Amanda Esch

Decoupling of the PI3K pathway via mutation necessitates combinatorial treatment in HER2+ breast cancer

We report here on experimental and theoretical efforts to determine how best to combine drugs that inhibit HER2 and AKT in HER2+ breast cancers. We accomplished this by measuring cellular and molecular responses to lapatinib and the AKT inhibitors (AKTi) GSK690693 and GSK2141795 in a panel of 22 HER2+ breast cancer cell lines carrying wild type or mutant PIK3CA. We observed that combinations of lapatinib plus AKTi were synergistic in HER2+/PIK3CAmut cell lines but not in HER2+/PIK3CAwt cell lines. We measured changes in phospho-protein levels in 15 cell lines after treatment with lapatinib, AKTi or lapatinib + AKTi to shed light on the underlying signaling dynamics. This revealed that p-S6RP levels were less well attenuated by lapatinib in HER2+/PIK3CAmut cells compared to HER2+/PIK3CAwt cells and that lapatinib + AKTi reduced p-S6RP levels to those achieved in HER2+/PIK3CAwt cells with lapatinib alone. We also found that that compensatory up-regulation of p-HER3 and p-HER2 is blunted in PIK3CAmut cells following lapatinib + AKTi treatment. Responses of HER2+ SKBR3 cells transfected with lentiviruses carrying control or PIK3CAmut sequences were similar to those observed in HER2+/PIK3CAmut cell lines but not in HER2+/PIK3CAwt cell lines. We used a nonlinear ordinary differential equation model to support the idea that PIK3CA mutations act as downstream activators of AKT that blunt lapatinib inhibition of downstream AKT signaling and that the effects of PIK3CA mutations can be countered by combining lapatinib with an AKTi. This combination does not confer substantial benefit beyond lapatinib in HER2+/PIK3CAwt cells.

Combating subclonal evolution of resistant cancer phenotypes

Metastatic breast cancer remains challenging to treat, and most patients ultimately progress on therapy. This acquired drug resistance is largely due to drug-refractory sub-populations (subclones) within heterogeneous tumors. Here, we track the genetic and phenotypic subclonal evolution of four breast cancers through years of treatment to better understand how breast cancers become drug-resistant. Recurrently appearing post-chemotherapy mutations are rare. However, bulk and single-cell RNA sequencing reveal acquisition of malignant phenotypes after treatment, including enhanced mesenchymal and growth factor signaling, which may promote drug resistance, and decreased antigen presentation and TNF-α signaling, which may enable immune system avoidance. Some of these phenotypes pre-exist in pre-treatment subclones that become dominant after chemotherapy, indicating selection for resistance phenotypes. Post-chemotherapy cancer cells are effectively treated with drugs targeting acquired phenotypes. These findings highlight cancer’s ability to evolve phenotypically and suggest a phenotype-targeted treatment strategy that adapts to cancer as it evolves.In metastatic breast cancer, subclonal evolution can drive drug resistance. Here, the authors genetically and transcriptionally follow the evolution of four breast cancers over time and treatment, and suggest a phenotype-targeted treatment strategy to adapt to cancer as it evolves.

PEG-lipid micelles enable cholesterol efflux in Niemann-Pick Type C1 disease-based lysosomal storage disorder

2-Hydroxy-propyl-β-cyclodextrin (HPβCD), a cholesterol scavenger, is currently undergoing Phase 2b/3 clinical trial for treatment of Niemann Pick Type C-1 (NPC1), a fatal neurodegenerative disorder that stems from abnormal cholesterol accumulation in the endo/lysosomes. Unfortunately, the extremely high doses of HPβCD required to prevent progressive neurodegeneration exacerbates ototoxicity, pulmonary toxicity and autophagy-based cellular defects. We present unexpected evidence that a poly (ethylene glycol) (PEG)-lipid conjugate enables cholesterol clearance from endo/lysosomes of Npc1 mutant (Npc1−/−) cells. Herein, we show that distearyl-phosphatidylethanolamine-PEG (DSPE-PEG), which forms 12-nm micelles above the critical micelle concentration, accumulates heavily inside cholesterol-rich late endosomes in Npc1−/− cells. This potentially results in cholesterol solubilization and leakage from lysosomes. High-throughput screening revealed that DSPE-PEG, in combination with HPβCD, acts synergistically to efflux cholesterol without significantly aggravating autophagy defects. These well-known excipients can be used as admixtures to treat NPC1 disorder. Increasing PEG chain lengths from 350 Da-30 kDa in DSPE-PEG micelles, or increasing DSPE-PEG content in an array of liposomes packaged with HPβCD, improved cholesterol egress, while Pluronic block copolymers capable of micelle formation showed slight effects at high concentrations. We postulate that PEG-lipid based nanocarriers can serve as bioactive drug delivery systems for effective treatment of lysosomal storage disorders.

Genome co-amplification upregulates a mitotic gene network activity that predicts outcome and response to mitotic protein inhibitors in breast cancer

BackgroundHigh mitotic activity is associated with the genesis and progression of many cancers. Small molecule inhibitors of mitotic apparatus proteins are now being developed and evaluated clinically as anticancer agents. With clinical trials of several of these experimental compounds underway, it is important to understand the molecular mechanisms that determine high mitotic activity, identify tumor subtypes that carry molecular aberrations that confer high mitotic activity, and to develop molecular markers that distinguish which tumors will be most responsive to mitotic apparatus inhibitors.MethodsWe identified a coordinately regulated mitotic apparatus network by analyzing gene expression profiles for 53 malignant and non-malignant human breast cancer cell lines and two separate primary breast tumor datasets. We defined the mitotic network activity index (MNAI) as the sum of the transcriptional levels of the 54 coordinately regulated mitotic apparatus genes. The effect of those genes on cell growth was evaluated by small interfering RNA (siRNA).ResultsHigh MNAI was enriched in basal-like breast tumors and was associated with reduced survival duration and preferential sensitivity to inhibitors of the mitotic apparatus proteins, polo-like kinase, centromere associated protein E and aurora kinase designated GSK462364, GSK923295 and GSK1070916, respectively. Co-amplification of regions of chromosomes 8q24, 10p15-p12, 12p13, and 17q24-q25 was associated with the transcriptional upregulation of this network of 54 mitotic apparatus genes, and we identify transcription factors that localize to these regions and putatively regulate mitotic activity. Knockdown of the mitotic network by siRNA identified 22 genes that might be considered as additional therapeutic targets for this clinically relevant patient subgroup.ConclusionsWe define a molecular signature which may guide therapeutic approaches for tumors with high mitotic network activity.

Pathway-enriched gene signature associated with 53BP1 response to PARP inhibition in triple-negative breast cancer

Effective treatment of patients with triple-negative (ER-negative, PR-negative, HER2-negative) breast cancer remains a challenge. Although PARP inhibitors are being evaluated in clinical trials, biomarkers are needed to identify patients who will most benefit from anti-PARP therapy. We determined the responses of three PARP inhibitors (veliparib, olaparib, and talazoparib) in a panel of eight triple-negative breast cancer cell lines. Therapeutic responses and cellular phenotypes were elucidated using high-content imaging and quantitative immunofluorescence to assess markers of DNA damage (53BP1) and apoptosis (cleaved PARP). We determined the pharmacodynamic changes as percentage of cells positive for 53BP1, mean number of 53BP1 foci per cell, and percentage of cells positive for cleaved PARP. Inspired by traditional dose–response measures of cell viability, an EC50 value was calculated for each cellular phenotype and each PARP inhibitor. The EC50 values for both 53BP1 metrics strongly correlated with IC50 values for each PARP inhibitor. Pathway enrichment analysis identified a set of DNA repair and cell cycle–associated genes that were associated with 53BP1 response following PARP inhibition. The overall accuracy of our 63 gene set in predicting response to olaparib in seven breast cancer patient-derived xenograft tumors was 86%. In triple-negative breast cancer patients who had not received anti-PARP therapy, the predicted response rate of our gene signature was 45%. These results indicate that 53BP1 is a biomarker of response to anti-PARP therapy in the laboratory, and our DNA damage response gene signature may be used to identify patients who are most likely to respond to PARP inhibition. Mol Cancer Ther; 16(12); 2892–901. ©2017 AACR.

Biological indicators of response and resistance to PARP inhibition in BRCA wild-type breast cancer.

125 Background: Although PARP inhibitors are currently being tested in clinical trials in breast cancer patients, it is not well understood which subgroup of patients will best respond to such therapy or how to combine anti-PARP therapy to improve therapeutic response. METHODS We assessed the response of a panel of 8 HER2-negative breast cancer cell lines against three PARP inhibitors: veliparib, olaparib, and BMN 673. Efficacy of carboplatin as a single agent and in combination with PARP inhibitors was determined. Therapeutic response and cellular phenotype were elucidated using live-cell imaging, high-content imaging and immunofluorescence. We computed IC50 as the measure of sensitivity to each PARP inhibitor across the panel of cell lines. Cellular phenotype was ascertained by quantifying endpoints for apoptosis and DNA damage. RESULTS BMN 673 demonstrated the highest potency, with IC50 values in the nanomolar range, while the IC50 values of olaparib and veliparib were in the micromolar range. Drug sensitivity was independent of BRCA mutational status. Biomarkers of apoptosis and DNA damage varied with the PARP inhibitor and across different cell lines. The resistant cell lines could be classified into two groups that varied in DNA damage response and cell death due to DNA damage. A combined DNA damage - cell death (DDD) Response Score was developed and correlated strongly with IC50 values in BRCA wild-type breast cancer cell lines. The DDD Response Score identified cell lines that were highly sensitive to single-agent PARP inhibitors or carboplatin. A Response Score that identified intermediate sensitivity may predict which cell lines may benefit from the combination of anti-PARP therapy and carboplatin. CONCLUSIONS Overall, a spectrum of response to three different PARP inhibitors was identified in a panel of eight breast cancer lines. Cellular phenotype can help classify resistant cancer cell lines. We have developed an experimental approach that may help to inform which breast cancer subtypes may benefit from PARP inhibitors and carboplatin, both as single agents, and in combination.

Publisher Correction: Combating subclonal evolution of resistant cancer phenotypes

The originally published version of this Article contained an error in Figure 4. In panel a, grey boxes surrounding the subclones associated with patients #2 and #4 obscured adjacent portions of the heatmap. This error has now been corrected in both the PDF and HTML versions of the Article.

Correction: Decoupling of the PI3K pathway via mutation necessitates combinatorial treatment in HER2+ breast cancer (PLoS ONE (2015) 10:7 (e0133219) DOI: 10.1371/journal.pone.0133219)

[This corrects the article DOI: 10.1371/journal.pone.0133219.].

The Biomechanical Environment and Impact on Tissue Fibrosis

The implantation of materials into the body elicits a foreign body response (FBR) that includes formation of a fibrous capsule around the implanted material. The formation of the fibrous capsule has many similarities to fibrotic responses to other insults or stressors. A number of biochemical factors are known to promote a fibrotic response including growth factors, cytokines, and hormones. Much less is known regarding the role of biomechanical forces in tissue fibrosis. The biomechanical environment plays a fundamental role in embryonic development, tissue maintenance, and pathogenesis. Mechanical forces play particularly important roles in the regulation of connective tissues including not only bone and cartilage but also the interstitial tissues of most organs. In vivo studies have correlated changes in mechanical load to modulation of the extracellular matrix and have indicated that increased mechanical force contributes to the enhanced expression and deposition of extracellular matrix components or fibrosis. A variety of in vitro models have been utilized to evaluate the effects of mechanical force on extracellular matrix-producing cells. In general, application of mechanical stretch, fluid flow, and compression results in enhanced expression and deposition of extracellular matrix components. More recent studies have indicated that tissue rigidity also provides profibrotic signals to cells. This is particularly relevant to implants as the implanted material generally alters the local biomechanical environment, which may promote fibrosis or the formation of the fibrous capsule. The mechanisms whereby cells detect mechanical signals and transduce them into biochemical responses have received considerable attention. Cell surface receptors for extracellular matrix components and intracellular signaling pathways are instrumental in the mechanotransduction process. Understanding the effects of the biomechanical environment and the mechanisms, whereby mechanical forces are transduced into biochemical and molecular signals in the cell, will provide important insight into tissue fibrosis and fibrous capsule formation.

Abstract B24: Genomic prediction of response to PARP inhibition in breast cancer

Efficacy of PARP inhibition has been demonstrated in several cancer types including prostate, ovarian, and breast. Most recently, anti-PARP therapy was shown to be effective in combination with carboplatin in triple-negative breast cancer patients in the neoadjuvant setting. However, it is not yet well known which subset of triple-negative breast cancers will benefit from single-agent anti-PARP therapy. We determined the therapeutic efficacy of three PARP inhibitors: veliparib, olaparib, and BMN 673, in a panel of eight triple-negative breast cancer cell lines. We used a 10-day in-vitro assay, after which we fixed the cells and determined 53BP1 expression using immunofluorescence and high-content imaging. We used cell counts to derive IC50 values and enumerated 53BP1 foci per cell to determine EC50 values. We used pre-treatment whole-transcriptome data to identify genes associated with 53BP1 response using gene set enrichment and pathway enrichment analysis. We determined the prevalence of these genes in a dataset of triple-negative breast cancer patients, and performed survival analysis. We found PARP inhibition to be effective in both BRCA-mutant and BRCA wild-type breast cancer cell lines. BMN 673 was the most potent PARP inhibitor, with the lowest concentrations required for DNA damage (measured by 53BP1 expression) and cell kill (measured by cell count), followed by olaparib, and then veliparib. We found a strong correlation between the IC50 values for cell count and the EC50 values for 53BP1 response. We identified a gene set associated with 53BP1 response, which was involved with three major pathways: DNA repair, cell cycle, and programmed cell death. These genes were found to be downregulated in triple-negative breast cancer patients. Patients with aberrations in these genes demonstrated poorer overall survival (P = 0.03). In conclusion, we identified a gene set involved with DNA repair, cell-cycle, and programmed cell death, which was associated with poor outcomes in triple-negative breast cancer patients that could potentially benefit from anti-PARP therapy. Citation Format: Saima Hassan, Amanda Esch, Laura M. Heiser, Joe W. Gray. Genomic prediction of response to PARP inhibition in breast cancer [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B24.