Hear Pancreatic Cancer Stem Cells ROR

In this issue of Cell, Lytle et al. (2019) integrate functional genomic approaches to identify molecular dependencies of pancreatic cancer stem cells that may be exploited therapeutically. The comprehensive analysis reveals an unexpected role for retinoic acid receptor-related orphan receptor gamma (RORγ), a T-cell-associated transcription factor, in defining the stemness and the aggressive behavior of pancreatic cancer. In this issue of Cell, Lytle et al. (2019) integrate functional genomic approaches to identify molecular dependencies of pancreatic cancer stem cells that may be exploited therapeutically. The comprehensive analysis reveals an unexpected role for retinoic acid receptor-related orphan receptor gamma (RORγ), a T-cell-associated transcription factor, in defining the stemness and the aggressive behavior of pancreatic cancer. Cancer stem cells (CSCs) are characterized by a sustained capacity for self-renewal and an inherent phenotypic plasticity for differentiation into heterogeneous lineages of that replenish the non-CSC compartment of tumors. While representing only a minor population of quiescent tumor cells, these potent properties explain many facets of the natural history of cancers, including disease recurrence, metastatic progression, and acquired drug resistance. Indeed, in preclinical models, CSCs exhibit enhanced spheroid formation and augmented tumor-initiating potential. Since their initial isolation (Li et al., 2007Li C. Heidt D.G. Dalerba P. Burant C.F. Zhang L. Adsay V. Wicha M. Clarke M.F. Simeone D.M. Identification of pancreatic cancer stem cells.Cancer Res. 2007; 67: 1030-1037Crossref PubMed Scopus (2753) Google Scholar), a multitude of markers have been used to define CSC-like cells in pancreatic ductal adenocarcinoma (PDAC). For example, studies have suggested that a rare population of Doublecortin-like kinase-1 (Dclk1)-positive cells, which promote injury-induced regeneration, delineate a putative CSC compartment facilitating tumor initiation in the pancreas (Bailey et al., 2014Bailey J.M. Alsina J. Rasheed Z.A. McAllister F.M. Fu Y.Y. Plentz R. Zhang H. Pasricha P.J. Bardeesy N. Matsui W. et al.DCLK1 marks a morphologically distinct subpopulation of cells with stem cell properties in preinvasive pancreatic cancer.Gastroenterology. 2014; 146: 245-256Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, Westphalen et al., 2016Westphalen C.B. Takemoto Y. Tanaka T. Macchini M. Jiang Z. Renz B.W. Chen X. Ormanns S. Nagar K. Tailor Y. et al.Dclk1 Defines Quiescent Pancreatic Progenitors that Promote Injury-Induced Regeneration and Tumorigenesis.Cell Stem Cell. 2016; 18: 441-455Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). More recently, a Musashi (Msi)-positive subpopulation has been identified in human and mouse PDAC, which exhibit CSC properties in a preclinical setting (Fox et al., 2016Fox R.G. Lytle N.K. Jaquish D.V. Park F.D. Ito T. Bajaj J. Koechlein C.S. Zimdahl B. Yano M. Kopp J. et al.Image-based detection and targeting of therapy resistance in pancreatic adenocarcinoma.Nature. 2016; 534: 407-411Crossref PubMed Scopus (94) Google Scholar). While the overlap between these various marker-defined populations in PDAC remains to be defined, cumulatively, the evidence strongly supports the notion that CSCs play a role in tumor initiation and maintenance and likely contribute to the near ubiquitous treatment resistance observed in this disease. Therefore, understanding the key functional effectors within CSCs also offers a facile opportunity for treating PDAC, a recalcitrant cancer in dire need of therapeutic advances. While the Msi proteins MSI1 and MSI2 are well established as regulators of neural and hemopoietic stem cells, they are also overexpressed in a variety of solid tumors and implicated in the maintenance of CSCs. Building upon their previous characterization of Msi+ PDAC CSCs (Fox et al., 2016Fox R.G. Lytle N.K. Jaquish D.V. Park F.D. Ito T. Bajaj J. Koechlein C.S. Zimdahl B. Yano M. Kopp J. et al.Image-based detection and targeting of therapy resistance in pancreatic adenocarcinoma.Nature. 2016; 534: 407-411Crossref PubMed Scopus (94) Google Scholar), in this issue of Cell, Lytle et al., 2019Lytle N.K. Ferguson L.P. Rajbhandari N. Gilroy K. Fox R.G. Deshpande A. Schürch C.M. Hamilton M. Robertson N. Lin W. et al.A multiscale map of the stem cell state in pancreatic adenocarcinoma.Cell. 2019; 177 (this issue): 572-586Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar comprehensively map the transcriptional, epigenetic, and functional genomic landscape of the Msi2+ CSC population (Figure 1A), generating a unique toolbox for interpreting therapeutic resistance and discovering new vulnerability nodes in PDAC (Lytle et al., 2019Lytle N.K. Ferguson L.P. Rajbhandari N. Gilroy K. Fox R.G. Deshpande A. Schürch C.M. Hamilton M. Robertson N. Lin W. et al.A multiscale map of the stem cell state in pancreatic adenocarcinoma.Cell. 2019; 177 (this issue): 572-586Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In comparison to Msi2 negative tumor cells, Msi2-expressing cells derived from PDAC autochthonous models harbor not only the developmental/stem cell and cellular quiescence signatures anticipated within this compartment, but also the transcriptional signatures observed in treatment refractory or relapsed cancers, highlighting the correlation of PDAC CSCs with adverse outcomes. It is noteworthy that such transcriptional programs are largely driven by a unique super enhancer (SE) landscape that distinguishes the PDAC CSC population from the remaining subsets, in accordance with the fact that SEs are central to the maintenance of CSC identity. Importantly, the genome-wide CRISPR screen indicates that the SE-associated transcripts unique to CSCs demonstrate a significant correlation with functional dependencies within said CSC. These findings have important therapeutic implications, as agents blocking co-activators critical for enabling SE-associated transcription, such as bromodomain-containing protein 4 (BRD4), are already in clinical trials. In fact, a BRD4 inhibitor used in combination with another epigenetic agent has previously demonstrated robust anti-tumor effect in PDAC preclinical models (Mazur et al., 2015Mazur P.K. Herner A. Mello S.S. Wirth M. Hausmann S. Sánchez-Rivera F.J. Lofgren S.M. Kuschma T. Hahn S.A. Vangala D. et al.Combined inhibition of BET family proteins and histone deacetylases as a potential epigenetics-based therapy for pancreatic ductal adenocarcinoma.Nat. Med. 2015; 21: 1163-1171Crossref PubMed Scopus (281) Google Scholar), providing additional rationale to broadly target the SE landscape in PDAC CSCs. The genome-wide CRISPR screen also elucidates several well-established as well as novel transcripts or signaling pathways whose sustained expression appears to be required for PDAC CSC survival (Figure 1A). The latter includes a “treasure trove” of potential targets—such as genes encoding for G-protein coupled receptors (GPCRs) or metabolic proteins, which have only undergone perfunctory orthogonal validation in this study—and will form the basis for future experiments. One of the more unexpected transcriptional programs discovered within PDAC CSCs is comprised of immune-regulatory transcripts, including the respective canonical receptors for two cytokine ligands, interleukin IL-10 and colony stimulating factor-1 (CSF-1), which are typically expressed by immune cells within the tumor microenvironment (TME) but instead appear to be “hijacked” by the neoplastic epithelium. As a corollary, genetic knockdown of either IL10Rβ or CSF1R block sphere formation in vitro and tumor growth in vivo, underscoring a novel paradigm of co-option of inflammatory ligand-receptor networks within the PDAC CSC compartment. Interestingly, CSF1R is overexpressed in tumor-associated macrophages in PDAC, and a CSF1R small molecule antagonist has shown promising anti-tumor effect in PDAC preclinical models (Candido et al., 2018Candido J.B. Morton J.P. Bailey P. Campbell A.D. Karim S.A. Jamieson T. Lapienyte L. Gopinathan A. Clark W. McGhee E.J. et al.CSF1R+ Macrophages Sustain Pancreatic Tumor Growth through T Cell Suppression and Maintenance of Key Gene Programs that Define the Squamous Subtype.Cell Rep. 2018; 23: 1448-1460Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Therefore, the finding that these immune-related networks also function in a cell-autonomous manner in CSCs raises the possibility that therapies aimed at overcoming the prevalent immune suppression within the PDAC TME have the potential to also impact tumor cell propagation through “on-target” effects. Along these lines, another unexpected discovery in this study is the identification of retinoic-acid-related orphan receptor gamma (RORγ) as a key regulatory element in PDAC CSCs (Figure 1B). Although the established function of RORγ is to orchestrate the differentiation of Th17 T cells, here, Lytle et al., 2019Lytle N.K. Ferguson L.P. Rajbhandari N. Gilroy K. Fox R.G. Deshpande A. Schürch C.M. Hamilton M. Robertson N. Lin W. et al.A multiscale map of the stem cell state in pancreatic adenocarcinoma.Cell. 2019; 177 (this issue): 572-586Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar provide robust molecular and functional evidence for sustained RORγ activity as a requirement in PDAC CSCs. RORγ binding is preferentially enriched within PDAC CSC-associated SEs compared to the non-CSC compartment, implying that RORγ governs CSC identity as an upstream regulator of a SE-linked transcriptional network. Further, genetic or pharmacological blockade of RORγ function inhibits tumor growth and propagation in preclinical models of both murine and human PDAC, including direct visualization of the depletion of Msi2+ fluorescent CSC by intravital imaging in treated autochthonous mice with late-stage disease. The finding with greatest translational import pertains to the combination therapy experiments performed using the RORγ inverse agonist SR2211, which demonstrate the ability of this agent to overcome resistance to a commonly used cytotoxic therapy (gemcitabine) offered to PDAC patients in the clinic. Although the impact of targeting RORγ is mainly attributed to its cell-autonomous requirement in CSCs, and not to its purported role in the differentiation of pro-tumorigenic Th17 T cells, one of the canonical receptors for IL-17 (IL-17RC) is, in fact, also expressed within the CSC compartment of PDAC and its precursor lesions (McAllister et al., 2014McAllister F. Bailey J.M. Alsina J. Nirschl C.J. Sharma R. Fan H. Rattigan Y. Roeser J.C. Lankapalli R.H. Zhang H. et al.Oncogenic Kras activates a hematopoietic-to-epithelial IL-17 signaling axis in preinvasive pancreatic neoplasia.Cancer Cell. 2014; 25: 621-637Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, Zhang et al., 2018Zhang Y. Zoltan M. Riquelme E. Xu H. Sahin I. Castro-Pando S. Montiel M.F. Chang K. Jiang Z. Ling J. et al.Immune Cell Production of Interleukin 17 Induces Stem Cell Features of Pancreatic Intraepithelial Neoplasia Cells.Gastroenterology. 2018; 155: 210-223 e213Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), highlighting yet another example of promiscuity in ligand-receptor networks previously considered to be restricted to the immune compartment. Current standard-of-care regimens for PDAC using cytotoxic agents are largely geared toward “tumor debulking” rather than eradication of CSCs. These agents, while prolonging survival, have rarely resulted in long-term disease remission even in the subset of PDAC patients that have undergone apparently curative surgery. Of note, residual PDAC cells that survive cytotoxic therapy, and thus represent the most probable source of post-treatment disease progression, harbor numerous phenotypic and functional similarities with CSCs (Viale and Draetta, 2016Viale A. Draetta G.F. Metabolic Features of Cancer Treatment Resistance.Recent Results Cancer Res. 2016; 207: 135-156Crossref PubMed Scopus (24) Google Scholar). Therefore, envisioning the possibility of a “cure” will likely require combining cytotoxic regimens with agents capable of depleting PDAC CSCs while sparing tissue-resident stem cells. The work by Lytle et al., 2019Lytle N.K. Ferguson L.P. Rajbhandari N. Gilroy K. Fox R.G. Deshpande A. Schürch C.M. Hamilton M. Robertson N. Lin W. et al.A multiscale map of the stem cell state in pancreatic adenocarcinoma.Cell. 2019; 177 (this issue): 572-586Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar identifies a multitude of such actionable targets that harbor the potential for reduction to practice. Moreover, by leveraging common regulatory networks that exist within CSCs and the immune microenvironment (RORγ being the pivotal example of such a shared mechanism), one can envision an “all-hands-on-deck” therapeutic approach in PDAC that might overcome the pervasive treatment resistance observed in this disease. The authors declare no competing interests. A Multiscale Map of the Stem Cell State in Pancreatic AdenocarcinomaLytle et al.CellApril 4, 2019In BriefPancreatic cancer stem cells co-opt immuno-regulatory pathways, a vulnerability that could be exploited therapeutically by agents currently in trials for autoimmune diseases. Full-Text PDF Open Archive