Haley Ellis

Convergent loss of PTEN leads to clinical resistance to a PI(3)Kα inhibitor

Broad and deep tumour genome sequencing has shed new light on tumour heterogeneity and provided important insights into the evolution of metastases arising from different clones. There is an additional layer of complexity, in that tumour evolution may be influenced by selective pressure provided by therapy, in a similar fashion to that occurring in infectious diseases. Here we studied tumour genomic evolution in a patient (index patient) with metastatic breast cancer bearing an activating PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha, PI(3)Kα) mutation. The patient was treated with the PI(3)Kα inhibitor BYL719, which achieved a lasting clinical response, but the patient eventually became resistant to this drug (emergence of lung metastases) and died shortly thereafter. A rapid autopsy was performed and material from a total of 14 metastatic sites was collected and sequenced. All metastatic lesions, when compared to the pre-treatment tumour, had a copy loss of PTEN (phosphatase and tensin homolog) and those lesions that became refractory to BYL719 had additional and different PTEN genetic alterations, resulting in the loss of PTEN expression. To put these results in context, we examined six other patients also treated with BYL719. Acquired bi-allelic loss of PTEN was found in one of these patients, whereas in two others PIK3CA mutations present in the primary tumour were no longer detected at the time of progression. To characterize our findings functionally, we examined the effects of PTEN knockdown in several preclinical models (both in cell lines intrinsically sensitive to BYL719 and in PTEN-null xenografts derived from our index patient), which we found resulted in resistance to BYL719, whereas simultaneous PI(3)K p110β blockade reverted this resistance phenotype. We conclude that parallel genetic evolution of separate metastatic sites with different PTEN genomic alterations leads to a convergent PTEN-null phenotype resistant to PI(3)Kα inhibition.

Single-Cell RNA Sequencing Identifies Extracellular Matrix Gene Expression by Pancreatic Circulating Tumor Cells

SUMMARY Circulating tumor cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.

PI3K inhibition results in enhanced estrogen receptor function and dependence in hormone receptor–positive breast cancer

Inhibition of the PI3K/AKT pathway results in induction of ER-dependent transcriptional activity and susceptibility to anti-estrogen therapy in ER-positive breast cancer. PIKing the correct therapeutic combination Mutations in a gene called PIK3CA are very common in estrogen receptor–positive breast cancers, and drugs that inhibit PI3K, the protein product of this gene, are already in clinical development. Unfortunately, these drugs are not always effective, and this study by Bosch et al. demonstrates a reason for this problem and a practical way to overcome it. By studying both mouse models and human patients’ tumors, the authors discovered that inhibition of PI3K often stimulates the activity of the estrogen receptor, which then drives tumor growth. By combining PI3K inhibitors with clinically available drugs that inhibit the estrogen receptor, the authors were able to overcome treatment resistance and effectively induce tumor regression in mouse models. Activating mutations of PIK3CA are the most frequent genomic alterations in estrogen receptor (ER)–positive breast tumors, and selective phosphatidylinositol 3-kinase α (PI3Kα) inhibitors are in clinical development. The activity of these agents, however, is not homogeneous, and only a fraction of patients bearing PIK3CA-mutant ER-positive tumors benefit from single-agent administration. Searching for mechanisms of resistance, we observed that suppression of PI3K signaling results in induction of ER-dependent transcriptional activity, as demonstrated by changes in expression of genes containing ER-binding sites and increased occupancy by the ER of promoter regions of up-regulated genes. Furthermore, expression of ESR1 mRNA and ER protein were also increased upon PI3K inhibition. These changes in gene expression were confirmed in vivo in xenografts and patient-derived models and in tumors from patients undergoing treatment with the PI3Kα inhibitor BYL719. The observed effects on transcription were enhanced by the addition of estradiol and suppressed by the anti-ER therapies fulvestrant and tamoxifen. Fulvestrant markedly sensitized ER-positive tumors to PI3Kα inhibition, resulting in major tumor regressions in vivo. We propose that increased ER transcriptional activity may be a reactive mechanism that limits the activity of PI3K inhibitors and that combined PI3K and ER inhibition is a rational approach to target these tumors.

Antagonism of EGFR and HER3 Enhances the Response to Inhibitors of the PI3K-Akt Pathway in Triple-Negative Breast Cancer

Predictions regarding drug resistance mechanisms and treatment strategies in triple-negative breast cancer are confirmed in tumors from patients. From Models to Breast Cancer Treatments Patients with triple-negative breast cancer (TNBC), a particularly aggressive form, have few treatment options. Targeting either the phosphatidylinositol 3-kinase to Akt (PI3K-Akt) pathway or epidermal growth factor receptor (EGFR) inhibits tumor growth in some patients, but durable responses are rare. Modeling studies using cell lines predict that the EGFR family member HER3 (human epidermal growth factor receptor 3) may confer drug resistance. Now, Tao et al. provide evidence from patient tumors to support those predictions. Treatment with PI3K-Akt pathway inhibitors increased the abundance of both total and activated HER3 in TNBC cells in culture and TNBC xenografts in mice. Residual tumors from patients treated with EGFR inhibitors had increased abundance and activation of HER3. Combining inhibitors of the PI3K-Akt pathway with a dual inhibitor of EGFR and HER3 substantially suppressed tumor growth in mice with TNBC xenografts derived from either cell lines or patients, suggesting that this combined strategy may improve therapeutic outcome in TNBC patients. Both abundant epidermal growth factor receptor (EGFR or ErbB1) and high activity of the phosphatidylinositol 3-kinase (PI3K)–Akt pathway are common and therapeutically targeted in triple-negative breast cancer (TNBC). However, activation of another EGFR family member [human epidermal growth factor receptor 3 (HER3) (or ErbB3)] may limit the antitumor effects of these drugs. We found that TNBC cell lines cultured with the EGFR or HER3 ligand EGF or heregulin, respectively, and treated with either an Akt inhibitor (GDC-0068) or a PI3K inhibitor (GDC-0941) had increased abundance and phosphorylation of HER3. The phosphorylation of HER3 and EGFR in response to these treatments was reduced by the addition of a dual EGFR and HER3 inhibitor (MEHD7945A). MEHD7945A also decreased the phosphorylation (and activation) of EGFR and HER3 and the phosphorylation of downstream targets that occurred in response to the combination of EGFR ligands and PI3K-Akt pathway inhibitors. In culture, inhibition of the PI3K-Akt pathway combined with either MEHD7945A or knockdown of HER3 decreased cell proliferation compared with inhibition of the PI3K-Akt pathway alone. Combining either GDC-0068 or GDC-0941 with MEHD7945A inhibited the growth of xenografts derived from TNBC cell lines or from TNBC patient tumors, and this combination treatment was also more effective than combining either GDC-0068 or GDC-0941 with cetuximab, an EGFR-targeted antibody. After therapy with EGFR-targeted antibodies, some patients had residual tumors with increased HER3 abundance and EGFR/HER3 dimerization (an activating interaction). Thus, we propose that concomitant blockade of EGFR, HER3, and the PI3K-Akt pathway in TNBC should be investigated in the clinical setting.

PDK1-SGK1 Signaling Sustains AKT-Independent mTORC1 Activation and Confers Resistance to PI3Kα Inhibition

Summary PIK3CA, which encodes the p110α subunit of PI3K, is frequently mutated and oncogenic in breast cancer. PI3Kα inhibitors are in clinical development and despite promising early clinical activity, intrinsic resistance is frequent among patients. We have previously reported that residual downstream mTORC1 activity upon treatment with PI3Kα inhibitors drives resistance to these agents. However, the mechanism underlying this phenotype is not fully understood. Here we show that in cancer cells resistant to PI3Kα inhibition, PDK1 blockade restores sensitivity to these therapies. SGK1, which is activated by PDK1, contributes to the maintenance of residual mTORC1 activity through direct phosphorylation and inhibition of TSC2. Targeting either PDK1 or SGK1 prevents mTORC1 activation, restoring the antitumoral effects of PI3Kα inhibition in resistant cells.

Masculine Epigenetic Sex Marks of the CYP19A1/Aromatase Promoter in Genetically Male Chicken Embryonic Gonads Are Resistant to Estrogen-Induced Phenotypic Sex Conversion

ABSTRACT Sex of birds is genetically determined through inheritance of the ZW sex chromosomes (ZZ males and ZW females). Although the mechanisms of avian sex determination remains unknown, the genetic sex is experimentally reversible by in ovo exposure to exogenous estrogens (ZZ-male feminization) or aromatase inhibitors (ZW-female masculinization). Expression of various testis- and ovary-specific marker genes during the normal and reversed gonadal sex differentiation in chicken embryos has been extensively studied, but the roles of sex-specific epigenetic marks in sex differentiation are unknown. In this study, we show that a 170-nt region in the promoter of CYP19A1/aromatase, a key gene required for ovarian estrogen biosynthesis and feminization of chicken embryonic gonads, contains highly quantitative, nucleotide base-level epigenetic marks that reflect phenotypic gonadal sex differentiation. We developed a protocol to feminize ZZ-male chicken embryonic gonads in a highly quantitative manner by direct injection of emulsified ethynylestradiol into yolk at various developmental stages. Taking advantage of this experimental sex reversal model, we show that the epigenetic sex marks in the CYP19A1/aromatase promoter involving DNA methylation and histone lysine methylation are feminized significantly but only partially in sex-converted gonads even when morphological and transcriptional marks of sex differentiation show complete feminization, being indistinguishable from gonads of normal ZW females. Our study suggests that the epigenetic sex of chicken embryonic gonads is more stable than the morphologically or transcriptionally characterized sex differentiation, suggesting the importance of the nucleotide base-level epigenetic sex in gonadal sex differentiation.

High-throughput applicable genomic sex typing of chicken by TaqMan real-time quantitative polymerase chain reaction

Genetic sex typing of vertebrate animals is an essential technique for research on reproductive phenomena such as sex determination of embryonic tissues. Polymerase chain reaction amplification of genomic DNA segments in the Z and W sex chromosomes has been widely used as a standard laboratory method to determine genetic sex of the chicken (Gallus gallus domesticus). However, conventional protocols for PCR determination of avian sex typically involve tedious steps of genomic DNA isolation, which often require relatively large amounts of tissue samples, and the purity of genomic DNA specimens significantly affects PCR efficiency. Moreover, detection of sex chromosome-specific PCR products by gel electrophoresis is prone to misjudgment caused by amplification of contaminating genomic DNA segments derived from tissue or DNA samples as well as previously generated PCR products. Thus, the credibility of genetic sex typing by conventional PCR-based methods that measure the relative amounts of the end product DNA amplicons critically depends on several experimental steps that are potentially vulnerable to errors. Here, we describe an optimized protocol of chicken genetic sex typing by TaqMan real-time quantitative PCR amplification of markers on the sex chromosomes. This TaqMan sex typing method accurately quantifies relative amounts of the Z and W sex chromosome markers directly from only 0.5 to 2 microL of total blood lysate without nucleic acid purification. The real-time amplification curves of the quantitative PCR reaction readily distinguished truly homozygous (ZZ) and heterozygous (ZW) sex chromosomes from contamination of the sex chromosomal DNA, ensuring highly credible sex determination. Thus, the TaqMan typing of chicken genetic sex has several advantageous features for high-throughput operation compared with conventional methods.

MACC1 expression levels as a novel prognostic marker for colorectal cancer

Metastasis-associated in colon cancer-1 (MACC1) is key in promoting tumor proliferation and invasion, and is mediated by the hepatocyte growth factor (HGF) and mesenchymal-epithelial transition factor. Previous reports have revealed that MACC1 is a novel oncogene that is expressed in various types of gastrointestinal cancer. The present study comprised of 174 patients who underwent curative surgery for colorectal cancer (CRC). The correlation between gene expression and clinical parameters of the patients was assessed. It was identified that patients exhibiting high MACC1 expression levels were statistically more susceptible to distant metastases and a poor prognosis, and those exhibiting low MACC1 expression showed improved disease-free and overall survival than those with high expression. Therefore, the present data indicates that MACC1 expression levels may present as a prognostic factor in CRC patients.

Abstract LB-327: Loss of PTEN leads to clinical resistance to the PI3Kα inhibitor BYL719 and provides evidence of convergent evolution under selective therapeutic pressure

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Activating mutations of PIK3CA, the gene encoding the p110α subunit of PI3K, are frequent in breast cancer and selective inhibitors of this enzyme have shown promising clinical activity in breast tumors harboring these mutations. We studied the case of a patient with metastatic breast cancer harboring a PIK3CA mutation that was treated in a clinical trial with BYL719, a highly selective PI3Kα inhibitor. The treatment resulted in a robust partial response that lasted 7 months followed by rapid progression and death. A rapid autopsy was performed with collection of tissue samples from 16 different metastatic sites. We compared by whole genome and exome sequencing the original primary tumor, a rapidly progressing lung metastasis and a periaortic lesion that was still responding to BYL719 at time of death. Besides several common alterations, PTEN loss and a missense mutation were detected only in the lung metastasis. Using targeted exome sequencing we analyzed all the other available samples. Strikingly, we observed a consistent loss in PTEN (via different mechanisms such as deletion, splice site mutation and frameshift mutations) in all the lesions refractory to BYL719 but not in the responding ones. In every case, the loss of PTEN was also documented by lack of protein expression by immunohistochemistry (IHC). Finally, we were able to build a dendrogram showing the phylogenetic evolution of the lesions and the evolutionary convergence of the PTEN alterations. To validate PTEN loss as a possible mechanism of acquired resistance to selective PI3Kα inhibition, we generated doxycycline-inducible PTEN shRNA stable clones starting from three different BYL719-sensitive cell lines. In all the studied models, induction of PTEN shRNA resulted in resistance to BYL719. Since PTEN deficient genetic models have been shown to rely on the β subunit of the PI3K holoenzyme, we tested whether the concomitant inhibition of both p110α and p110β was sufficient to revert the resistant phenotype. BKM120 (a pan-PI3K inhibitor) or the addition of AZD6482 (p110β inhibitor) to BYL719 re-sensitized the cells to BYL719. To expand our findings in vivo, we generated a patient-derived xenograft (PDX) model from a PTEN-null non-responding lesion (lung). Consistently, this PDX model was refractory to the antitumor activity of BYL719 but conserved sensitivity to BKM120 or the combination of AZD6482 and BYL719. In both cases, IHC analysis revealed a decrease in PI3K/AKT downstream effectors pPRAS40 (246) and pS6 (240/4) staining with BKM120 or AZD6482+BYL719, but not with BYL719 alone. Preliminary analyses of other samples collected from patients treated with BYL719 suggest that PTEN loss is a relatively frequent event upon therapy progression. Taken together, the different mechanisms that inactivate PTEN in the tumor treated with BYL719 can be explained by convergent phenotypic evolution in a heterogeneous tumor and highlight the importance of PTEN and PI3Kβ in acquired resistance to PI3Kα inhibitors. Citation Format: Pau Castel, Dejan Juric, Helen Won, Benjamin Ainscough, Haley Ellis, Saya Ebbesen, Malachi Griffith, Obi Griffith, Iyer Gopakumar, Dennis Sgroi, Steven Isakoff, Elaine Mardis, David Solit, Scott Lowe, Cornelia Quadt, Malte Peters, Michael Berger, Maurizio Scaltriti, Jose Baselga. Loss of PTEN leads to clinical resistance to the PI3Kα inhibitor BYL719 and provides evidence of convergent evolution under selective therapeutic pressure. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-327. doi:10.1158/1538-7445.AM2014-LB-327

Abstract NG04: Diversity of circulating tumor cells in a mouse pancreatic cancer model identified by single cell RNA sequencing

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal adult malignancy due to the propensity of this disease to metastasize. Circulating tumor cells (CTCs) are thought to be enriched for cells with metastatic potential and their characterization offers a means to understand the biological underpinnings of the distal spread of cancer. However, CTCs are rare cells in the blood and their isolation has posed a signifcant technological challenge. Multiple platforms have emerged in capturing these cells with most relying on a positive antibody based selection process (e.g. EpCAM). Our initial molecular characterization of pancreatic CTCs in the genetically engineered LSL-KrasG12D, Trp53flox/flox or +, Pdx1-Cre (KPC) mouse model utilized a microfluidic anti-EpCAM device followed by RNA sequencing to identify aberrant non-canonical WNT signaling in CTC populations (Yu M*, Ting DT*, et al. Nature 2012). Wnt2 was found to increase metastatic potential by enhancing anoikis resistance, a requirement of CTC survival, through activation of Tak1 kinase. However, this study was limited in only providing a partial CTC signature given the variable purity of these purified cell populations and analysis of bulk CTC populations could not provide the resolution to truly assess heterogeneity of these cells. Furthermore, a recent study has shown that cells from the mouse model may disseminate into circulation at an early point in PDAC development through epithelial-to-mesenchymal transition (EMT), which would generate CTCs with very low EpCAM expression and therefore would be missed in an EpCAM based capture device (Rhim AD et al. Cell 2012). We have overcome these barriers by employing a novel microfluidic isolation device to capture high numbers of CTCs that can be isolated as single cells (Ozkumur E*, Shah AM*, et al. Science Translational Medicine 2013). This device achieves high efficiency negative depletion of normal blood cells providing an enriched population of CTCs in solution that are not biased by a particular extracellular epitope and without any antibody interactions that could affect expression profiles. This has provided a method to truly understand the key transcriptional programs that differentiate CTCs from their primary tumors on an isogenic mouse background. Five tumor-bearing KPC mice generated a total of 168 single CTCs that were morphologically intact and subjected to a modified single cell RNA sequencing protocol (Tang F et al., Nature Protocols 2010). A total of 75 (45%) of these single CTCs were of sufficient quality for RNA sequencing indicating that the majority (55%) of intact CTCs selected likely had lost viability in the process of hematogenous transit. Single cell RNA-sequencing was also performed on 12 normal leukocytes (WBCs) from a control mouse, 12 mouse embryonic fibroblasts (MEFs), 16 single cells from the mouse NB508 pancreatic cancer cell line, and 34 (min 8 replicates) samples from primary tumors matched to the CTCs. Unsupervised hierarchical clustering of single cell samples demonstrated clear separation of MEFs, the NB508 pancreatic cancer cell line, and normal WBCs supporting the technical validity of the sequencing approach. Analysis of candidate CTCs identified three major CTC clusters, which were all distinct form matched primary tumors as well as from the NB508 cancer cell line. The must abundant CTC cluster comprised 41 of 75 cells (55%) and was defined by presence of epithelial markers (Krt7, Krt8, Krt18, Krt19) consistent with a “classical” CTC phenotype (CTC-c). The second CTC cluster was defined by enrichment of platelet markers CD41 (Itga2b) and CD61 (Itgb3) (CTC-plt) and a third having enrichment of cellular proliferation genes including Mki67 (CTC-pro). Single cell heterogeneity was assessed by intra-cluster correlation coeffficients, where lower values reflect higher heterogeneity. Not surprisingly, single cell heterogeneity was much higher in CTCs (mean correlation coefficient 0.42, 95% CI 0.36-0.47) compared to cancer cell lines (mean 0.86, 95% CI 0.80-0.91, p-value 1.2 x 10-15), but was notably similar comparing CTCs to single primary tumor cells (mean 0.38, 95% CI 0.28-0.47). Focusing on the dominant CTC-c cells, we used a non-parametric differential gene expression analysis including a rank product (RP) methodology suitable for large variations in absolute transcript levels found in single cell expression data (Breitling R et al. FEBS Letters 2004). Using a stringent FDR of ≤ 0.01, CTC-c cells had 878 genes with increased expression and 774 genes with reduced expression when compared with matched primary tumors. CTC-c cells were enriched for MAPK, as well as WNT, TGF-β, Neurotrophin, Toll-like receptor, and B-cell receptor signaling pathways. Analysis of a panel of EMT genes with significant differential expression revealed that CTC-c cells were in a biphenotypic state with universal loss of the epithelial markers E-cadherin (Cdh1) and Muc1, while the mesenchymal genes Cdh11 and Vim were found to be expressed much more heterogeneously amongst individual CTCs. Proposed pancreatic cancer stem cell genes were also evaluated and the Aldh1a1 and Aldh1a2 genes were found to be significantly enriched in CTC-c. Expression of both Aldh1a1 and Aldh1a2 in matched primary tumors was done through RNA in situ hybridization (RNA-ISH) revealing a heterogeneous distribution of these stem cell genes in both the stromal and epithelial compartments of the tumor. This highlighted the potential relevance of these stem cell markers in tumor cells dynamically shifting between epithelial and non-epithelial states. To provide further insight into the potential region from which CTCs emanate from the primary tumor, we selected the most highly enriched CTC transcripts found in ≥ 90% of all classical CTCs. Three genes met these criteria, which were decorin (Dcn), insulin-like growth factor binding protein 5 (Igfbp5), and Kruppel-like factor 4 (Klf4). Each of these genes has been previously implicated in pancreatic cancer development and were evaluated by RNA-ISH in primary tumor specimens to determine if they colocalized in particular tumor cells. Dcn is an extracellular matrix proteoglycan known to be expressed in a wide range of tumor stroma and by RNA-ISH was found primarily in the stromal elements of the tumor. However, both Igfbp5 and Klf4 were found to be focally expressed in cells at the epithelial-stromal interface. Although these genes are co-expressed in a minority of primary tumor cells, they are co-expressed at high levels in 85% of all classical CTCs. Together with the mixed epithelial/mesenchymal marks and enrichment of Aldh1a2 cells in stromal elements, these data point to the majority of viable CTCs emanating from the epithelial/stromal interface. In summary, we have successfully purified individual pancreatic CTCs using a novel microfluidic device and provided the first comprehensive single cell transcriptome analysis of these rare but exceptional cells. Three major classes of CTCs have been identified that would not have been possible without a single cell approach and we have characterized the major pathways that define these different subsets. The most abundant CTCs were found to have robust expression of keratin genes and are defined by a mixed E/M state with enrichment of Aldh1a1 and Aldh1a2 stem cell genes. These classical CTCs are marked by the co-expression of Igfbp5 and Klf4, which appear to localize to the epithelial-stromal interface in primary tumors. Ultimately, CTC cultures and functional testing will determine the contribution of these genes to CTC metastatic potential. This deep analysis of CTCs at single cell resolution has provided new biological insight into the metastatic cascade that will inform the development of novel therapies to treat this deadly disease. Citation Format: David T. Ting, Ben S. Wittner, Ajay M. Shah, David T. Miyamoto, Brian W. Brannigan, Kristina Xega, Jordan Ciciliano, Olivia C. MacKenzie, Julie Trautwein, Mohammad Shahid, Haley L. Ellis, Na Qu, Nabeel Bardeesy, Miguel N. Rivera, Ravi Kapur, Sridhar Ramaswamy, Toshi Shioda, Mehmet Toner, Shyamala Maheswaran, Daniel A. Haber. Diversity of circulating tumor cells in a mouse pancreatic cancer model identified by single cell RNA sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr NG04. doi:10.1158/1538-7445.AM2014-NG04