Waging war against pancreatic cancer: an interview with David Tuveson

David Tuveson, Director of the Cancer Center at Cold Spring Harbor Laboratory, is a clinician-scientist with a longstanding interest in understanding and treating pancreatic cancer. Since developing the first mouse model of pancreatic cancer in 2002, the Tuveson lab has made a series of discoveries that shed light on the molecular drivers of this disease and provide promising therapeutic avenues for a malignancy that is notoriously challenging to treat. In collaboration with Hans Clevers, David developed the first pancreatic cancer organoids, which revolutionized the field by providing a powerful model system for basic discoveries and advancement of personalized medicine. Here, David talks to Ross Cagan about his path from chemistry student to world-renowned oncologist, highlighting how his colleagues, mentors and patient interactions shaped his research interests and unique approach to scientific discovery. As well as discussing the story behind some of his breakthroughs, he provides tips on running a lab and succeeding in or outside academia

Emerging concepts in pancreatic cancer medicine: targeting the tumor stroma

Pancreatic ductal adenocarcinoma is a stroma-rich and highly challenging cancer to treat. Over recent years, it has become increasingly evident that the complex network of soluble cytokines, growth factors, proteases, and components of the extracellular matrix collaboratively interact within the tumor microenvironment, sustaining and driving cancer cell proliferation, invasion, and early metastasis. More recently, the tumor microenvironment has also been appreciated to mediate therapeutic resistance in pancreatic ductal adenocarcinoma, thus opening numerous avenues for novel therapeutic explorations. Inert and soluble components of the tumor stroma have been targeted in order to break down the extracellular matrix scaffold, relieve vessel compression, and increase drug delivery to hypovascular tumors. Moreover, targeting of antiapoptotic, immunosuppressive, and pro-proliferative effects of the tumor stroma provides novel vantage points of attack. This review focuses on current and future developments in pancreatic cancer medicine, with a particular emphasis on biophysical and biochemical approaches that target the tumor microenvironment

SCRIB expression is deregulated in human prostate cancer, and its deficiency in mice promotes prostate neoplasia

Loss of cellular polarity is a hallmark of epithelial cancers, raising the possibility that regulators of polarity have a role in suppressing tumorigenesis. The Scribble complex is one of at least three interacting protein complexes that have a critical role in establishing and maintaining epithelial polarity. In human colorectal, breast, and endometrial cancers, expression of the Scribble complex member SCRIB is often mislocalized and deregulated. Here, we report that Scrib is indispensable for prostate homeostasis in mice. Scrib heterozygosity initiated prostate hyperplasia, while targeted biallelic Scrib loss predisposed mice to prostate intraepithelial neoplasia. Mechanistically, Scrib was shown to negatively regulate the MAPK cascade to suppress tumorigenesis. Further analysis revealed that prostate-specific loss of Scrib in mice combined with expression of an oncogenic Kras mutation promoted the progression of prostate cancer that recapitulated the human disease. The clinical significance of the work in mice was highlighted by our observation that SCRIB deregulation strongly correlated with poor survival in human prostate cancer. These data suggest that the polarity network could provide a new avenue for therapeutic intervention

Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer

An autochthonous model of pancreatic ductal adenocarcinoma (PDA) permitted the analysis of why immunotherapy is ineffective in this human disease. Despite finding that PDA-bearing mice had cancer cell-specific CD8+ T cells, the mice, like human patients with PDA, did not respond to two immunological checkpoint antagonists that promote the function of T cells: anti-cytotoxic T-lymphocyte-associated protein 4 (α-CTLA-4) and α-programmed cell death 1 ligand 1 (α-PD-L1). Immune control of PDA growth was achieved, however, by depleting carcinoma-associated fibroblasts (CAFs) that express fibroblast activation protein (FAP). The depletion of the FAP+ stromal cell also uncovered the antitumor effects of α-CTLA-4 and α-PD-L1, indicating that its immune suppressive activity accounts for the failure of these T-cell checkpoint antagonists. Three findings suggested that chemokine (C-X-C motif) ligand 12 (CXCL12) explained the overriding immunosuppression by the FAP+ cell: T cells were absent from regions of the tumor containing cancer cells, cancer cells were coated with the chemokine, CXCL12, and the FAP+ CAF was the principal source of CXCL12 in the tumor. Administering AMD3100, a CXCL12 receptor chemokine (C-X-C motif) receptor 4 inhibitor, induced rapid T-cell accumulation among cancer cells and acted synergistically with α-PD-L1 to greatly diminish cancer cells, which were identified by their loss of heterozygosity of Trp53 gene. The residual tumor was composed only of premalignant epithelial cells and in flammatory cells. Thus, a single protein, CXCL12, from a single stromal cell type, the FAP+ CAF, may direct tumor immune evasion in a model of human PDA

CTGF antagonism with mAb FG-3019 enhances chemotherapy response without increasing drug delivery in murine ductal pancreas cancer

Pancreatic ductal adenocarcinoma (PDA) is characterized by abundant desmoplasia and poor tissue perfusion. These features are proposed to limit the access of therapies to neoplastic cells and blunt treatment efficacy. Indeed, several agents that target the PDA tumor microenvironment promote concomitant chemotherapy delivery and increased antineoplastic response in murine models of PDA. Prior studies could not determine whether chemotherapy delivery or microenvironment modulation per se were the dominant features in treatment response, and such information could guide the optimal translation of these preclinical findings to patients. To distinguish between these possibilities, we used a chemical inhibitor of cytidine deaminase to stabilize and thereby artificially elevate gemcitabine levels in murine PDA tumors without disrupting the tumor microenvironment. Additionally, we used the FG-3019 monoclonal antibody (mAb) that is directed against the pleiotropic matricellular signaling protein connective tissue growth factor (CTGF/CCN2). Inhibition of cytidine deaminase raised the levels of activated gemcitabine within PDA tumors without stimulating neoplastic cell killing or decreasing the growth of tumors, whereas FG-3019 increased PDA cell killing and led to a dramatic tumor response without altering gemcitabine delivery. The response to FG-3019 correlated with the decreased expression of a previously described promoter of PDA chemotherapy resistance, the X-linked inhibitor of apoptosis protein. Therefore, alterations in survival cues following targeting of tumor microenvironmental factors may play an important role in treatment responses in animal models, and by extension in PDA patients

CD20 and CD19 targeted vectors induce minimal activation of resting B lymphocytes

B lymphocytes are an important cell population of the immune system. However, until recently it was not possible to transduce resting B lymphocytes with retro- or lentiviral vectors, making them unsusceptible for genetic manipulations by these vectors. Lately, we demonstrated that lentiviral vectors pseudotyped with modified measles virus (MV) glycoproteins hemagglutinin, responsible for receptor recognition, and fusion protein were able to overcome this transduction block. They use either the natural MV receptors, CD46 and signaling lymphocyte activation molecule (SLAM), for cell entry (MV-LV) or the vector particles were further modified to selectively enter via the CD20 molecule, which is exclusively expressed on B lymphocytes (CD20-LV). It has been shown previously that transduction by MV-LV does not induce B lymphocyte activation. However, if this is also true for CD20-LV is still unknown. Here, we generated a vector specific for another B lymphocyte marker, CD19, and compared its ability to transduce resting B lymphocytes with CD20-LV. The vector (CD19ds-LV) was able to stably transduce unstimulated B lymphocytes, albeit with a reduced efficiency of about 10% compared to CD20-LV, which transduced about 30% of the cells. Since CD20 as well as CD19 are closely linked to the B lymphocyte activation pathway, we investigated if engagement of CD20 or CD19 molecules by the vector particles induces activating stimuli in resting B lymphocytes. Although, activation of B lymphocytes often involves calcium influx, we did not detect elevated calcium levels. However, the activation marker CD71 was substantially up-regulated upon CD20-LV transduction and most importantly, B lymphocytes transduced with CD20-LV or CD19ds-LV entered the G1b phase of cell cycle, whereas untransduced or MV-LV transduced B lymphocytes remained in G0. Hence, CD20 and CD19 targeting vectors induce activating stimuli in resting B lymphocytes, which most likely renders them susceptible for lentiviral vector transduction

TLR9 ligation in pancreatic stellate cells promotes tumorigenesis

Modulation of Toll-like receptor (TLR) signaling can have protective or protumorigenic effects on oncogenesis depending on the cancer subtype and on specific inflammatory elements within the tumor milieu. We found that TLR9 is widely expressed early during the course of pancreatic transformation and that TLR9 ligands are ubiquitous within the tumor microenvironment. TLR9 ligation markedly accelerates oncogenesis, whereas TLR9 deletion is protective. We show that TLR9 activation has distinct effects on the epithelial, inflammatory, and fibrogenic cellular subsets in pancreatic carcinoma and plays a central role in cross talk between these compartments. Specifically, TLR9 activation can induce proinflammatory signaling in transformed epithelial cells, but does not elicit oncogene expression or cancer cell proliferation. Conversely, TLR9 ligation induces pancreatic stellate cells (PSCs) to become fibrogenic and secrete chemokines that promote epithelial cell proliferation. TLR9-activated PSCs mediate their protumorigenic effects on the epithelial compartment via CCL11. Additionally, TLR9 has immune-suppressive effects in the tumor microenvironment (TME) via induction of regulatory T cell recruitment and myeloid-derived suppressor cell proliferation. Collectively, our work shows that TLR9 has protumorigenic effects in pancreatic carcinoma which are distinct from its influence in extrapancreatic malignancies and from the mechanistic effects of other TLRs on pancreatic oncogenesis

Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer

Pancreatic stellate cells (PSCs) differentiate into cancer-associated fibroblasts (CAFs) that produce desmoplastic stroma, thereby modulating disease progression and therapeutic response in pancreatic ductal adenocarcinoma (PDA). However, it is unknown whether CAFs uniformly carry out these tasks or if subtypes of CAFs with distinct phenotypes in PDA exist. We identified a CAF subpopulation with elevated expression of alpha-smooth muscle actin (alphaSMA) located immediately adjacent to neoplastic cells in mouse and human PDA tissue. We recapitulated this finding in co-cultures of murine PSCs and PDA organoids, and demonstrated that organoid-activated CAFs produced desmoplastic stroma. The co-cultures showed cooperative interactions and revealed another distinct subpopulation of CAFs, located more distantly from neoplastic cells, which lacked elevated alphaSMA expression and instead secreted IL6 and additional inflammatory mediators. These findings were corroborated in mouse and human PDA tissue, providing direct evidence for CAF heterogeneity in PDA tumor biology with implications for disease etiology and therapeutic development

Suppression of Lung Adenocarcinoma Progression by Nkx2-1

Despite the high prevalence and poor outcome of patients with metastatic lung cancer the mechanisms of tumour progression and metastasis remain largely uncharacterized. Here we modelled human lung adenocarcinoma, which frequently harbours activating point mutations in KRAS and inactivation of the p53 pathway, using conditional alleles in mice. Lentiviral-mediated somatic activation of oncogenic Kras and deletion of p53 in the lung epithelial cells of Kras[superscript LSL-G12D/+];p53[superscript flox/flox] mice initiates lung adenocarcinoma development4. Although tumours are initiated synchronously by defined genetic alterations, only a subset becomes malignant, indicating that disease progression requires additional alterations. Identification of the lentiviral integration sites allowed us to distinguish metastatic from non-metastatic tumours and determine the gene expression alterations that distinguish these tumour types. Cross-species analysis identified the NK2-related homeobox transcription factor Nkx2-1 (also called Ttf-1 or Titf1) as a candidate suppressor of malignant progression. In this mouse model, Nkx2-1 negativity is pathognomonic of high-grade poorly differentiated tumours. Gain- and loss-of-function experiments in cells derived from metastatic and non-metastatic tumours demonstrated that Nkx2-1 controls tumour differentiation and limitsmetastatic potential in vivo. Interrogation of Nkx2-1-regulated genes, analysis of tumours at defined developmental stages, and functional complementation experiments indicate that Nkx2-1 constrains tumours in part by repressing the embryonically restricted chromatin regulator Hmga2. Whereas focal amplification of NKX2-1 in a fraction of human lung adenocarcinomas has focused attention on its oncogenic function, our data specifically link Nkx2-1 downregulation to loss of differentiation, enhanced tumour seeding ability and increased metastatic proclivity. Thus, the oncogenic and suppressive functions ofNkx2-1 in the sametumourNational Institutes of Health (U.S.) (grant U01-CA84306 )National Institutes of Health (U.S.) (grant K99-CA151968)Howard Hughes Medical InstituteLudwig Center for Molecular OncologyNational Cancer Institute (U.S.) (Cancer Center Support (core) grant P30-CA14051

Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities.

The RAS/MAPK (mitogen-activated protein kinase) signalling pathway is frequently deregulated in non-small-cell lung cancer, often through KRAS activating mutations. A single endogenous mutant Kras allele is sufficient to promote lung tumour formation in mice but malignant progression requires additional genetic alterations. We recently showed that advanced lung tumours from Kras(G12D/+);p53-null mice frequently exhibit Kras(G12D) allelic enrichment (Kras(G12D)/Kras(wild-type) > 1) (ref. 7), implying that mutant Kras copy gains are positively selected during progression. Here we show, through a comprehensive analysis of mutant Kras homozygous and heterozygous mouse embryonic fibroblasts and lung cancer cells, that these genotypes are phenotypically distinct. In particular, Kras(G12D/G12D) cells exhibit a glycolytic switch coupled to increased channelling of glucose-derived metabolites into the tricarboxylic acid cycle and glutathione biosynthesis, resulting in enhanced glutathione-mediated detoxification. This metabolic rewiring is recapitulated in mutant KRAS homozygous non-small-cell lung cancer cells and in vivo, in spontaneous advanced murine lung tumours (which display a high frequency of Kras(G12D) copy gain), but not in the corresponding early tumours (Kras(G12D) heterozygous). Finally, we demonstrate that mutant Kras copy gain creates unique metabolic dependences that can be exploited to selectively target these aggressive mutant Kras tumours. Our data demonstrate that mutant Kras lung tumours are not a single disease but rather a heterogeneous group comprising two classes of tumours with distinct metabolic profiles, prognosis and therapeutic susceptibility, which can be discriminated on the basis of their relative mutant allelic content. We also provide the first, to our knowledge, in vivo evidence of metabolic rewiring during lung cancer malignant progression.We thank T. Jacks (Kras^LSL-G12D), A. Berns (p53^Fx) and the NIH Mouse repository for mice. We also thank Sam Kleeman and Patricia Ogger for assistance with redox cell profiling and cell viability assays, respectively. We are very thankful to CRUK CI BRU staff for support with in vivo work and all the members of the Martins lab for critical comments and advice. This work was supported by the Medical Research Council.This is the author accepted manuscript. The final version is available at http://www.nature.com/nature/journal/v531/n7592/full/nature16967.html