Cecile de la Cruz

A Rare Population of CD24+ITGB4+Notchhi Cells Drives Tumor Propagation in NSCLC and Requires Notch3 for Self-Renewal

Sustained tumor progression has been attributed to a distinct population of tumor-propagating cells (TPCs). To identify TPCs relevant to lung cancer pathogenesis, we investigated functional heterogeneity in tumor cells isolated from Kras-driven mouse models of non-small-cell lung cancer (NSCLC). CD24(+)ITGB4(+)Notch(hi) cells are capable of propagating tumor growth in both a clonogenic and an orthotopic serial transplantation assay. While all four Notch receptors mark TPCs, Notch3 plays a nonredundant role in tumor cell propagation in two mouse models and in human NSCLC. The TPC population is enriched after chemotherapy, and the gene signature of mouse TPCs correlates with poor prognosis in human NSCLC. The role of Notch3 in tumor propagation may provide a therapeutic target for NSCLC.

Blocking NRG1 and Other Ligand-Mediated Her4 Signaling Enhances the Magnitude and Duration of the Chemotherapeutic Response of Non–Small Cell Lung Cancer

Inhibition of Her4 signaling enhances the response to chemotherapy and delays tumor regrowth after cessation of treatment. Regaining the Yellow Jersey Professional sports—from cycling to football and even baseball—are now cracking down on doping. The use of performance-enhancing drugs is thought to give an unfair advantage, and regulatory agencies are trying to return everyone to even ground. But whereas athletes who dope become pariahs, in some fights it’s better not to play fair. Now, Hegde et al. suggest a way to enhance chemotherapy in the fight against non–small cell lung cancer (NSCLC). Chemotherapy is a first-line treatment for NSCLC but, in some cases, cannot either adequately remove the tumor or prevent recurrence. The authors use multiple models of NSCLC and find that residual tumor cells after chemotherapy express high levels of neuregulin 1 (NRG1), which is a ligand for human epidermal growth factor receptor 3 and 4 (HER3/4). Inhibited NRG1 signaling had only variable effects on primary tumor growth, but significantly enhanced the magnitude and duration of tumor response to chemotherapy. NRG1 inhibition in combination with chemotherapy greatly impeded relapse. Although this combination remains to be tested in the clinic, this study suggests that when it comes to a competition between NSCLC and chemotherapy, all’s fair. Although standard chemotherapies are commonly used to treat most types of solid tumors, such treatment often results in inadequate response to, or relapse after, therapy. This is particularly relevant for lung cancer because most patients are diagnosed with advanced-stage disease and are treated with frontline chemotherapy. By studying the residual tumor cells that remain after chemotherapy in several in vivo non–small cell lung cancer models, we found that these cells have increased levels of human epidermal growth factor receptor (HER) signaling due, in part, to the enrichment of a preexisting NRG1HI subpopulation. Neuregulin 1 (NRG1) signaling in these models can be mediated by either the HER3 or HER4 receptor, resulting in the differential activation of downstream effectors. Inhibition of NRG1 signaling inhibits primary tumor growth and enhances the magnitude and duration of the response to chemotherapy. Moreover, we show that inhibition of ligand-mediated Her4 signaling impedes disease relapse in cases where NRG1 inhibition is insufficient. These findings demonstrate that ligand-dependent Her4 signaling plays an important role in disease relapse.

Intratumoral Heterogeneity: From Diversity Comes Resistance

Tumors consist of a heterogeneous mixture of functionally distinct cancer cells. These functional differences can be caused by varying levels of receptor activity, differentiation, and distinct metabolic and epigenetic states. Intratumoral heterogeneity can lead to interdependence among different subpopulations of cells for sustained tumor growth. In addition, subpopulations can vary widely in their responses to therapeutic agents. As such, it is believed that intratumoral heterogeneity may underlie incomplete treatment responses, acquired and innate resistance, and disease relapse observed in the clinic in response to conventional chemotherapy and targeted agents. Clin Cancer Res; 21(13); 2916–23. ©2015 AACR.

Residual Tumor Cells That Drive Disease Relapse after Chemotherapy Do Not Have Enhanced Tumor Initiating Capacity

Although chemotherapy is used to treat most advanced solid tumors, recurrent disease is still the major cause of cancer-related mortality. Cancer stem cells (CSCs) have been the focus of intense research in recent years because they provide a possible explanation for disease relapse. However, the precise role of CSCs in recurrent disease remains poorly understood and surprisingly little attention has been focused on studying the cells responsible for re-initiating tumor growth within the original host after chemotherapy treatment. We utilized both xenograft and genetically engineered mouse models of non-small cell lung cancer (NSCLC) to characterize the residual tumor cells that survive chemotherapy treatment and go on to cause tumor regrowth, which we refer to as tumor re-initiating cells (TRICs). We set out to determine whether TRICs display characteristics of CSCs, and whether assays used to define CSCs also provide an accurate readout of a cell’s ability to cause tumor recurrence. We did not find consistent enrichment of CSC marker positive cells or enhanced tumor initiating potential in TRICs. However, TRICs from all models do appear to be in EMT, a state that has been linked to chemoresistance in numerous types of cancer. Thus, the standard CSC assays may not accurately reflect a cell’s ability to drive disease recurrence.

Therapeutic Targeting of the CBP/p300 Bromodomain Blocks the Growth of Castration-Resistant Prostate Cancer

Resistance invariably develops to antiandrogen therapies used to treat newly diagnosed prostate cancers, but effective treatments for castration-resistant disease remain elusive. Here, we report that the transcriptional coactivator CBP/p300 is required to maintain the growth of castration-resistant prostate cancer. To exploit this vulnerability, we developed a novel small-molecule inhibitor of the CBP/p300 bromodomain that blocks prostate cancer growth in vitro and in vivo Molecular dissection of the consequences of drug treatment revealed a critical role for CBP/p300 in histone acetylation required for the transcriptional activity of the androgen receptor and its target gene expression. Our findings offer a preclinical proof of concept for small-molecule therapies to target the CBP/p300 bromodomain as a strategy to treat castration-resistant prostate cancer. Cancer Res; 77(20); 5564-75. ©2017 AACR.

The Hippo pathway effector TAZ induces TEAD-dependent liver inflammation and tumors

Transcriptional cofactors YAP and TAZ have distinct roles in promoting proinflammatory cytokine production and tumorigenesis. TAZ drives inflammation Key effectors of the Hippo pathway, YAP and TAZ, are overexpressed in various cancers. Loss of upstream kinases that inhibit the activity of these transcriptional coactivators promotes inflammation. In patient-derived xenografts and TCGA data sets, Hagenbeek et al. found that only TAZ expression correlated strongly with inflammatory cytokine transcript abundance. Expression of hyperactivated TAZ, but not YAP, in the livers of mice augmented transcription factor TEAD-mediated systemic inflammation and tissue infiltration by myeloid cells. RNA-seq analysis identified distinct gene signatures in tumor cells driven by activated YAP or TAZ, suggesting that these Hippo pathway effectors have nonredundant functions. The Hippo signaling pathway regulates organ size and plays critical roles in maintaining tissue growth, homeostasis, and regeneration. Dysregulated in a wide spectrum of cancers, in mammals, this pathway is regulated by two key effectors, YAP and TAZ, that may functionally overlap. We found that TAZ promoted liver inflammation and tumor development. The expression of TAZ, but not YAP, in human liver tumors positively correlated with the expression of proinflammatory cytokines. Hyperactivated TAZ induced substantial myeloid cell infiltration into the liver and the secretion of proinflammatory cytokines through a TEAD-dependent mechanism. Furthermore, tumors with hyperactivated YAP and TAZ had distinct transcriptional signatures, which included the increased expression of inflammatory cytokines in TAZ-driven tumors. Our study elucidated a previously uncharacterized link between TAZ activity and inflammatory responses that influence tumor development in the liver.

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

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 266: Notch3 is a marker of tumor-propagating cells in non-small cell lung cancer and is required for their self-renewal.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC We investigated tumor cell heterogeneity in mouse models of non-small cell lung cancer (NSCLC) that recapitulate key genetic events in this disease. In tumors driven by simultaneous expression of an oncogenic Kras allele and loss of the tumor suppressor Trp53, only a relatively rare subset of tumor cells is capable of propagating tumor growth in both a clonogenic in vitro assay and an in vivo orthotopic transplantation assay. The tumor-propagating cell (TPC) population was enriched by sorting for the combination of the cell surface markers CD24, ITGB4 and Notch(1-4). We demonstrate a critical and specific role for Notch3 signaling in the maintenance of TPCs in the KrasG12D; Trp53fl/fl mouse model. Knock-down of Notch3, but not Notch 1, 2 or 4, decreased self-renewal of TPCs in vitro. In addition, knock-down of Notch3 was critical for tumor propagation in vivo. The relevance of these studies to human NSCLC was confirmed by demonstrating a significant effect of Notch inhibition on the self-renewal of primary human lung adenocarcinoma cells in ex vivo cultures established directly from patients. These findings identify a novel subpopulation of tumor cells with distinct functional capabilities, underscoring the importance of interrogating heterogeneity in NSCLC. The unique role of Notch3 in tumor propagation may provide a novel therapeutic target for the treatment of non-small cell lung cancer.   Citation Format: Yanyan Zheng, Cecile de la Cruz, Leanne Sayles, Chris Alleyne-Chin, Yue Xu, Chuong D. Hoang, Joseph B. Shrager, Erica Jackson, Alejandro Sweet-Cordero. Notch3 is a marker of tumor-propagating cells in non-small cell lung cancer and is required for their self-renewal. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 266. doi:10.1158/1538-7445.AM2013-266