Senescence-Induced Vascular Remodeling Creates Therapeutic Vulnerabilities in Pancreas Cancer.

KRAS mutant pancreatic ductal adenocarcinoma (PDAC) is characterized by a desmoplastic response that promotes hypovascularity, immunosuppression, and resistance to chemo- and immunotherapies. We show that a combination of MEK and CDK4/6 inhibitors that target KRAS-directed oncogenic signaling can suppress PDAC proliferation through induction of retinoblastoma (RB) protein-mediated senescence. In preclinical mouse models of PDAC, this senescence-inducing therapy produces a senescence-associated secretory phenotype (SASP) that includes pro-angiogenic factors that promote tumor vascularization, which in turn enhances drug delivery and efficacy of cytotoxic gemcitabine chemotherapy. In addition, SASP-mediated endothelial cell activation stimulates the accumulation of CD8+ T cells into otherwise immunologically "cold" tumors, sensitizing tumors to PD-1 checkpoint blockade. Therefore, in PDAC models, therapy-induced senescence can establish emergent susceptibilities to otherwise ineffective chemo- and immunotherapies through SASP-dependent effects on the tumor vasculature and immune system.

p53 Represses the Mevalonate Pathway to Mediate Tumor Suppression.

There are still gaps in our understanding of the complex processes by which p53 suppresses tumorigenesis. Here we describe a novel role for p53 in suppressing the mevalonate pathway, which is responsible for biosynthesis of cholesterol and nonsterol isoprenoids. p53 blocks activation of SREBP-2, the master transcriptional regulator of this pathway, by transcriptionally inducing the ABCA1 cholesterol transporter gene. A mouse model of liver cancer reveals that downregulation of mevalonate pathway gene expression by p53 occurs in premalignant hepatocytes, when p53 is needed to actively suppress tumorigenesis. Furthermore, pharmacological or RNAi inhibition of the mevalonate pathway restricts the development of murine hepatocellular carcinomas driven by p53 loss. Like p53 loss, ablation of ABCA1 promotes murine liver tumorigenesis and is associated with increased SREBP-2 maturation. Our findings demonstrate that repression of the mevalonate pathway is a crucial component of p53-mediated liver tumor suppression and outline the mechanism by which this occurs.

Integrating the Study of Polyploidy Across Organisms, Tissues, and Disease.

Polyploidy is a cellular state containing more than two complete chromosome sets. It has largely been studied as a discrete phenomenon in either organismal, tissue, or disease contexts. Increasingly, however, investigation of polyploidy across disciplines is coalescing around common principles. For example, the recent Polyploidy Across the Tree of Life meeting considered the contribution of polyploidy both in organismal evolution over millions of years and in tumorigenesis across much shorter timescales. Here, we build on this newfound integration with a unified discussion of polyploidy in organisms, cells, and disease. We highlight how common polyploidy is at multiple biological scales, thus eliminating the outdated mindset of its specialization. Additionally, we discuss rules that are likely common to all instances of polyploidy. With increasing appreciation that polyploidy is pervasive in nature and displays fascinating commonalities across diverse contexts, inquiry related to this important topic is rapidly becoming unified.

Mutant p53 drives pancreatic cancer metastasis through cell-autonomous PDGF receptor ß signaling.

Missense mutations in the p53 tumor suppressor inactivate its antiproliferative properties but can also promote metastasis through a gain-of-function activity. We show that sustained expression of mutant p53 is required to maintain the prometastatic phenotype of a murine model of pancreatic cancer, a highly metastatic disease that frequently displays p53 mutations. Transcriptional profiling and functional screening identified the platelet-derived growth factor receptor b (PDGFRb) as both necessary and sufficient to mediate these effects. Mutant p53 induced PDGFRb through a cell-autonomous mechanism involving inhibition of a p73/NF-Y complex that represses PDGFRb expression in p53-deficient, noninvasive cells. Blocking PDGFRb signaling by RNA interference or by small molecule inhibitors prevented pancreatic cancer cell invasion in vitro and metastasis formation in vivo. Finally, high PDGFRb expression correlates with poor disease-free survival in pancreatic, colon, and ovarian cancer patients, implicating PDGFRb as a prognostic marker and possible target for attenuating metastasis in p53 mutant tumors.

Ordered and deterministic cancer genome evolution after p53 loss.

Although p53 inactivation promotes genomic instability1 and presents a route to malignancy for more than half of all human cancers2,3, the patterns through which heterogenous TP53 (encoding human p53) mutant genomes emerge and influence tumorigenesis remain poorly understood. Here, in a mouse model of pancreatic ductal adenocarcinoma that reports sporadic p53 loss of heterozygosity before cancer onset, we find that malignant properties enabled by p53 inactivation are acquired through a predictable pattern of genome evolution. Single-cell sequencing and in situ genotyping of cells from the point of p53 inactivation through progression to frank cancer reveal that this deterministic behaviour involves four sequential phases-Trp53 (encoding mouse p53) loss of heterozygosity, accumulation of deletions, genome doubling, and the emergence of gains and amplifications-each associated with specific histological stages across the premalignant and malignant spectrum. Despite rampant heterogeneity, the deletion events that follow p53 inactivation target functionally relevant pathways that can shape genomic evolution and remain fixed as homogenous events in diverse malignant populations. Thus, loss of p53-the 'guardian of the genome'-is not merely a gateway to genetic chaos but, rather, can enable deterministic patterns of genome evolution that may point to new strategies for the treatment of TP53-mutant tumours.

A gene-environment-induced epigenetic program initiates tumorigenesis.

Tissue damage increases the risk of cancer through poorly understood mechanisms1. In mouse models of pancreatic cancer, pancreatitis associated with tissue injury collaborates with activating mutations in the Kras oncogene to markedly accelerate the formation of early neoplastic lesions and, ultimately, adenocarcinoma2,3. Here, by integrating genomics, single-cell chromatin assays and spatiotemporally controlled functional perturbations in autochthonous mouse models, we show that the combination of Kras mutation and tissue damage promotes a unique chromatin state in the pancreatic epithelium that distinguishes neoplastic transformation from normal regeneration and is selected for throughout malignant evolution. This cancer-associated epigenetic state emerges within 48 hours of pancreatic injury, and involves an 'acinar-to-neoplasia' chromatin switch that contributes to the early dysregulation of genes that define human pancreatic cancer. Among the factors that are most rapidly activated after tissue damage in the pre-malignant pancreatic epithelium is the alarmin cytokine interleukin 33, which recapitulates the effects of injury in cooperating with mutant Kras to unleash the epigenetic remodelling program of early neoplasia and neoplastic transformation. Collectively, our study demonstrates how gene-environment interactions can rapidly produce gene-regulatory programs that dictate early neoplastic commitment, and provides a molecular framework for understanding the interplay between genetic and environmental cues in the initiation of cancer.

α-Ketoglutarate links p53 to cell fate during tumour suppression.

The tumour suppressor TP53 is mutated in the majority of human cancers, and in over 70% of pancreatic ductal adenocarcinoma (PDAC)1,2. Wild-type p53 accumulates in response to cellular stress, and regulates gene expression to alter cell fate and prevent tumour development2. Wild-type p53 is also known to modulate cellular metabolic pathways3, although p53-dependent metabolic alterations that constrain cancer progression remain poorly understood. Here we find that p53 remodels cancer-cell metabolism to enforce changes in chromatin and gene expression that favour a premalignant cell fate. Restoring p53 function in cancer cells derived from KRAS-mutant mouse models of PDAC leads to the accumulation of α-ketoglutarate (αKG, also known as 2-oxoglutarate), a metabolite that also serves as an obligate substrate for a subset of chromatin-modifying enzymes. p53 induces transcriptional programs that are characteristic of premalignant differentiation, and this effect can be partially recapitulated by the addition of cell-permeable αKG. Increased levels of the αKG-dependent chromatin modification 5-hydroxymethylcytosine (5hmC) accompany the tumour-cell differentiation that is triggered by p53, whereas decreased 5hmC characterizes the transition from premalignant to de-differentiated malignant lesions that is associated with mutations in Trp53. Enforcing the accumulation of αKG in p53-deficient PDAC cells through the inhibition of oxoglutarate dehydrogenase-an enzyme of the tricarboxylic acid cycle-specifically results in increased 5hmC, tumour-cell differentiation and decreased tumour-cell fitness. Conversely, increasing the intracellular levels of succinate (a competitive inhibitor of αKG-dependent dioxygenases) blunts p53-driven tumour suppression. These data suggest that αKG is an effector of p53-mediated tumour suppression, and that the accumulation of αKG in p53-deficient tumours can drive tumour-cell differentiation and antagonize malignant progression.

A preclinical platform for assessing antitumor effects and systemic toxicities of cancer drug targets.

Anticancer drug development campaigns often fail due to an incomplete understanding of the therapeutic index differentiating the efficacy of the agent against the cancer and its on-target toxicities to the host. To address this issue, we established a versatile preclinical platform in which genetically defined cancers are produced using somatic tissue engineering in transgenic mice harboring a doxycycline-inducible short hairpin RNA against the target of interest. In this system, target inhibition is achieved by the addition of doxycycline, enabling simultaneous assessment of efficacy and toxicity in the same animal. As proof of concept, we focused on CDK9­a cancer target whose clinical development has been hampered by compounds with poorly understood target specificity and unacceptable toxicities. We systematically compared phenotypes produced by genetic Cdk9 inhibition to those achieved using a recently developed highly specific small molecule CDK9 inhibitor and found that both perturbations led to robust antitumor responses. Remarkably, nontoxic levels of CDK9 inhibition could achieve significant treatment efficacy, and dose-dependent toxicities produced by prolonged CDK9 suppression were largely reversible upon Cdk9 restoration or drug withdrawal. Overall, these results establish a versatile in vivo target validation platform that can be employed for rapid triaging of therapeutic targets and lend support to efforts aimed at advancing CDK9 inhibitors for cancer therapy.

A combinatorial strategy for treating KRAS-mutant lung cancer.

Therapeutic targeting of KRAS-mutant lung adenocarcinoma represents a major goal of clinical oncology. KRAS itself has proved difficult to inhibit, and the effectiveness of agents that target key KRAS effectors has been thwarted by activation of compensatory or parallel pathways that limit their efficacy as single agents. Here we take a systematic approach towards identifying combination targets for trametinib, a MEK inhibitor approved by the US Food and Drug Administration, which acts downstream of KRAS to suppress signalling through the mitogen-activated protein kinase (MAPK) cascade. Informed by a short-hairpin RNA screen, we show that trametinib provokes a compensatory response involving the fibroblast growth factor receptor 1 (FGFR1) that leads to signalling rebound and adaptive drug resistance. As a consequence, genetic or pharmacological inhibition of FGFR1 in combination with trametinib enhances tumour cell death in vitro and in vivo. This compensatory response shows distinct specificities: it is dominated by FGFR1 in KRAS-mutant lung and pancreatic cancer cells, but is not activated or involves other mechanisms in KRAS wild-type lung and KRAS-mutant colon cancer cells. Importantly, KRAS-mutant lung cancer cells and patients' tumours treated with trametinib show an increase in FRS2 phosphorylation, a biomarker of FGFR activation; this increase is abolished by FGFR1 inhibition and correlates with sensitivity to trametinib and FGFR inhibitor combinations. These results demonstrate that FGFR1 can mediate adaptive resistance to trametinib and validate a combinatorial approach for treating KRAS-mutant lung cancer.

A modular and flexible ESC-based mouse model of pancreatic cancer.

Genetically engineered mouse models (GEMMs) have greatly expanded our knowledge of pancreatic ductal adenocarcinoma (PDAC) and serve as a critical tool to identify and evaluate new treatment strategies. However, the cost and time required to generate conventional pancreatic cancer GEMMs limits their use for investigating novel genetic interactions in tumor development and maintenance. To address this problem, we developed flexible embryonic stem cell (ESC)-based GEMMs that facilitate the rapid generation of genetically defined multiallelic chimeric mice without further strain intercrossing. The ESCs harbor a latent Kras mutant (a nearly ubiquitous feature of pancreatic cancer), a homing cassette, and other genetic elements needed for rapid insertion and conditional expression of tetracycline-controlled transgenes, including fluorescence-coupled shRNAs capable of efficiently silencing gene function by RNAi. This system produces a disease that recapitulates the progression of pancreatic cancer in human patients and enables the study and visualization of the impact of gene perturbation at any stage of pancreas cancer progression. We describe the use of this approach to dissect temporal roles for the tumor suppressor Pten and the oncogene c-Myc in pancreatic cancer development and maintenance.

Cancer-associated IDH2 mutants drive an acute myeloid leukemia that is susceptible to Brd4 inhibition.

Somatic mutations in the isocitrate dehydrogenase (IDH) genes IDH1 and IDH2 occur frequently in acute myeloid leukemia (AML) and other cancers. These genes encode neomorphic proteins that produce the presumed oncometabolite 2-hydroxyglutarate (2-HG). Despite the prospect of treating AML and other cancers by targeting IDH mutant proteins, it remains unclear how these mutants affect tumor development and maintenance in vivo, and no cancer models exist to study the action of IDH2 mutants in vivo. We show that IDH2 mutants can cooperate with oncogenic Flt3 or Nras alleles to drive leukemia in mice by impairing the differentiation of cells of the myeloid lineage. Pharmacologic or genetic inhibition of IDH2 triggers the differentiation and death of AML cells, albeit only with prolonged IDH2 inhibition. In contrast, inhibition of the bromodomain-containing protein Brd4 triggers rapid differentiation and death of IDH2 mutant AML. Our results establish a critical role for mutant IDH2 in leukemogenesis and tumor maintenance and identify an IDH-independent strategy to target these cancers therapeutically.

Nr5a2 maintains acinar cell differentiation and constrains oncogenic Kras-mediated pancreatic neoplastic initiation.

OBJECTIVES: Emerging evidence from mouse models suggests that mutant Kras can drive the development of pancreatic ductal adenocarcinoma (PDA) precursors from acinar cells by enforcing ductal de-differentiation at the expense of acinar identity. Recently, human genome-wide association studies have identified NR5A2, a key regulator of acinar function, as a susceptibility locus for human PDA. We investigated the role of Nr5a2 in exocrine maintenance, regeneration and Kras driven neoplasia. DESIGN: To investigate the function of Nr5a2 in the pancreas, we generated mice with conditional pancreatic Nr5a2 deletion (PdxCre(late); Nr5a2(c/c)). Using this model, we evaluated acinar differentiation, regeneration after caerulein pancreatitis and Kras driven pancreatic neoplasia in the setting of Nr5a2 deletion. RESULTS: We show that Nr5a2 is not required for the development of the pancreatic acinar lineage but is important for maintenance of acinar identity. Nr5a2 deletion leads to destabilisation of the mature acinar differentiation state, acinar to ductal metaplasia and loss of regenerative capacity following acute caerulein pancreatitis. Loss of Nr5a2 also dramatically accelerates the development of oncogenic Kras driven acinar to ductal metaplasia and PDA precursor lesions. CONCLUSIONS: Nr5a2 is a key regulator of acinar plasticity. It is required for maintenance of acinar identity and re-establishing acinar fate during regeneration. Nr5a2 also constrains pancreatic neoplasia driven by oncogenic Kras, providing functional evidence supporting a potential role as a susceptibility gene for human PDA.

Bmi1 is required for regeneration of the exocrine pancreas in mice.

BACKGROUND & AIMS: Bmi1 is a member of the Polycomb protein family and represses transcription by modifying chromatin organization at specific promoters. Bmi1 is implicated in the control of stem cell self-renewal and has been shown to regulate cell proliferation, tissue homeostasis, and differentiation. Bmi1 is present in a subpopulation of self-renewing pancreatic acinar cells and is expressed in response to pancreatic damage. We investigated the role of Bmi1 in regeneration of exocrine pancreas. METHODS: Acute pancreatitis was induced in Bmi1(-/-) mice with cerulein; pancreatic cell regeneration, differentiation, and apoptosis were assessed. Cultured Bmi1(-/-) and wild-type primary acini were analyzed in vitro to determine acinar-specific consequences of Bmi1 deletion. To investigate cell autonomous versus non-cell autonomous roles for Bmi1 in vivo, pancreatitis was induced in Bmi1(-/-) mice reconstituted with a wild-type hematopoietic system. RESULTS: Bmi1 expression was up-regulated in the exocrine pancreas during regeneration after cerulein-induced pancreatitis. Exocrine regeneration was impaired following administration of cerulein to Bmi1(-/-) mice. Pancreata of Bmi1(-/-) mice were hypoplastic, and the exocrine pancreas was replaced with ductal metaplasia that had increased apoptosis and decreased cell proliferation compared with that of wild-type mice. Expression of Cdkn2a and p53-dependent apoptotic genes was markedly up-regulated in Bmi1(-/-) pancreas compared with wild-type mice after injury. Furthermore, after transplantation of bone marrow from wild-type to Bmi1(-/-) mice, the chimeric mice had intermediate levels of pancreatic hypoplasia and significant but incomplete rescue of impaired exocrine regeneration after cerulein injury. CONCLUSIONS: Bmi1 contributes to regeneration of the exocrine pancreas after cerulein-induced injury through cell autonomous mechanisms, in part by regulating Cdkn2a expression, and non-cell autonomous mechanisms.

MLL3 regulates the CDKN2A tumor suppressor locus in liver cancer.

Mutations in genes encoding components of chromatin modifying and remodeling complexes are among the most frequently observed somatic events in human cancers. For example, missense and nonsense mutations targeting the mixed lineage leukemia family member 3 (MLL3, encoded by KMT2C) histone methyltransferase occur in a range of solid tumors, and heterozygous deletions encompassing KMT2C occur in a subset of aggressive leukemias. Although MLL3 loss can promote tumorigenesis in mice, the molecular targets and biological processes by which MLL3 suppresses tumorigenesis remain poorly characterized. Here, we combined genetic, epigenomic, and animal modeling approaches to demonstrate that one of the mechanisms by which MLL3 links chromatin remodeling to tumor suppression is by co-activating the Cdkn2a tumor suppressor locus. Disruption of Kmt2c cooperates with Myc overexpression in the development of murine hepatocellular carcinoma (HCC), in which MLL3 binding to the Cdkn2a locus is blunted, resulting in reduced H3K4 methylation and low expression levels of the locus-encoded tumor suppressors p16/Ink4a and p19/Arf. Conversely, elevated KMT2C expression increases its binding to the CDKN2A locus and co-activates gene transcription. Endogenous Kmt2c restoration reverses these chromatin and transcriptional effects and triggers Ink4a/Arf-dependent apoptosis. Underscoring the human relevance of this epistasis, we found that genomic alterations in KMT2C and CDKN2A were associated with similar transcriptional profiles in human HCC samples. These results collectively point to a new mechanism for disrupting CDKN2A activity during cancer development and, in doing so, link MLL3 to an established tumor suppressor network.

EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype to potentiate pancreatic cancer immune surveillance.

Immunotherapies that produce durable responses in some malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to rampant immune suppression and poor tumor immunogenicity. We and others have demonstrated that induction of the senescence-associated secretory phenotype (SASP) can be an effective approach to activate anti-tumor natural killer (NK) cell and T cell immunity. In the present study, we found that the pancreas tumor microenvironment suppresses NK cell and T cell surveillance after therapy-induced senescence through enhancer of zeste homolog 2 (EZH2)-mediated epigenetic repression of proinflammatory SASP genes. EZH2 blockade stimulated production of SASP chemokines CCL2 and CXCL9/10, leading to enhanced NK cell and T cell infiltration and PDAC eradication in mouse models. EZH2 activity was also associated with suppression of chemokine signaling and cytotoxic lymphocytes and reduced survival in patients with PDAC. These results demonstrate that EZH2 represses the proinflammatory SASP and that EZH2 inhibition combined with senescence-inducing therapy could be a powerful means to achieve immune-mediated tumor control in PDAC.

Expanded RNA-binding activities of mammalian Argonaute 2.

Mammalian Argonaute 2 (Ago2) protein associates with microRNAs (miRNAs) or small interfering RNAs (siRNAs) forming RNA-induced silencing complexes (RISCs/miRNPs). In the present work, we characterize the RNA-binding and nucleolytic activity of recombinant mouse Ago2. Our studies show that recombinant mouse Ago2 binds efficiently to miRNAs forming active RISC. Surprisingly, we find that recombinant mouse Ago2 forms active RISC using pre-miRNAs or long unstructured single stranded RNAs as guides. Furthermore, we demonstrate that, in vivo, endogenous human Ago2 binds directly to pre-miRNAs independently of Dicer, and that Ago2:pre-miRNA complexes are found both in the cytoplasm and in the nucleus of human cells.

Vitamin B6 Addiction in Acute Myeloid Leukemia.

Cancer cells rely on altered metabolism to support abnormal proliferation. We performed a CRISPR/Cas9 functional genomic screen targeting metabolic enzymes and identified PDXK-an enzyme that produces pyridoxal phosphate (PLP) from vitamin B6-as an acute myeloid leukemia (AML)-selective dependency. PDXK kinase activity is required for PLP production and AML cell proliferation, and pharmacological blockade of the vitamin B6 pathway at both PDXK and PLP levels recapitulated PDXK disruption effects. PDXK disruption reduced intracellular concentrations of key metabolites needed for cell division. Furthermore, disruption of PLP-dependent enzymes ODC1 or GOT2 selectively inhibited AML cell proliferation and their downstream products partially rescued PDXK disruption induced proliferation blockage. Our work identifies the vitamin B6 pathway as a pharmacologically actionable dependency in AML.

Altered RNA Splicing by Mutant p53 Activates Oncogenic RAS Signaling in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is driven by co-existing mutations in KRAS and TP53. However, how these mutations collaborate to promote this cancer is unknown. Here, we uncover sequence-specific changes in RNA splicing enforced by mutant p53 which enhance KRAS activity. Mutant p53 increases expression of splicing regulator hnRNPK to promote inclusion of cytosine-rich exons within GTPase-activating proteins (GAPs), negative regulators of RAS family members. Mutant p53-enforced GAP isoforms lose cell membrane association, leading to heightened KRAS activity. Preventing cytosine-rich exon inclusion in mutant KRAS/p53 PDACs decreases tumor growth. Moreover, mutant p53 PDACs are sensitized to inhibition of splicing via spliceosome inhibitors. These data provide insight into co-enrichment of KRAS and p53 mutations and therapeutics targeting this mechanism in PDAC.

Arid1a restrains Kras-dependent changes in acinar cell identity.

Mutations in members of the SWI/SNF chromatin remodeling family are common events in cancer, but the mechanisms whereby disruption of SWI/SNF components alters tumorigenesis remain poorly understood. To model the effect of loss of function mutations in the SWI/SNF subunit Arid1a in pancreatic ductal adenocarcinoma (PDAC) initiation, we directed shRNA triggered, inducible and reversible suppression of Arid1a to the mouse pancreas in the setting of oncogenic KrasG12D. Arid1a cooperates with Kras in the adult pancreas as postnatal silencing of Arid1a following sustained KrasG12D expression induces rapid and irreversible reprogramming of acinar cells into mucinous PDAC precursor lesions. In contrast, Arid1a silencing during embryogenesis, concurrent with KrasG12D activation, leads to retention of acinar cell fate. Together, our results demonstrate Arid1a as a critical modulator of Kras-dependent changes in acinar cell identity, and underscore an unanticipated influence of timing and genetic context on the effects of SWI/SNF complex alterations in epithelial tumorigenesis.