPTD) of MK-2206 when combined with either anastrozole, fulvestrant, or anastrozole/fulvestrant. EXPERIMENTAL DESIGN: ER(+) breast cancer cell lines were exposed in vitro to MK-2206 plus estrogen deprivation with or without fulvestrant and monitored for apoptosis. A standard 3+3 design was employed to first determine the maximum tolerated dose (MTD) of MK-2206 plus anastrozole based on cycle 1 toxicity. Each cycle was 28 days. The RPTD was determined on the basis of toxicities observed at MTD level during the first 3 cycles. Subsequent patients received MK-2206, at the RPTD determined above, plus fulvestrant or anastrozole/fulvestrant to define RPTD for these additional regimens. RESULTS: MK-2206 induced apoptosis in parental ER(+) but not in long-term estrogen-deprived cell lines, for which fulvestrant was required for apoptosis induction. Thirty-one patients enrolled. The RPTD was defined as MK-2206 150 mg orally weekly with prednisone prophylaxis for each combination. Grade 3 rash was dose limiting. 42% (95% CI, 23%-63%) patients derived clinical benefit without progression within 6 months. Response was not associated with tumor PIK3CA mutation. CONCLUSIONS: MK-2206 plus endocrine treatments were tolerable. MK-2206 in combination with anastrozole is being further evaluated in a phase II neoadjuvant trial for newly diagnosed ER(+)HER2(-) breast cancer. Clin Cancer Res; 22(11); 2650-8. ©2016 AACRSee related commentary by Jansen et al., p. 2599.

A Phase I Trial of BKM120 (Buparlisib) in Combination with Fulvestrant in Postmenopausal Women with Estrogen Receptor-Positive Metastatic Breast Cancer.

PURPOSE: This trial was conducted to determine the maximum tolerated dose (MTD) and preliminary efficacy of buparlisib, an oral pan-class I PI3K inhibitor, plus fulvestrant in postmenopausal women with metastatic estrogen receptor positive (ER(+)) breast cancer. EXPERIMENTAL DESIGN: Phase IA employed a 3+3 design to determine the MTD of buparlisib daily plus fulvestrant. Subsequent cohorts (phase IB and cohort C) evaluated intermittent (5/7-day) and continuous dosing of buparlisib (100 mg daily). No more than 3 prior systemic treatments in the metastatic setting were allowed in these subsequent cohorts. RESULTS: Thirty-one patients were enrolled. MTD was defined as buparlisib 100 mg daily plus fulvestrant. Common adverse events (AE) included fatigue (38.7%), transaminases elevation (35.5%), rash (29%), and diarrhea (19.4%). C-peptide was significantly increased during treatment, consistent with on-target effect of buparlisib. Compared with intermittent dosing, daily buparlisib was associated with more frequent early onset AEs and higher buparlisib plasma concentrations. Among the 29 evaluable patients, the clinical benefit rate was 58.6% (95% CI, 40.7%-74.5%). Response was not associated with PIK3CA mutation or treatment cohort; however, loss of PTEN, progesterone receptor (PgR) expression, or mutation in TP53 was most common in resistant cases, and mutations inAKT1 and ESR1 did not exclude treatment response. CONCLUSIONS: Buparlisib plus fulvestrant is clinically active with manageable AEs in patients with metastatic ER(+)breast cancer. Weekend breaks in buparlisib dosing reduced toxicity. Patients with PgR negative and TP53 mutation did poorly, suggesting buparlisib plus fulvestrant may not be adequately effective against tumors with these poor prognostic molecular features.

Combined targeting of mTOR and AKT is an effective strategy for basal-like breast cancer in patient-derived xenograft models.

Basal-like breast cancer is an aggressive disease for which targeted therapies are lacking. Recent studies showed that basal-like breast cancer is frequently associated with an increased activity of the phosphatidylinositol 3-kinase (PI3K) pathway, which is critical for cell growth, survival, and angiogenesis. To investigate the therapeutic potential of PI3K pathway inhibition in the treatment of basal-like breast cancer, we evaluated the antitumor effect of the mTOR inhibitor MK-8669 and AKT inhibitor MK-2206 in WU-BC4 and WU-BC5, two patient-derived xenograft models of basal-like breast cancer. Both models showed high levels of AKT phosphorylation and loss of PTEN expression. We observed a synergistic effect of MK-8669 and MK-2206 on tumor growth and cell proliferation in vivo. In addition, MK-8669 and MK-2206 inhibited angiogenesis as determined by CD31 immunohistochemistry. Biomarker studies indicated that treatment with MK-2206 inhibited AKT activation induced by MK-8669. To evaluate the effect of loss of PTEN on tumor cell sensitivity to PI3K pathway inhibition, we knocked down PTEN in WU-BC3, a basal-like breast cancer cell line with intact PTEN. Compared with control (GFP) knockdown, PTEN knockdown led to a more dramatic reduction in cell proliferation and tumor growth inhibition in response to MK-8669 and MK-2206 both in vitro and in vivo. Furthermore, a synergistic effect of these two agents on tumor volume was observed in WU-BC3 with PTEN knockdown. Our results provide a preclinical rationale for future clinical investigation of this combination in basal-like breast cancer with loss of PTEN.

A phase I trial of the IGF-1R antibody Cixutumumab in combination with temsirolimus in patients with metastatic breast cancer.

The mammalian target of rapamycin (mTOR) plays a critical role in promoting tumor cell growth and is frequently activated in breast cancer. In preclinical studies, the antitumor activity of mTOR inhibitors is attenuated by feedback up-regulation of AKT mediated in part by Insulin-like growth factor type 1 receptor (IGF-1R). We designed a phase I trial to determine the maximum-tolerated dose (MTD) and pharmacodynamic effects of the IGF-1R antibody Cixutumumab in combination with temsirolimus in patients with metastatic breast cancer refractory to standard therapies. A 3 + 3 Phase I design was chosen. Temsirolimus and Cixutumumab were administered intravenously on days 1, 8, 15, and 22 of a 4-week cycle. Of the 26 patients enrolled, four did not complete cycle 1 because of disease progression (n = 3) or comorbid condition (n = 1) and were replaced. The MTD was determined from the remaining 22 patients, aged 34-72 (median 48) years. Most patients (86 %) had estrogen receptor positive cancer. The median number of prior chemotherapy regimens for metastatic disease was 3. The MTD was determined to be Cixutumumab 4 mg/kg and temsirolimus 15 mg weekly. Dose-limiting toxicities (DLTs) included mucositis, neutropenia, and thrombocytopenia. Other adverse events included grade 1/2 fatigue, anemia, and hyperglycemia. No objective responses were observed, but four patients experienced stable disease that lasted for at least 4 months. Compared with baseline, there was a significant increase in the serum levels of IGF-1 (p < 0.001) and IGFBP-3 (p = 0.019) on day 2. Compared with day 2, there were significant increases in the serum levels of IGF-1 (p < 0.001), IGF-2 (p = 0.001), and IGFBP-3 (p = 0.019) on day 8. A phase II study in women with metastatic breast cancer is ongoing.