Mutant Breast Cancer Cell Lines Research Articles

Abstract GS03-10: Allosteric PI3K-alpha inhibition overcomes on-target resistance to orthosteric inhibitors mediated by secondary PIK3CA mutations

Abstract Background: PIK3CA mutations occur in ~40% of HR-positive breast cancers, where alpelisib, an orthosteric PI3Kα inhibitor, is FDA-approved in combination with fulvestrant. Although prior studies have identified potential resistance mechanisms, such as PTEN loss, clinical acquired resistance to orthosteric PI3Kα inhibitors and the role of next-generation allosteric PI3Kα inhibitors remain poorly understood. Methods: To identify on-target and off-target alterations potentially mediating resistance to PI3Kα inhibitors, we used a targeted next-generation sequencing assay (Guardant360; Guardant Health) to analyze ctDNA in serially collected plasma samples from 32 patients with PIK3CA-mutated advanced HR-positive, HER2-negative breast cancer treated with alpelisib and inavolisib. In addition, we performed whole exome sequencing (WES) of 100 tissue samples collected from 8 autopsy series from patients with metastatic, PIK3CA-mutant HR-positive, HER2-negative breast cancer previously treated with PI3Kα inhibitors. Acquired alterations were prioritized through a combination of structural modeling and free-energy perturbation simulation and validated in genomically engineered PIK3CA mutant breast cancer cell lines T47D (PIK3CA H1047R-mutant) or MCF7 (PIK3CA E545K-mutant). Results: We observed that 50% of patients acquire genomic alterations within the PI3K-pathway, including PTEN loss and activating AKT1 mutations. Notably, while secondary PIK3CA mutations were previously reported to increase sensitivity to PI3Ka-inhibitors, we identified emergent secondary resistance mutations in PIK3CA that alter the inhibitor binding pocket including PIK3CA Q859K and PIK3CA W780R. Some mutations had differential effects on PI3Ka-selective vs. pan-PI3K inhibitors, but resistance induced by all mutations could be overcome by the novel allosteric pan-mutant-selective PI3Ka-inhibitor RLY-2608. Conclusion: In one of the largest patient cohorts analyzed to date, this study defines the clinical landscape of acquired resistance to PI3Ka inhibitors. Genomic alterations within the PI3K pathway represent a major mode of resistance and identify a novel class of secondary PIK3CA resistance mutations that can be overcome by an allosteric PI3Ka inhibitor. Together, these findings provide insights to guide strategies to overcome resistance in PIK3CA-mutated cancers. Citation Format: Andreas Varkaris, Ferran Fece de la Cruz, Elizabeth Martin, Bryanna Nordern, Nicholas Chevalier, Ignaty Leshchiner, Anastasia Stavridi, Janice Kim, Alkistis Papatheodoridi, Hakan Gunaydin, Brian Danysh, Laxmi Parida, Ioannis Sanidas, Yongli Ji, Kayao Lau, Gerburg Wulf, Aditya Bardia, Laura Spring, Steven Isakoff, Jochen Lennerz, Levi Pierce, Ermira Pazolli, Gad Getz, Ryan Corcoran, Dejan Juric. Allosteric PI3K-alpha inhibition overcomes on-target resistance to orthosteric inhibitors mediated by secondary PIK3CA mutations [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr GS03-10.

Degradation of MYC by the mutant p53 reactivator drug, COTI-2 in breast cancer cells

TP53 (p53) and MYC are amongst the most frequently altered genes in cancer. Both are thus attractive targets for new anticancer therapies. Historically, however, both genes have proved challenging to target and currently there is no approved therapy against either. The aim of this study was to investigate the effect of the mutant p53 reactivating drug, COTI-2 on MYC. Total MYC, pSer62 MYC and pThr58 MYC were detected using Western blotting. Proteasome-mediated degradation was determined using the proteasome, inhibitor MG-132, while MYC half-life was measured using pulse chase experiments in the presence of cycloheximide. Cell proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Treatment of 5 mutant p53 breast cancer cell lines with COTI-2 resulted in dose-dependent MYC degradation. Addition of the proteasome inhibitor, MG132, rescued the degradation, suggesting that this proteolytic system was at least partly responsible for the inactivation of MYC. Using cycloheximide in pulse chase experiments, COTI-2 was found to reduce the half-life of MYC in 2 different mutant p53 breast cancer cell lines, i.e., from 34.8 to 18.6 min in MDA-MB-232 cells and from 29.6 to 20.3 min in MDA-MB-468 cells. Co-treatment with COTI-2 and the MYC inhibitor, MYCi975 resulted in synergistic growth inhibition in all 4 mutant p53 cell lines investigated. The dual ability of COTI-2 to reactivate mutant p53 and degrade MYC should enable this compound to have broad application as an anticancer drug.

Abstract P2-16-01: The Kinesin-like protein Kif11 is essential for the Survival of TP53-mutant Triple Negative Breast Cancer Cells

Abstract Background: Breast cancer is the most commonly diagnosed non-cutaneous malignancy in American women and one of the leading causes of cancer deaths. Breast cancer can be divided into several subtypes, the most aggressive of which is Triple-Negative Breast Cancer (TNBC), a disease with few targeted therapies. TP53 mutations are found in 80% or more of TNBCs. However, direct targeting of mutant TP53 has been difficult. To identify drugs that can specifically induce the death of TP53-mutant breast cancers, we conducted a drug screen in TP53-mutant and TP53-wild type breast cancer cells. Through this combined in silico and in vitro drug screen, we discovered that TP53 mutant TNBC cells have an increased sensitivity to KIF11 inhibition as compared to TP53 wild-type cells. Hypothesis: We hypothesize that TP53 mutational status confers sensitivity of triple-negative breast cancer cells to KIF11 inhibition. Methods: We obtained data on TP53 mutational status, KIF11 mRNA expression levels, and clinical characteristics from publicly-available TCGA, METABRIC, and CCLE datasets. To demonstrate the effect of KIF11 inhibition on cell growth, we treated TP53 mutant and wild-type breast cancer cell lines with the small molecule KIF11 inhibitor SB-743921 and KIF11 siRNAs. Cell counts were obtained through staining with DAPI or Hoechst nuclear stains and imaging on the MetaXPress PICO instrument. To determine protein expression of p53 and Kif11 across various cell lines, western blotting was performed. We then investigated the biological mechanisms of growth inhibition by DRAQ7 staining and flow cytometry analysis following Annexin V-PI staining. Results: Using cell growth assays we demonstrated that TP53 mutant cells are more sensitive to KIF11 inhibition. We next utilized cell lines in which TP53 mutations had been introduced into TP53 wild-type cells to show that overexpression of a TP53 mutant gene can sensitize cells to KIF11 inhibition. Using assays of proliferation and apoptosis, we showed that TP53 mutant breast cancer cells treated with a KIF11 inhibitor undergo cell death. Using publicly-available datasets of breast cancers, we showed that KIF11 is upregulated in TNBCs and TP53 mutant cancers, and that high expression of KIF11 in breast cancer is correlated with poorer clinical prognosis. Conclusions: Our results show that the Kif11 protein is essential for the survival of TP53 mutant TNBC cells. Inhibitors of Kif11 induce death of TP53 mutant TNBCs and thus, this kinesin-like protein involved in cell spindle mechanics is a potential target for the treatment of these aggressive cancers. Research reported in this publication was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Numbers TL1TR003169 and UL1TR003167. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported by the John Charles Cain Endowment. Citation Format: Amanda Lanier, Abhijit Mazumdar, William Tahaney, Powel Brown. The Kinesin-like protein Kif11 is essential for the Survival of TP53-mutant Triple Negative Breast Cancer Cells [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-16-01.

Upregulation of sphingosine kinase 1 in response to doxorubicin generates an angiogenic response via stabilization of Snail.

Sphingosine kinase 1 (SK1) converts the pro-death lipid sphingosine to the pro-survival sphingosine-1-phosphate (S1P) and is upregulated in several cancers. DNA damaging agents, such as the chemotherapeutic doxorubicin (Dox), have been shown to degrade SK1 protein in cancer cells, a process dependent on wild-type p53. As mutations in p53 are very common across several types of cancer, we evaluated the effects of Dox on SK1 in p53 mutant cancer cells. In the p53 mutant breast cancer cell line MDA-MB-231, we show that Dox treatment significantly increases SK1 protein and S1P. Using MDA-MB-231 cells with CRISPR-mediated knockout of SK1 or the selective SK1 inhibitor PF-543, we implicated SK1 in both Dox-induced migration and in a newly uncovered proangiogenic program induced by Dox. Mechanistically, inhibition of SK1 suppressed the induction of the cytokine BMP4 and of the EMT transcription factor Snail in response to Dox. Interestingly, induction of BMP4 by SK1 increased Snail levels following Dox treatment by stabilizing Snail protein. Furthermore, we found that SK1 was required for Dox-induced p38 MAP kinase phosphorylation and that active p38 MAPK in turn upregulated BMP4 and Snail, positioning p38 downstream of SK1 and upstream of BMP4/Snail. Modulating production of S1P by inhibition of de novo sphingolipid synthesis or knockdown of the S1P-degrading enzyme S1P lyase identified S1P as the sphingolipid activator of p38 in this model. This work establishes a novel angiogenic pathway in response to a commonly utilized chemotherapeutic and highlights the potential of SK1 as a secondary drug target for patients with p53 mutant cancer.

PLA2G7/PAF-AH as Potential Negative Regulator of the Wnt Signaling Pathway Mediates Protective Effects in BRCA1 Mutant Breast Cancer.

Past studies have confirmed that aberrant activation of the Wnt/β-catenin signaling is associated with tumorigenesis and metastasis in breast cancer, while the role of platelet-activating factor acetylhydrolase (PLA2G7/PAF-AH) in this signaling pathway remains unclear. In this study, we analyze the functional impact of PAF-AH on BRCA1 mutant breast cancer and explore its relationship to the Wnt signaling pathway. By performing immunohistochemistry, PAF-AH expression and β-catenin expression were examined in both BRCA1 WT and BRCA1 mutant breast cancer specimens. The BRCA1 mutant breast cancer cell line HCC1937 was used for in vitro experiments to assess the impact of PAF-AH on cellular functions. The intracellular distribution of β-catenin depending on PLA2G7/PAF-AH expression was investigated by immunocytochemistry. Significantly higher nuclear expression levels of PAF-AH were found in BRCA1 mutant tissue specimens than in BRCA1 WT samples. Cell viability, proliferation, and the motility rate of HCC1937 were significantly enhanced after PLA2G7 silencing, which indicated a protective role of PAF-AH in breast cancer. Nuclear PAF-AH expressed correlatedly with membranous β-catenin. PLA2G7 silencing provoked the β-catenin translocation from the membrane to the nucleus and activated Wnt signaling downstream genes. Our data showed a protective effect of high PAF-AH expression in BRCA1 mutant breast cancer. PAF-AH may achieve its protective effect by negatively regulating the Wnt pathway. In conclusion, our research sheds new light on the regulatory pathways in BRCA1 mutant breast cancer.

Genome engineering for estrogen receptor mutations reveals differential responses to anti-estrogens and new prognostic gene signatures for breast cancer

Mutations in the estrogen receptor (ESR1) gene are common in ER-positive breast cancer patients who progress on endocrine therapies. Most mutations localise to just three residues at, or near, the C-terminal helix 12 of the hormone binding domain, at leucine-536, tyrosine-537 and aspartate-538. To investigate these mutations, we have used CRISPR-Cas9 mediated genome engineering to generate a comprehensive set of isogenic mutant breast cancer cell lines. Our results confirm that L536R, Y537C, Y537N, Y537S and D538G mutations confer estrogen-independent growth in breast cancer cells. Growth assays show mutation-specific reductions in sensitivities to drugs representing three classes of clinical anti-estrogens. These differential mutation- and drug-selectivity profiles have implications for treatment choices following clinical emergence of ER mutations. Our results further suggest that mutant expression levels may be determinants of the degree of resistance to some anti-estrogens. Differential gene expression analysis demonstrates up-regulation of estrogen-responsive genes, as expected, but also reveals that enrichment for interferon-regulated gene expression is a common feature of all mutations. Finally, a new gene signature developed from the gene expression profiles in ER mutant cells predicts clinical response in breast cancer patients with ER mutations.

Abstract 2955: SMYD2 regulates chromatin modifier KMT2D in ER+/PIK3CA mutant breast cancer

Abstract Breast cancer leads current cancer diagnoses worldwide, accounting for 1 in 4 cancer cases and 1 in 6 cancer deaths in women. Over 70% of breast tumors are hormone receptor positive and can be treated with endocrine therapy, however tumor recurrence remains a major obstacle. Oncogenic mutations in the catalytic subunit of PI3K occur in up to 40% of ER+/HER2- negative breast tumors, making it a promising therapeutic target. Clinically, PI3K inhibitors have shown limited efficacy as monotherapy due to a number of resistance mechanisms, including an upregulation of ER-dependent signaling. Identification of the ER-PI3K signaling crosstalk led to clinical trials which resulted in the approval of the PI3K inhibitor alpelisib in combination with fulvestrant in patients with hormone-refractory ER+/PIK3CA-mutant breast cancer. Further work uncovered a key role for the chromatin modifier KMT2D in regulating ER-dependent transcription upon PI3K inhibition. PI3K effectors AKT1/SGK1 negatively regulate KMT2D through direct S1331 phosphorylation. When PI3K is inhibited, this downstream regulatory event is lost, leading to increased chromatin recruitment of KMT2D and an upregulation of ER-dependent transcription. We have identified a novel methylation site on KMT2D, at K1330 directly adjacent to S1331, catalyzed by the lysine methyltransferase SMYD2. Knockdown of SMYD2 sensitizes ER+/PIK3CA mutant breast cancer cell lines to PI3K inhibition. The combination of SMYD2 knockdown and alpelisib leads to a significant reduction in tumor growth compared to alpelisib alone in a xenograft model. Mechanistically, SMYD2 knockdown is able to attenuate alpelisib-induced KMT2D chromatin binding globally and at ER loci. Accordingly, loss of SMYD2 abrogates alpelisib-induced changes in gene expression, including ER-dependent transcription. Altogether our results demonstrate a role for epigenetic therapy using SMYD2 inhibitors in combination with alpelisib for the treatment of ER+/PIK3CA mutant breast cancer. Citation Format: Ryan Blawski, Mirna Sallaku, Xinyu Guo, Srushti Kittane, Maurizio Scaltriti, Minkui Luo, Eneda Toska. SMYD2 regulates chromatin modifier KMT2D in ER+/PIK3CA mutant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2955.

Abstract P3-09-12: Breast cancer derived GATA3 mutations disrupt luminal transcriptional network

Abstract GATA3 has been identified as one of the most frequently mutated genes in breast cancer. In the METABRIC cohort, among 1,980 patient cases, 230 breast cancers harbored GATA3 mutations (~12%). 75% of the GATA mutations were observed in luminal breast tumors (47% in luminal A, 28% in luminal B). The recent genomic data in metastatic breast cancer also showed that the frequency of GATA3 somatic mutations was even higher in the metastatic breast cancer cohort. Lung, lymph nodes, and brain metastases were observed in the GATA3 mutant breast cancer patients. Based on these patient genomic data, GATA3 mutations have been considered as cancer drivers, yet the functional consequences of GATA3 mutations are underexplored. We and other groups previously identified that patients carrying GATA3 mutations have diverse clinical features. More than 70% of cases are small nucleotide deletions or insertions (indel), while less than 30% are missense mutations. By classifying the GATA3 indel mutations into 4 groups, we observed distinct clinical features. Somatic mutations found in the GATA3 second zinc-finger domain (ZnFn2) are significantly associated with poorer patient outcomes including worse patient survival. ZnFn2 mutations are predominantly found in luminal B breast tumors, while splice site mutations are frequently found in luminal A breast tumors and associated with better patient survival. These distinct clinical features clearly suggest the differential impacts of GATA3 mutations on breast cancer cells. To dissect the function of GATA3 mutants, we developed a GATA3 mutant cell line (R330fs/WT T47D cells) by CRISPR, which endogenously expresses a heterozygous R330 frame-shift (R330fs) mutant. R330fs mutations are found in multiple data cohorts. The majority (>90%) of GATA3 mutations are heterozygous. Therefore, this R330fs mutant T47D cell line mimics the type of alteration found in patients. Importantly, we found that the R330fs mutation induces transcriptional reprogramming in T47D luminal breast cancer cells leading to more aggressive phenotypes such as faster tumor growth. In the mutant cells, many epithelial marker genes (such as progesterone receptor and TFF1) were down-regulated while mesenchymal marker genes (such as TWIST1 and SNAI2) are up-regulated suggesting that R330fs induces a partial EMT in T47D cells. We also found that transcriptional changes in the R330fs mutant cells were strongly associated with redistribution of GATA3, Estrogen Receptor alpha, and FOXA1. Gene expression profiles in the other GATA3 mutant breast cancer cell lines also suggest similar genomic alterations. These results suggest the active roles of GATA3 mutations during breast cancer development. Citation Format: Motoki Takaku, Mika Saotome, Renju Nair. Breast cancer derived GATA3 mutations disrupt luminal transcriptional network [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-12.

Abstract P215: The MDM2 inhibitor milademetan induces synthetic lethality in GATA3 mutant, ER positive breast cancer

Abstract GATA3 is mutated, predominantly via frameshift (fs) mutations, in 15-18% of estrogen receptor (ER)-positive breast cancers and GATA3 mutations define a subset of patients with poor response to hormonal therapy. Importantly, GATA3 mutations are not currently targetable. Computational analysis of the data from the large-scale, deep RNAi screen project DRIVE led to the identification of MDM2 as a synthetic lethal partner in a GATA3 mutant ER+ breast cancer cell line. The objective of this study was to evaluate the activity of milademetan (RAIN-32), an orally available, small molecule inhibitor of MDM2 in GATA3 mutant ER+ breast cancer. Here, we evaluated the effect of MDM2 silencing in ER+, GATA3 mutant breast cancer cell line MCF-7 harboring a loss-of-function mutation (p.D335Gfs) that has been recurrently observed in breast cancer patients and tested the antitumoral activity of milademetan in vitro (MCF-7 cell line) ex vivo (patient derived organoids; PDO) and in vivo (cell line-derived xenograft and patient-derived xenograft (PDX) models). Additionally, we hypothesized that the synthetic lethality is p53 dependent and to this aim we explored the p53 dependent effects of milademetan inhibition by determining the effect of MDM2 loss in the absence of p53 and analyzing the expression of p53-target genes. Finally, we evaluated the impact of GATA3 mutations on survival in breast cancer using TCGA and AACR Genie datasets.MDM2 silencing significantly reduced cell proliferation in MCF-7. This effect was shown to be p53 dependent as p53 knockdown reversed the effect of MDM2 loss. MCF-7 cell line was also sensitive to milademetan in vitro. Finally, we tested milademetan in ER+ breast cancer models (PDO and a fulvestrant-resistant PDX) harboring a GATA3fs mutation and wild type TP53. Treatment of these models displayed dose-dependent anti-tumor activity to milademetan ex vivo and in vivo. Sequencing data analyses of the publicly available datasets revealed lower survival in GATA3 mutant breast cancer and mutual exclusivity with TP53 mutations. In conclusion, MDM2 deficiency leads to synthetic lethality in GATA3 mutant breast cancers. Milademetan shows evidence of antitumor activity in genetically selected ER+ breast tumors with GATA3 mutations. These data provide evidence for clinical exploration of milademetan in ER+, GATA3 mutant breast cancer refractory to current therapies. Citation Format: Vijaya G. Tirunagaru, Gaia Bianco, Charlotte K. Y. Ng, Elisabetta Marangoni, Salvatore Piscuoglio, François-Clément Bidard, Robert C. Doebele. The MDM2 inhibitor milademetan induces synthetic lethality in GATA3 mutant, ER positive breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P215.

Abstract 1236: Preclinical characterization of LY3484356, a novel, potent and orally bioavailable selective estrogen receptor degrader (SERD)

Abstract Nearly 70% of newly diagnosed breast cancers are estrogen receptor alpha (ERα) positive, for which endocrine therapy is a primary treatment. However, approximately 40% of those patients on endocrine therapy develop resistance, which includes mutations in ERα (ESR1) that drive constitutive activation of the receptor. One approach to overcome this resistance is to develop a potent degrader and pure antagonist of the estrogen receptor, which can effectively suppress estrogen receptor signaling. Fulvestrant, a pure antagonist and selective estrogen receptor degrader (SERD) is approved for the treatment of HR+/HER2- metastatic breast cancer. However, fulvestrant is limited by poor pharmacokinetics properties, limited exposure and sub-optimal in vivo estrogen receptor degradation. Here we describe the preclinical profile of LY3484356, an oral SERD and pure estrogen receptor antagonist, with potent activity against the wild type and mutant estrogen receptor. LY3484356 has Ki values of 0.64 nM and 2.8 nM against wild type ERα and Y537S mutant ERα proteins, respectively. It is a potent and highly efficient degrader of wild type ERα and Y537N mutant ERα proteins in cells, with IC50 values 3.0 nM and 9.6 nM, respectively. LY3484356 is also a potent inhibitor of ERα-mediated transcription in vitro and in vivo. It inhibits cell proliferation in wild type ERα and ESR1 Y537N mutant breast cancer cell lines, with average IC50 values of 3 nM and 17 nM, respectively. In a panel of breast cancer cell lines, 11 out of 12 ER+ breast cancer cell lines were sensitive to LY3484356 (IC50 less than 100 nM), whereas all ER- cell lines tested were insensitive. LY3484356 has demonstrated sustained and prolonged target inhibition (>75% inhibition of PGR transcription up to 96h after last dose) in ESR1 wild type (MCF7) and ESR1 Y537S mutant (ST941/C) xenograft tumors. Consistent with its profile as a pure antagonist, immature rat studies demonstrated no significant effect on uterine wet weight. LY3484356 demonstrated significant tumor growth inhibition and tumor regressions in wild type ESR1 breast cancer xenograft models such as MCF7, T47D and ZR-75-1, as well as ESR1 mutant breast cancer PDX models. LY3484356 has shown synergy or additivity in combination with CDK4/6 inhibitor abemaciclib, mTOR inhibitor everolimus and PIK3CA inhibitor alpelisib in inhibiting cell proliferation in ER+ breast cancer cell lines in vitro and tumor growth inhibition in relevant xenograft or PDX models in vivo. The first-in-human Phase 1/2 clinical trial of LY3484356 (EMBER, ClinicalTrials.gov NCT04188548) is currently ongoing and a window-of-opportunity study evaluating the pharmacodynamic effects of LY3484356 in early stage breast cancer is expected to begin early 2021. Citation Format: Shripad V. Bhagwat, Baohui Zhao, Weihua Shen, Cecilia Mur, Robert Barr, Lisa J. Kindler, Almudena Rubio, Jolie A. Bastian, Jeffrey D. Cohen, Brian E. Mattioni, Eunice Yuen, Thomas K. Baker, Mark A. Castanares, Dongling Fei, Jason R. Manro, Maria Jose Lallena, Sheng-Bin Peng, Alfonso de Dios. Preclinical characterization of LY3484356, a novel, potent and orally bioavailable selective estrogen receptor degrader (SERD) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1236.

Distinct Classes of Flavonoids and Epigallocatechin Gallate, Polyphenol Affects an Oncogenic Mutant p53 Protein, Cell Growth and Invasion in a TNBC Breast Cancer Cell Line.

Mutant p53(s) are widely considered as oncogenes and promote several gain-of-function oncogenic activities. p53 mutations correlate with higher rates of metastasis and poor survival; therefore, it is paramount to inhibit mutant p53 protein either directly or indirectly. Although some compounds have been developed, none of them have achieved a desirable level of specificity. Some of these compounds only targeted specific mutations. In search of less-toxic compounds, we tested plant-derived compounds on mutant p53 triple-negative breast cancer cell lines. Here, we show that the compounds tested reduced the protein levels of one of the more frequent oncogenic p53 mutants (R249S; hot spot mutation), and its important targets that promote invasion and metastasis, including GMPS and IMPDH1. All compounds tested perturbed the invasion potential of the breast cancer cell line. These compounds downregulated several nucleotide metabolism genes (NMGs) which are essential for cell cycle progression. We observed S-phase arrest correlating to reduced cell proliferation and increased replication stress. Moreover, we also show a reduction of key ETS transcription family members including ETS2, ETS1, ETV1, and ETV4, which are involved in invasion and metastasis. We propose that these compounds may inhibit invasion by interfering with multiple pathways. Our findings exemplify that these tested compounds could inhibit invasion and cell growth in TNBC in a nucleotide-dependent manner.

A novel series of nitrofuran derivatives produces an anti‐tumor effect via a p53‐dependent mechanism

Nitrofurantoin (NF) is an antibacterial that acts by inducing DNA damage. Increased selectivity towards bacterial DNA is conferred through the rapid activation by prokaryotic reductases. In cancer therapy, radiation and anti‐neoplastic drugs induce DNA damage to activate the p53‐dependent pathway, provoking apoptosis in cancer cells. In this study, a novel series of nitrofuran derivatives was designed with a presumed enhanced eukaryotic activity. The safety and the anti‐tumor efficacy of this series was assessed. Anticancer effects were assessed on colorectal cancer (Caco‐2), breast cancer (MCF‐7), cervical cancer (Hela), and liver cancer cells (HepG‐2). Cytotoxicity was estimated by MTT assays. Three compounds, NF‐5, NPO, and NS‐1 were shown to have the highest cytotoxic activity against cancer cells at IC50 values that are several‐fold lower that those observed for non‐malignant lung fibroblasts. The abilities to induce oxidative stress and apoptosis were tested by flow cytometry to measure dihydrodichlorofluorescein fluorescence and nuclear annexin‐V staining, and compared to that of NF and the reference chemotherapeutic drug (5‐FU). Other apoptosis markers measured include caspase3/7 activity and Bax/Bcl2 ratio. NF‐5, NPO, and NS‐1 induced oxidative DNA damage in cancer cells in vitro with subsequent cellular apoptosis. Furthermore, the three derivatives induced more than 50% increase in reactive oxygen species when compared to 5‐FU. Cells treated with these drugs showed an increased Bax/Bcl2 ratio and caspase 3/7 activity compared to the traditional chemotherapeutic drug. A positive linear correlation was shown between the oxidative load produced in the cells and the increase in apoptotic markers. Interestingly, despite equivalent induction of DNA damage demonstrated by similar pATM nuclear shuttling, NF‐5, NPO, and NS‐1 produced more cytotoxicity in MCF‐7 cells compared to the p53 mutant breast cancer cell line MDA‐MB‐231. Moreover, the effect of these derivatives on the p53 interaction with its negative modulator Murine Double Minute 2 (MDM2) was assessed using a multi‐spot sandwich ELISA kit. NF‐5, NPO, and NS‐1 promoted p53 release from MDM2 at concentrations that are 2–3 orders of magnitude lower than their cytotoxic IC50 values. These findings were further confirmed by in silico docking studies demonstrating possible binding sites on the protein. Further experiments are underway to assess the ability of lower, non‐cytotoxic concentrations of these drugs to improve the radio‐sensitivity of cancer cells via activation of p53‐dependnet pathways.Support or Funding InformationFunded by: AUB‐FM MPP grant #320148

Abstract P3-11-23: GDC-0077 is a selective PI3K alpha inhibitor with robust efficacy in PIK3CA mutant hormone-positive breast cancer models

Abstract The phosphatidylinositol 3kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway is a major regulator of tumor cell growth, proliferation and survival. Hotspot mutations of PIK3CA are common in all subtypes of breast cancer with a prevalence of approximately 30%. GDC-0077 is a potent ATP-competitive inhibitor of PI3K alpha (IC50 = 0.038 ± 0.003 nM), thereby inhibiting the phosphorylation of membranebound PIP2 to PIP3. Biochemically, GDC-0077 is more than 300fold selective over the other Class I PI3K isoforms, beta, delta, and gamma, and more than 2000-fold more selective over PI3K class II and III family members. Importantly, GDC-0077 is more selective for mutant versus wild-type PI3K alpha in cell-based assays. Mechanism of action studies indicate that GDC-0077 treatment leads to degradation of the mutant PI3K alpha protein in a proteasome dependent fashion, without changing WT PI3K alpha levels. Drug treatment results in sustained reduction of PI3K pathway biomarkers pAkt and pPRAS40, inhibition of cell proliferation, and increased apoptosis in human PIK3CA mutant breast cancer cell lines, to a greater extent than other non-degrading inhibitors such as alpelisib. Combination studies of GDC-0077 with the CDK4/6 inhibitor palbociclib demonstrate a stronger effect in estrogen-depleted HR+ PIK3CA mutant breast cell lines, compared to cells grown in the presence of estrogen. Daily dosing of GDC-0077 in PIK3CA mutant breast cancer patient derived xenograft (PDX) models (n=4) resulted in tumor regressions, induction of apoptosis and a reduction of pAkt, pPRAS40, and pS6RP in a dose-dependent fashion. In vivo combination efficacy studies of GDC-0077 with fulvestrant and palbociclib also indicate the triple combination results in the greatest efficacy. Collectively, our preclinical data support evaluation of GDC-0077 in a triple combination with endocrine therapy and CDK4/6 inhibition in the clinic, in patients with locally advanced or metastatic hormone receptor positive breast cancer whose tumors harbor mutant PIK3CA. Citation Format: Kyle Edgar, Rebecca Hong, Kyung Song, Steven Schmidt, Marc Hafner, Alfonso Arrazate, Erin Williams, Cecile De La Cruz, Jason Oeh, Deepak Sampath, Steve Staben, Lori Friedman. GDC-0077 is a selective PI3K alpha inhibitor with robust efficacy in PIK3CA mutant hormone-positive breast cancer models [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-23.

Targeting the PI3K/Akt/mTOR pathway with the pan-Akt inhibitor GDC-0068 in PIK3CA-mutant breast cancer brain metastases.

Activating mutations in the pathway of phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) occur in 43-70% of breast cancer brain metastasis patients. To date, the treatment of these patients presents an ongoing challenge, mainly because of the lack of targeted agents that are able to sufficiently penetrate the blood-brain barrier. GDC-0068 is a pan-Akt inhibitor that has shown to be effective in various preclinical tumor models as well as in clinical trials. The purpose of this study was to analyze the efficacy of GDC-0068 in a breast cancer brain metastases model. In in vitro studies, antitumor activity of GDC-0068 was assessed in breast cancer cells of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)-mutant and PIK3CA-wildtype breast cancer cell lines using cell viability and apoptosis assays, cell cycle analysis, and western blots. In vivo, the efficacy of GDC-0068 was analyzed in a PIK3CA-mutant breast cancer brain metastasis orthotopic xenograft mouse model and evaluated by repeated bioluminescent imaging and immunohistochemistry. GDC-0068 decreased cell viability, induced apoptosis, and inhibited phosphorylation of proline rich Akt substrate 40 kDa and p70 S6 kinase in a dose-dependent manner in PIK3CA-mutant breast cancer brain metastatic cell lines compared with PIK3CA-wildtype cell lines. In vivo, treatment with GDC-0068 notably inhibited the growth of PIK3CA-mutant tumors and resulted in a significant survival benefit compared with sham, whereas no effect was detected in a PIK3CA-wildtype model. This study suggests that the Akt inhibitor GDC-0068 may be an encouraging targeted treatment strategy for breast cancer brain metastasis patients with activating mutations in the PI3K pathway. These data provide a rationale to further evaluate the efficacy of GDC-0068 in patients with brain metastases.

BRCA1 participates in the expression of noncoding XIST RNA

Introduction . Noncoding RNA of XIST gene (X inactivation-specific transcript) initiates inactivation of one of X chromosomes in cells of female organism. Further stages of this process include chromatin epigenetic modifications leading to the inhibition of the most genes on X chromosome. Recently the data were obtained that tumor suppressor BRCA1 is associated with inactive X chromosome (Xi) participating in XIST RNA localization on Xi and influencing XIST RNA expression.Objective: to reveal the role of BRCA1 in XIST RNA expression.Materials and methods . The objects of the study were mutant breast cancer cell lines (BRCA1–/–): HCC1395, HCC1937, SUM149PT, and, as controls – cell lines containing wild type of BRCA1 gene (BRCA1+/+): IMR90 и 293T. Method of reverse transcription coupled with polymerase chain reaction (RT-PCR) was used for the analysis of XIST RNA expression.Results . In the clone of doxycycline-inducible HCC1937 breast cancer cell line XIST RNA expression was observed upon BRCA1 induction. In HCC1395, HCC1937 and SUM149PT breast cancer cell lines containing mutant BRCA1 gene (BRCA1–/–) and nonfunctional BRCA1 protein the absence of XIST RNA expression was observed using RT-PCR. This observation indicates the indispensable role of functional BRCA1 protein in XIST RNA expression.Conclusion . Altogether, the data obtained in this study confirm the role of BRCA1 in the expression of noncoding inhibiting XIST RNA and suggest the involvement of BRCA1 in the inhibition of gene expression on Xi.

Abstract 3934: Determination of the PI3Kα selective inhibitor alpelisib mechanism of action and efficacy in ER+/ PIK3CA mutant breast cancer preclinical models

Abstract Over the past years, a key focus has been on identifying inhibitors against components of pathways that drive tumor cell proliferation, survival, and metastasis such as the PI3K/mTOR pathway, known to be implicated in many human cancers through various mechanisms, such as, somatic PIK3CA missense mutations that occur at high frequency in a number of common solid tumors including ~40% of ER+ breast cancer patients. Based on these findings, cancer-specific mutants of PI3Kα appear to be ideal targets for PI3Kα selective inhibitors such as alpelisib which is unique in its relative selectivity for the α-isoform and previously reported to be efficacious in PIK3CA mutant cancer models. Recently, using the ER-negative/HER2-positive/PIK3CA mutant HCC1954 breast cancer cell line, the PI3K β-sparing inhibitor taselisib has been described to have a dual mechanism of action, both blocking PI3K signaling and inducing a decrease in p110α protein levels. Here, we investigated whether such dual mechanism of action could be associated with PI3Kα selective inhibition in ER+/PIK3CA mutant breast cancer pre-clinical models specifically. In vitro, using the T47D ER+/PIK3CA mutant breast cancer cell line, alpelisib displays dual MoA by inhibiting p-Akt and inducing a decrease of p110a protein levels in a dose-dependent manner. This effect is observed upon 24 hours of compound treatment and is maintained for at least 96 hours upon treatment. Interestingly, for both alpelisib and taselisib, p110α degradation becomes more pronounced at concentrations which produce strong PI3K pathway inhibition (>80% p-Akt inhibition). In contrast to T47D cells, we do not observe p110α degradation upon 24 hours treatment in 4 additional ER+/PIK3CA mutant breast cancer cell lines tested, despite a dose-dependent and robust inhibition of the PI3K pathway, suggesting the effect might be specific to certain cell lines or restricted to certain PIK3CA mutations or might require longer compound treatment periods. In the EFM19 cells, the PI3K inhibition-mediated p110α degradation occurs upon minimum 48 hours treatment for both alpelisib and taselisib. In vitro, when alpelisib and taselisib are then compared at concentrations that equally inhibit the PI3K pathway and equally degrade p110α, both compounds display similar inhibitory effects on T47D and EFM19 cell proliferation and survival. In vivo, in an ER+/ PIK3CA mutant breast cancer patient derived xenograft (PTX) model, alpelisib and taselisib show comparable anti-tumoral efficacy when administered at doses that are equally well tolerated and induced comparable PI3K pathway modulation. Overall, the data demonstrate that in ER+/ PIK3CA mutant breast cancer pre-clinical models, selective PI3Kα inhibition can induce a dual mechanism of action and produces robust efficacy. Citation Format: Christine Fritsch, Estelle Pfister, Nicolas Ebel, Daniel Guthy, Christian Schnell, Francesco Hofmann. Determination of the PI3Kα selective inhibitor alpelisib mechanism of action and efficacy in ER+/ PIK3CA mutant breast cancer preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3934.

Abstract 156: Preclinical characterization of GDC-0077, a specific PI3K alpha inhibitor in early clinical development

Abstract The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) signaling pathway is a major regulator of tumor cell growth, proliferation and survival. Dysregulation of the PI3K/Akt/mTOR signaling pathway through multiple mechanisms has been described in solid tumor malignancies, including activating and transforming “hotspot” mutations as well as amplification of PIK3CA that encodes the p110 alpha subunit of PI3K. Hotspot mutations of PIK3CA mutation are frequently observed in breast cancer with a prevalence of approximately 30%. GDC-0077 is a potent inhibitor of PI3K alpha (IC50 = 0.038 + 0.003 nM) and exerts its activity by binding to the ATP binding site of PI3K, thereby inhibiting the phosphorylation of PIP2 to PIP3. Biochemically, GDC-0077 is more than 300-fold selective over other Class I PI3K isoforms such as beta, delta, and gamma and more than 2000 fold more selective over PI3K class II and III family members. Importantly, GDC-0077 is more selective for mutant versus wild-type PI3K alpha in cell based assays. Compared to the PI3K inhibitor, taselisib, the improved biochemical selectivity of GDC-0077 against PI3K delta is demonstrated in human CD69+ B-cells, which are primarily dependent on PI3K delta for proliferation and survival and were more sensitive to taselisib than GDC-0077. Mechanism of action studies indicate that GDC-0077 induces depletion of mutant PI3K alpha protein resulting in reduction of PI3K pathway biomarkers such as pAkt and pPRAS40, inhibition of cell proliferation and increased apoptosis in human PIK3CA mutant breast cancer cell lines to a greater extent when compared to PIK3CA wild-type cells. In vivo, daily oral treatment with GDC-0077 in cell-culture-derived and patient derived PIK3CA mutant breast cancer xenograft models, resulted in tumor regressions, induction of apoptosis and a reduction of pAkt, pPRAS40, and pS6RP in a dose-dependent fashion. In vivo efficacy in a PIK3CA-mutant human breast cancer xenograft model was also improved when GDC-0077 was combined with standard-of-care therapies for hormone-receptor positive (HR+) breast cancer such as anti-estrogens (fulvestrant) or CDK4/6 inhibitor (palbociclib). Collectively, the preclinical data provide rationale for evaluating GDC-0077, a PI3K alpha mutant selective inhibitor, as a single agent and in combination with standard-of-care endocrine and targeted therapies that may provide additional benefit to patients that harbor PIK3CA mutations. Citation Format: Kyle Edgar, Emily Hanan, Steven Staben, Stephen Schmidt, Rebecca Hong, Kyung Song, Amy Young, Patricia Hamilton, Alfonso Arrazate, Cecile de la Cruz, Marcia Belvin, Michelle Nannini, Lori S. Friedman, Deepak Sampath. Preclinical characterization of GDC-0077, a specific PI3K alpha inhibitor in early clinical development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 156. doi:10.1158/1538-7445.AM2017-156

Abstract 2475: In vitro and in vivo assessment of the mechanism of action of the PARP inhibitor rucaparib

Abstract Background: Rucaparib (CO-338) is an oral small molecule inhibitor of poly (ADP-ribose) polymerase (PARP) that is being developed for patients with BRCA1 and BRCA2 mutated and homologous recombination deficient ovarian cancer (Swisher et al., 2016; Lancet Oncol. Epub). The mechanism of action (MOA) of rucaparib was characterized in the studies reported here. Methods: Enzymatic and cellular reporter assay profiling were performed by BPS Biosciences. Rucaparib potency was assessed using 6-day cell viability assays (CellTiter-Glo). In vivo PK/PD and efficacy studies in the MDA-MB-436 (BRCA1 mutant) and the HBCx-17 (BRCA2 mutant) TNBC models were performed at Crown Biosciences and XenTech, respectively. Results: In biochemical assays rucaparib inhibited PARP-1, PARP-2 and PARP-3, with IC50 values of 0.8, 0.5, and 28 nM, respectively. Rucaparib weakly inhibited PARP5a (tankyrase 1[TNKS1]) and PARP5b (tankyrase 2[TNKS2]) with an IC50 of 796 and 486 nM; however rucaparib did not inhibit TNKS1/2 in a cellular reporter assay evaluating Wnt pathway signaling (IC50 >10 µM). Rucaparib cytotoxicity was evaluated in the BRCA1 mutant (UWB1.289) and wild-type (UWB1.289+BRCA1) isogenic pair, and a panel (n=26) of breast and ovarian cancer cell lines. The UWB1.289 cell line was more sensitive to rucaparib (IC50 = 375 nM) than the UWB1.289+BRCA1 cell line (IC50 = 5430 nM). Rucaparib also decreased the cell viability of several BRCA1 and BRCA2 mutant ovarian and breast cancer cell lines. Mechanistic analyses in the UWB1.289 cell line demonstrated a dose dependent decrease in poly (ADP) ribosylation (PAR; IC50 = 2.8 nM), and an increase in DNA damage and apoptosis. Taken together, these results are consistent with the concept of synthetic lethality, where simultaneous inhibition of multiple DNA repair pathways results in irreparable DNA damage and cell death. In vivo PK/PD and efficacy studies assessed the MOA and activity of rucaparib in the MDA-MD-436 (BRCA1 mutant) xenograft model. In a PK/PD study, dose dependent inhibition of PAR was observed in the tumors of mice treated with 15, 50 and 150 mg/kg BID rucaparib, resulting in statistically significant (p < 0.05) inhibition of 45%, 86% and 96%, respectively. An inverse and dose dependent correlation between PAR and rucaparib levels in the plasma and tumor was observed, and the levels of rucaparib in the tumor were consistently higher (> 2-fold) than the levels of rucaparib measured in plasma. Dose dependent and statistically significant tumor growth inhibition was observed in both subcutaneous and orthotopic xenograft studies evaluating the anti-tumor activity of rucaparib in the MDA-MB-436 model. Rucaparib also demonstrated potent activity in the BRCA2 mutant HBCx-17 patient-derived xenograft model. Conclusion: These results demonstrate that rucaparib is a potent and selective inhibitor of PARP1, PARP-2, and PARP-3 with in vitro and in vivo activity in BRCA1/2 deficient tumors. Citation Format: Liliane Robillard, Minh Nguyen, Thomas C. Harding, Andrew D. Simmons. In vitro and in vivo assessment of the mechanism of action of the PARP inhibitor rucaparib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2475. doi:10.1158/1538-7445.AM2017-2475

Abstract 1546: Contribution of heterozygous loss of ribosomal protein L5 as general tumor suppressor in cancer

Abstract The association between ribosome defects and cancer became clear with the recent discovery of somatic mutations in ribosomal protein genes in several cancers, such as lymphoid leukemia’s and glioblastoma (Nat Genet. 2013 Feb;45(2):186-90 & Blood. 2016 Feb 25;127(8):1007-16 & Nature. 2014 Jan 23;505(7484):495-501). To further delineate the role of ribosomal proteins in human cancer, we systematically screened the TCGA database for genetic lesions in ribosomal protein genes, confirmed the contributing capacity of the most common somatic ribosomal protein defect in cancer pathogenesis by molecular interference, and explored the mode of action of this ribosomal protein defect. Systematic analysis of TCGA mutation and copy number data of respectively 4926 and 7322 patients representing 16 cancer types for defects in all 81 ribosomal protein genes. After stringent filtering, six ribosomal protein encoding genes (RPL5, RPL11, RPL23A, RPS5, RPS20 and RPSA) were significantly altered and identified as candidate cancer driver genes. RPL5 was located at a significant peak of heterozygous deletion on chromosome 1p22 and showed significant mutations and deletions in 11% of glioblastoma (GBM), 28% of melanoma, 34% of breast cancer (BRCA), and in 20% of multiple myeloma cases, thereby supporting heterozygous RPL5 inactivation to be the most common somatic ribosomal protein defect in human cancer. Human specific RPL5 doxycycline inducible knockdown (sh-hRPL5) in TP53 WT and TP53 mutant human breast cancer cell lines (MCF7 and MDA-MB-231) and in a TP53 mutant human glioblastoma cell line (U-118 MG) proved that RPL5 knockdown accelerated in vivo tumor progression in NSG mice. This acceleration was associated with reduced phosphorylation of CDK1tyr15, which is required for cell cycle progression from G2 to mitosis. Whereas RPL5 has been implicated in TP53 and MYC regulation, no consistent effects of RPL5 downregulation on these proteins could be detected in TP53 WT and TP53 homozygous R280K mutant breast cancer models (sh-hRPL5). Interestingly, partial RPL5 inactivation was found to be associated with PTEN protein suppression in these tumors. This observation was confirmed in various cell models; in normal mouse neural stem/progenitor cells and mouse bone marrow cells (RPL5+/lox by INFβ and poly I:C), and also in GBM cancer pre-disposition mouse neural stem/progenitor cell model (sh-mRPL5 TP53 -/-). RPL5 downregulation also accelerated tumor formation in the PTEN deficient GBM cell line U-118 MG, underscoring undefined PTEN-independent mechanisms of RPL5. Overall, we identified RPL5 as a new tumor suppressor that shows heterozygous inactivation in 11-34% of multiple human cancer types. Partial molecular inactivation of RPL5 supported a tumor suppressor function for RPL5 in accelerating breast cancer and glioblastoma progression in vivo, which may be, in part, due to a new function of RPL5 in regulating PTEN. Citation Format: Kim R. Kampen, Laura Fancello, Jelle Verbeeck, Kim De Keersmaecker. Contribution of heterozygous loss of ribosomal protein L5 as general tumor suppressor in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1546. doi:10.1158/1538-7445.AM2017-1546

31P - Taselisib enhances effects of anti-microtubule chemotherapic agents in phosphatidylinositol 3-kinase (PI3K&agr;) mutant breast cancer cell lines

31P - Taselisib enhances effects of anti-microtubule chemotherapic agents in phosphatidylinositol 3-kinase (PI3K&agr;) mutant breast cancer cell lines