MYC Instructs and Maintains Pancreatic …...MYC Instructs and Maintains Pancreatic Adenocarcinoma...

588 | CANCER DISCOVERY APRIL 2020 AACRJournals.org

MYC Instructs and Maintains Pancreatic Adenocarcinoma Phenotype Nicole M. Sodir 1 , Roderik M. Kortlever 1 , Valentin J.A. Barthet 1 , Tania Campos 1 , Luca Pellegrinet 1 , Steven Kupczak 2 , Panayiotis Anastasiou 1 , Lamorna Brown Swigart 3 , Laura Soucek 4 , Mark J. Arends 5 , Trevor D. Littlewood 1 , and Gerard I. Evan 1

RESEARCH ARTICLE

ABSTRACT The signature features of pancreatic ductal adenocarcinoma (PDAC) are its fi bro-infl ammatory stroma, poor immune activity, and dismal prognosis. We show that

acute activation of Myc in indolent pancreatic intraepithelial neoplasm (PanIN) epithelial cells in vivo is, alone, suffi cient to trigger immediate release of instructive signals that together coordinate changes in multiple stromal and immune-cell types and drive transition to pancreatic adenocarcinomas that share all the characteristic stromal features of their spontaneous human counterpart. We also demonstrate that this Myc -driven PDAC switch is completely and immediately reversible: Myc deactivation/inhibition triggers meticulous disassembly of advanced PDAC tumor and stroma and concomitant death of tumor cells. Hence, both the formation and deconstruction of the complex PDAC phenotype are continuously dependent on a single, reversible Myc switch.

SIGNIFICANCE: We show that Myc activation in indolent Kras G12D -induced PanIN epithelium acts as an immediate pleiotropic switch, triggering tissue-specifi c signals that instruct all the diverse signature stromal features of spontaneous human PDAC. Subsequent Myc deactivation or inhibition immediately triggers a program that coordinately disassembles PDAC back to PanIN.

See related commentary by English and Sears, p. 495.

1 Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom. 2 Cambridge Research Institute, Li Ka Shing Centre, Cambridge, United Kingdom. 3 Department of Laboratory Medicine, Univer-sity of California, San Francisco, San Francisco, California. 4 Vall d’Hebron Institute of Oncology (VHIO), Barcelona, Spain. 5 Division of Pathology, Cancer Research UK Edinburgh Centre, University of Edinburgh, Edinburgh, Scotland, United Kingdom. Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/). V.J.A. Barthet and T. Campos contributed equally to this article.

Current address for V.J.A. Barthet: Cancer Research UK Beatson Institute, Glasgow, United Kingdom. Corresponding Author: Gerard I. Evan, University of Cambridge, Sanger Building, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom. Phone: 4412-2376-5944; Fax: 4412-2376-6082; E-mail: [email protected] . Cancer Discov 2020;10:588–607 doi: 10.1158/2159-8290.CD-19-0435 ©2020 American Association for Cancer Research.

Research. on July 30, 2020. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from

Published OnlineFirst January 15, 2020; DOI: 10.1158/2159-8290.CD-19-0435

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APRIL 2020 CANCER DISCOVERY | 589

INTRODUCTION

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a 5-year survival of less than 9% (1). Its dismal prognosis is in great part due to its typically late presenta-tion and its profound resistance to all forms of conventional chemotherapy and radiotherapy. Studies of resected tumors suggest that malignant ductal adenocarcinoma evolves from pancreatic intraepithelial neoplasms (PanIN), indolent precursors that are histologically classified into four stages (PanIN1A, 1B, 2, and 3) based on increasing degrees of nuclear and architectural atypia (2–4). Frank PDAC is char-acterized by its striking fibroinflammatory stroma, absent from normal pancreas, whose attendant dense desmoplasia typically constitutes some 90% of tumor bulk (5, 6). This fibroinflammatory stroma is a product of complex interplay between tumor cells and adjacent mesenchymal, endothe-lial, inflammatory, and immune cells and is thought to contribute to PDAC therapeutic recalcitrance by impeding vascular perfusion and oxygenation (7, 8) and suppressing antitumor immunity (9).

At the molecular level, PDAC is associated with a core of recurring, presumed causal, oncogenic mutations. Of these, activating mutations in KRAS are present in more than 90% of both PanINs and PDAC and are thought to be founder drivers. Other frequently recurring signature mutations inac-tivate CDKN2A (90%–95%), TP53 (50%–84%), and SMAD4/DPC4 (49%–55%), while recent, more granular, analyses reveal a plethora of additional sporadic PDAC mutations encom-passing great functional diversity (10–12). As KRAS muta-tion is the most commonly recurring oncogenic mutation, many of the canonical features of PDAC are attributed to precocious KRAS signaling. KRAS is a multifunctional com-munication node with diverse outputs that encompass many downstream intracellular effectors (e.g., MAPK, PI3K, p38, JAK–STAT, Hippo, and NFκB) and a host of extracellular signals and their receptors [e.g., WNT, Notch, SHH, CXCL1, 2, and 5, CXCL8 (IL8), IL6, GM-CSF, and IL17RA]. In this way, KRAS mutations can influence diverse aspects of PDAC pathology, including tumor initiation, maintenance, drug sensitivity, metabolism, macropinocytosis, metastasis, desmo-plasia, inflammation, and immunity (13, 14). Oncogenic Kras




Sodir et al.RESEARCH ARTICLE


has been used as the initiating driver in multiple pancreatic cancer mouse models (15–23). However, all concur that, without cooperating oncogenic mutations, KRAS oncogenic potential is very poor, stalling at indolent PanIN with little desmoplasia, inflammation, or lymphocytic suppression (15, 24). In addition to its role in pancreatic tumor initiation (15), oncogenic KRAS also plays a role in maintenance of estab-lished PDAC (15, 17, 24, 25), where it appears to provide an obligate necessary platform upon which subsequent cooper-ating lesions exert their oncogenic impacts.

The prototypical cooperating oncogenic partner of KRAS is MYC (26), a pleiotropic transcription factor whose putative role is to mediate expression of the thousands of genes that, together, coordinate the diverse genetic programs needed for somatic cell proliferation. In normal cells, MYC serves as a functionally nonredundant and essential transcriptional node and is tightly controlled by mitogen availability (27). In contrast, aberrantly persistent MYC activation is implicated in the majority of, perhaps all, cancers, although it seldom involves mutation of the MYC gene itself and is more com-monly an indirect consequence of its relentless induction by “upstream” oncoproteins—notable examples being WNT, Notch, and RAS itself (28–34). During pancreatic organogen-esis and early postnatal growth, MYC is expressed in a popu-lation of multipotent pancreatic progenitor cells and, like its upstream activators β-catenin (34–36) and Notch (33), is required for exocrine pancreas development, maturation, and neonatal growth (32, 37–40). MYC is not appreciably expressed in normal adult, uninjured, nonproliferating pancreas, but a majority of PDACs exhibit demonstrable expression of MYC and its canonical target gene signature. A complication, how-ever, is that endogenous MYC is obligatorily expressed in all normal proliferating somatic cells, so its mere expression in proliferating cancer cells is not itself evidence of oncogenic causation. However, overt amplification of the MYC gene is frequent in especially aggressive tumors (20, 32, 41–43) and multiple in vivo mouse studies indicate that deregulated MYC is causally oncogenic in exocrine pancreas (reviewed in ref. 32). For example, classic transgenic overexpression of MYC targeted to mature acinar cells via an elastase promoter induces widespread acinar-to-ductal metaplasia (ADM; ref. 7), whereas pdx1 promoter–directed overexpression of MYC in pancreatic progenitor cells through organ development elic-its rapid emergence of ductal PanIN (44). Conversely, pdx1-Cre–driven excision of endogenous Myc concurrently with KrasG12D activation blocks transition of PanINs to PDAC. Studies also indicate a persistent maintenance role for MYC in established PDAC. MYC knockdown inhibits proliferation of human PDAC cell lines in vitro (45), and direct (44) or indi-rect (46) inactivation of MYC triggers macroscopic regression of established PDAC in mouse models in vivo (47), albeit with persistence of dormant tumor cells (44). Taken together, these data indicate a critical role for MYC in both the progression from PanIN to PDAC and in subsequent PDAC maintenance (33). However, the nature of this role remains obscure, espe-cially because MYC has no obvious functional overlap with any of the canonical CDKN2A, TP53, and SMAD4 mutants that typically associate with KRAS mutation in PDAC.

Here we use a rapidly and reversibly switchable genetic mouse model whereby Myc may be engaged or disengaged, at

will, in indolent KrasG12D-induced PanINs to address key ques-tions concerning the mechanistic role of MYC in PDAC. Does Myc activation drive immediate transition of KrasG12D-driven indolent PanIN to PDAC or, instead, merely raise the likeli-hood that happenstance progression eventually occurs? What is the phenotype of the pancreatic tumors that Myc activation elicits, and do they resemble spontaneous PDACs? Finally, we address why Myc activation is required for the maintenance of KrasG12D-driven PDAC and by what mechanism such tumors regress upon Myc deactivation.

RESULTSMyc Deregulation Drives Immediate Progression of KrasG12D-Induced PanINs to Adenocarcinoma

Conditional expression of oncogenic KrasG12D in pancreas progenitor cells drives protracted and sporadic outgrowth of indolent PanIN lesions (18). Rarely, these progress to overt adenocarcinoma through sporadic accretion of additional lesions. Because deregulated and/or overexpressed MYC is a frequent feature of advanced PDAC, we first asked whether MYC deregulation is sufficient to drive progression of indo-lent KrasG12D-driven PanINs to PDAC using the well-char-acterized pdx1-Cre;LSL-KrasG12D/+ model (Kpdx1) in which Cre recombinase expression, driven by the pdx/IPF1 (Pancreas/duodenum homeobox protein 1) promoter in embryonic pan-creatic and duodenal progenitor cells from around E8 (19), triggers expression of a single allele of KrasG12D driven from the endogenous Kras2 promoter. Kpdx1 mice homozygous for Rosa26-LSL-MycERT2 (48) were generated. In the resulting pdx1-Cre;LSL-KrasG12D;Rosa26LSL-MycERT2 (KMpdx1) mice, activation of Cre recombinase in pancreatic progenitor cells induces expression of both KrasG12D, driven at physiologic levels from its endogenous promoter, and the conditionally regulatable MycERT2 variant of Myc (48–51). Of special note, MycERT2 in KMpdx1 pancreatic epithelia is driven at quasi-physiologic levels from the promiscuously active but weak Rosa26 promoter (48), levels comparable to that of endogenous Myc in proliferat-ing neonatal mouse pancreas cells (Supplementary Fig. S1A and S1B). Hence, both KrasG12D and Myc in KMpdx1 mice are expressed at physiologic levels throughout.

As previously described (18), by 15 weeks of age Kpdx1 con-trol mice (i.e., KrasG12D alone) each exhibited a small number of early-stage PanIN-1A and PanIN-1B lesions, with the vast majority of pancreatic ducts appearing histologically normal. These PanIN lesions were small, with little or no desmoplastic stroma (Fig. 1A). Activation of MycERT2 alone for 3 weeks in the absence of KrasG12D (Mpdx1 mice) had no significant histologic impact (Fig. 1A) save for a very modest increase in proliferative index (data not shown). In contrast, sustained activation of MycERT2 for 3 weeks in KMpdx1 mice (i.e., expression of KrasG12D and Myc together) induced dramatic progression of invasive adenocarcinomas (Fig. 1A) that bore the stereotypical features of spontaneous mouse and human PDAC (Fig. 1B): highly proliferative nests of epithelial tumor cells of predominantly ductal phenotype embedded in extensive α-smooth muscle actin (α-SMA)–positive desmoplastic stroma. Tamoxifen alone had no discernible impact on pancreas architecture (Fig. 1B).

Unexpectedly, activation of MycERT2 proved rapidly lethal in KMpdx1 mice with only a few surviving to 3 weeks of




MYC and Pancreatic Adenocarcinoma Phenotype RESEARCH ARTICLE


Figure 1. MYC drives progression from PanIN to pancreatic adenocarcinoma in KMpdx1 mice (related to Supplementary Figs. S1–S3). A, Left, repre-sentative hematoxylin and eosin (H&E)–stained sections of pancreata harvested from 15-week-old Kpdx1 (KrasG12D only; left), Mpdx1 (MycERT2 only; second column), and KMpdx1 (KrasG12D and MycERT2; right two columns) mice treated with either oil control (top row) or tamoxifen (Tam; bottom row) for 3 weeks. Scale bars apply across each row. Right, percentage tumor burden relative to total pancreas in KMpdx1 mice treated with either oil (control) or tamoxifen is shown. Each point refers to an individual mouse. Results represent mean ± SD, n = 6 mice per treatment group. The unpaired Student t test was used to analyze tumor burden. ****, P < 0.0001. B, Left, representative IHC staining for proliferation (Ki-67, top row), α-smooth muscle actin (α-SMA; second row), and collagen deposition by Gomori Trichrome staining (blue-stained collagen, bottom row) in sections of pancreata harvested from 15-week-old wild-type (WT) control mice treated with tamoxifen alone (No Myc, first column) versus age-matched KMpdx1 mice treated with either oil (Myc OFF, second column) or tamoxifen (Myc ON, third and fourth columns) for 3 weeks. Column 3 shows examples of early invasive PDAC and advanced PanIN. Column 4 shows examples of frank PDAC. Scale bars apply across each row. Right, box-and-whisker quantitation plots showing upper extreme, upper quartile, median, lower quartile, lower extreme. n = 5–7 mice for each treatment group. ****, P < 0.0001. Data were analyzed using unpaired t test. FOV, field of view.

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sustained Myc activation. Lethality coincided with extensive MYC-induced overgrowth of KMpdx1 duodenal epithelium, severely disrupted crypt–villus architecture, and emergence of multiple intestinal neoplasms. We attribute this to the known activity of the pdx1 promoter in relatively uncommit-ted E8 foregut progenitor cells that have yet to commit to pancreatic versus duodenal specification. To circumvent this

problem, we confirmed our KMpdx1 studies using the com-plementary p48-Cre;LSL-KrasG12D; Rosa26LSL-MycERT2 (KMp48) mouse PDAC model, in which Cre expression is driven rather later and in more pancreas-specific progenitors by the p48/PTF1 promoter (18, 52). The histologic impacts of activating Myc in pancreata of KMpdx1 and KMp48 models were indistin-guishable (Supplementary Fig. S2A). By 3 weeks of sustained






Myc activation, both KMpdx1 and KMp48 pancreata exhibited an abundance of advanced PanIN3 lesions together with early invasive PDAC consisting of irregular malignant glands and solid clusters of neoplastic cells invading the stroma. Frank, highly invasive morphologically heterogenous pancre-atic adenocarcinomas were also evident in some 30% of both KMpdx1 and KMp48 mice, rising to 90% of KMp48 mice after 6 weeks of sustained Myc activation (Fig. 1; Supplementary Figs. S2A–S2C and S3A–S3D). No intestinal neoplasia was ever evident in KMp48 mice, and any occasional lethality of KMp48 mice after protracted Myc activation was attributed to the burden of grossly expanded, highly desmoplastic PDAC (Supplementary Fig. S2B and S2C). Accordingly, KMp48 mice were used thereafter.

Although MycERT2 is restricted solely to the PanIN epi-thelial cells in both KMpdx1 and KMp48 mice, Myc activation elicited widespread changes in stromal hematopoietic, endothelial, and mesenchymal cells. To address whether this MYC-induced stromal phenotype is an immediate, instruc-tive consequence of MYC action or, instead, a delayed result of passive channeling by the host pancreatic tissue, we made use of the inherent rapidity and synchrony of MycERT2 activa-tion by tamoxifen in vivo (50, 51, 53, 54). Myc was activated in KMp48 mice by systemic administration of tamoxifen for 0, 1, or 3 days, and pancreata were then harvested and analyzed. Within 24 hours of Myc activation, we observed a dramatic increase in proliferation (Ki-67) of the MycERT2-expressing epithelial tumor compartment of all PanIN lesions accom-panied by diverse stromal changes (Fig. 2): specifically, a marked influx of F4/80+ and CD206+ macrophages, Ly-6B.2+ neutrophils, and, to the periphery of each tumor, B220+ B lymphocytes, concurrent with loss of CD3+ T cells and induc-tion of α-SMA in proximal stellate and fibroblastic cells, the latter indicative of myofibroblastic transdifferentiation. By 72 hours, progressive deposition of dense desmoplasia was evi-dent together with loss of blood-vessel patency and upregula-tion of HIF1α, a marker of hypoxia, all presumed secondary consequences of prior stellate cell activation. Identical MYC-induced phenotypic changes, with identical kinetics, were observed in pancreata of the complementary KMpdx1 switch-able Myc model (Supplementary Fig. S4). Taken together, our data indicate that all the diverse stromal changes that MYC induces, all of them features of aggressive spontaneous PDAC seen in spontaneous mouse and human PDAC, are instructed by MYC from the outset.

Specific Signals Instruct the MYC-Induced PDAC Transition

As MycERT2 is activated only in the pancreatic epithelial compartments of KMpdx1 and KMp48 mice, the immediate stromal changes that MYC elicits must be instructed by

paracrine signals originating in those MycERT2-expressing tumor epithelial cells. To identify such signals, we activated MycERT2 in KMp48 mice with a single systemic dose of tamox-ifen, harvested pancreata 6 hours later, and then interrogated tissue extracts with growth factor/cytokine arrays (Fig. 3A and B) and isolated mRNA by RT-PCR (Fig. 3C). Various growth factors and regulators of inflammatory and immune cells were demonstrably induced. Some of these are already documented. For example, SHH was previously shown to be critical for PDAC desmoplastic deposition and maintenance (8). Similarly, macrophage influx into PanINs, clearly evident by 24 hours post Myc activation, was presaged by induction at 6 hours of the well-documented macrophage chemoattractant CCL9 (MIP1γ). MYC-induced influx of Ly-6B+ neutrophils was preceded at 6 hours by induction of CXCL5 (Fig. 3A), a well-characterized neutrophil attractant (55, 56) and one of several CXCR2 (IL8RB) receptor ligands. Myc activation also induced immediate upregulation of GAS6 (Fig. 3B), an AXL kinase ligand previously implicated in an unspecified role in PDAC-stromal signaling (57). GAS6 induction correlated with appearance of pAXL immunoreactivity in a subset of PanIN epithelial cells (Fig. 4A). Blockade of AXL kinase with a specific inhibitor, TP-0903 (ref. 58; Fig. 4B), profoundly inhibited MYC-driven neutrophil influx into tumors and B-cell influx into tumor peripheries and markedly suppressed induction of α-SMA in local stellate and fibroblastic cells (Fig. 4B). Comparable changes were observed following inhi-bition of GAS6 with low-dose warfarin (refs. 59, 60; Sup-plementary Fig. S5A). However, GAS6–AXL inhibition had only a modest inhibitory impact on macrophage influx and no discernible impact on MYC-induced depletion of intra-tumoral CD3+ T cells (Fig. 4B). Overall, blocking GAS6–AXL signaling profoundly retarded early MYC-driven tumor pro-gression (reducing the prevalence of PanIN2 and 3 lesions in favor of PanIN1 and 2), reduced tumor burden, and inhibited proliferation of both tumor and stroma (Fig. 4C), in the main by suppressing proliferating neutrophils and fibroblastic cells (Supplementary Fig. S5B).

We recently showed in lung adenomas that MYC activation induces immediate T-cell expulsion via a mechanism requiring macrophage-specific expression of PD-L1 (also known as B7H1, encoded by CD274), a ligand for the PD-1 (CD279) receptor whose activation quells T lymphocyte immune functions. Myc activation rapidly induced PD-L1 expression on KMp48 PanIN epithelial cells in vivo (Supplementary Fig. S6A), while systemic blockade of PD-L1 completely abrogated MYC-induced T-cell depletion in pancreatic tumors (Supplementary Fig. S6B and S6C), confirming a causal role for PD-L1 induction in driving exclusion of CD3+ T cells during the MYC-induced transition from PanIN to PDAC. However, PD-L1 blockade and consequent persistence of CD3+ T cells had no discernible inhibitory

Figure 2. MYC instructs immediate onset of transition to pancreatic adenocarcinoma in KMp48 mice (related to Supplementary Fig. S4). IHC and immu-nofluorescence (IF) analyses of immediate pancreatic tumor–stromal changes at 0, 1, and 3 days post Myc activation in KMp48 mice. Left, representative staining for proliferation (Ki-67 IHC), macrophages (F4/80/EMR1 and CD206/MRC1 IF), neutrophils (Ly-6B IF), T cells (CD3 IHC), B cells (B220 IHC: red arrows indicate B220+ cells, broken line marks tumor border), activated fibroblasts/stellate cells (α-SMA IHC), collagen (Gomori Trichrome), vascular endothelial cells (CD31/PECAM IF), and hypoxia (HIF1α IF) of pancreata harvested from 12-week-old KMp48 mice before Myc activation (0 days, left column) or tamoxifen for 1 or 3 days (Myc ON, second and third columns, respectively). Scale bars apply across all images. Quantifications are shown to the right. n = 5–7 for each treatment group. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant. Data were analyzed using unpaired t test. FOV, field of view.






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Figure 3. Myc activation in PanIN epithelium of KMp48 mice rapidly induces specific signaling molecules. A and B, Representative dot blots of whole pan-creas protein lysates from 12-week-old KMp48 mice treated for 6 hours with oil (Myc OFF, left) or tamoxifen (Myc ON, right) probed with 40 inflammation-associated target arrays (A) and 97 cytokine target arrays (B). Corresponding quantitation of fold change in CXCL5, CCL9, and GAS6 signals derived from protein arrays. Each data point is generated from a pancreas extract from an individual mouse. n = 3 for each cohort. Ratio paired t test is used to analyze the data. *, P < 0.05; **, P < 0.01. C, qRT-PCR analysis of Cxcl5, Ccl9, Gas6, and Shh mRNA isolated from 12-week-old KMp48 pancreata treated for 6 hours with either oil (Myc OFF, blue bars) or tamoxifen (Myc ON, red bars). TBP was used as the internal amplification control. The unpaired t test with was used to analyze TaqMan expression data. n = 3 for each cohort. The mean ± SD is shown. *, P < 0.05; **, P < 0.01.

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Figure 4. Inhibition of the GAS6–AXL pathway selectively blocks a subset of MYC-induced stromal changes in KMp48 pancreatic tumors (related to Supplementary Fig. S5). A, Top row, IF of phospho-AXL (pAXL; green) and its quantification in pancreata of 12-week-old KMp48 mice treated with oil (Myc OFF, top left) or tamoxifen for 1 day (middle) or 3 days (right; Myc ON), together with corresponding quantitation. The absence of detectable pAXL stain-ing in section of a KrasG12D-driven lung adenoma in which Myc had been activated for 3 days (74) is shown for comparison (extreme right). Bottom row, representative colocalization of pAXL (green) and pan-keratin (red, epithelial cells). Scale bar applies to all images. Percentage of epithelial cells positive for pAXL is quantified. n = 5–7 per group. *, P < 0.05; ***, P < 0.001. B, Representative IF staining for neutrophils (Ly-6B), macrophages (F4/80 and CD206) and IHC staining of T cells (CD3), B cells (B220), and myofibroblasts (α-SMA) of pancreata sections from mice treated with tamoxifen (Myc ON) for 5 days in either the absence (top row) or presence (bottom row) of the pAXL inhibitor TP-0903. Inset shows boxed region at higher magnification. Scale bar applies to all images. Relevant quantitation is provided under each column. n = 5–6 per group. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (continued on next page)

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impact on the MYC-driven PDAC transition or subsequent tumor growth (Supplementary Fig. S6D). Myc activation also induced PD-L1 expression on KMpdx1 PanIN epithelial cells in vivo (Supplementary Fig. S7A) and isolated KMpdx1 PDAC cells in vitro (Supplementary Fig. S7B) while chromatin immu-noprecipitation (ChIP) analysis located MycERT2 protein on the CD274 gene (Supplementary Fig. S7C), all indicating that MYC-dependent PD-L1 induction in PanIN epithelium is cell-autonomous and that the CD274 gene is most likely a direct MYC target in pancreatic tumors.

The Myc-Driven Transition from PanIN to Adenocarcinoma Is Rapidly Reversible

We next addressed whether persistent Myc activity is required to maintain PDAC. MycERT2 was activated for 3

weeks in 12-week-old KMpdx1 or KMp48 animals, inducing widespread multifocal aggressive PDAC in all mice. Subse-quent MycERT2 deactivation triggered ostensibly complete regression of all adenocarcinomas (Fig. 5A), culminating in pancreata largely indistinguishable from those in which Myc had never been activated. Even grossly expanded pancreatic tumors, resulting from 4 to 6 weeks of MycERT2 sustained activation, underwent rapid regression following Myc deacti-vation (Supplementary Fig. S8A and S8B), although the gross architectures of the residual pancreata were often marred by extensive scarring and cyst-like dysmorphias (Supplementary Fig. S8C).

To confirm that MYC dependency is not solely an arte-factual peculiarity of pancreatic adenocarcinomas driven directly by MYC, we also determined the consequence of






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Figure 4. (Continued) C, Representative H&E staining and Ki-67 IHC of pancreata from 12-week-old KMp48 mice either untreated (top row) or treated (bottom row) with AXL inhibitor TP-0903 during Myc activation (Myc ON) for 5 days. Insets show higher magnifications. Black arrows indicate PanIN 1, 2, and 3. Scale bars apply across each column. Corresponding quantitation of tumor burden, tumor score, and Ki-67 staining in epithelial and stromal compartments (bottom row) in pancreata of mice described above. The tumor score is calculated as follows: score 1 with <10% of the lesions are PanIN 3, score 2 with 10% to 25% of the lesions are PanIN 3, score 3 with >25% to 45% of the lesions are PanIN 3, score 4 with >45% of the lesions are PanIN 3. n = 6 mice per treatment group. ***, P < 0.001; ****, P < 0.0001. FOV, field of view.

inhibiting endogenous MYC in spontaneously evolved KrasG12D-driven PDAC using the Omomyc dominant-negative MYC dimerization mutant to block endogenous MYC function (61). For this, we used pdx1-Cre;LSL-KrasG12D;Trp53ERTAM KI; TRE-OmoMyc;CMVrtTA (KPO) mice in which pdx1-Cre-driven activation of oncogenic KrasG12D is combined with a back-ground in which the endogenous p53 is replaced with the conditional Trp53ERTAM allele (62). Because p53ERTAM is func-tionally inactive in the absence of tamoxifen, KPO animals without tamoxifen are effectively p53null, which greatly accel-erates spontaneous transition of KrasG12D-driven PanIN to PDAC (18, 19). KPO animals in addition harbor a doxycycline- inducible transgene encoding the dominant-negative MYC dimerization mutant Omomyc (61, 63). Accordingly, 10-week-old KPO mice harboring extensive, highly desmoplastic, spon-taneous PDAC were treated with doxycycline for 4 weeks to induce Omomyc and inhibit endogenous MYC. As with deactivation of MycERT2 in KMpdx1 and KMp48 mice, inhibi-tion of endogenous MYC by Omomyc triggered quantitative regression of all spontaneous PDAC tumors, including their extensive attendant fibroinflammatory stroma (Fig. 5B).

To investigate the mechanics underlying the initiation of tumor regression post Myc deactivation, MycERT2 was acti-vated for 3 weeks in 12-week-old KMp48 mice to generate early invasive PDAC and then acutely deactivated by tamoxifen withdrawal for 3 days (64). Myc deactivation triggered imme-diate tumor cell proliferative arrest (Fig. 6A) and initiated pro-gressive reversal of stromal changes that Myc activity initially induced—specifically, rapid efflux of macrophages and neu-trophils, rapid reinfiltration of CD3+ T cells concurrent with reduced expression of PD-L1, and deactivation of stromal stellate and fibroblastic cells (Fig. 6B and C). Of note, these stromal reversions all initiated after Myc deactivation with an

immediacy comparable to their initial MYC-driven induction. Equivalent Myc deactivation–induced stromal reversal was also observed following Myc deactivation in KMpdx1 animals (Supplementary Fig. S9).

One possible mechanism by which Myc deactivation might lead to overt loss of PDAC cells is by phenotypic reversal of neoplastic, predominantly ductal-like, dysplastic state back to their original acinar state and ductal organization. To investigate the potential for such ductal-to-acinar transdif-ferentiation, we used RNA-sequencing (RNA-seq) analysis to compare the transcriptional outputs of control pancreata isolated from 12-week-old KMp48 mice in which Myc was never activated, versus KMp48 pancreata in which Myc had been activated continuously for 2 weeks, versus pancreata in which Myc was activated for 2 weeks and then deactivated for 3 days (Supplementary Fig. S10). This clearly showed a marked induction of ductal markers (Egfr, Krt20, Tgfa), together with corresponding repression of acinar markers (Amy2b, Bhlha15, Ptf1a) in response to Myc activation and reversal back to acinar status upon subsequent Myc deactivation (Supple-mentary Fig. S10A). This was verified with KMpdx1 cells in vitro using the amylase and cytokeratin as immunocytochemical markers of, respectively, acinar (65) and ductal (66) states (Supplementary Fig. S10B). Hence, Myc reversibly toggles PDAC epithelial cells between ductal (Myc ON) and acinar (Myc OFF) states.

Nonetheless, tumor regression induced by Myc deacti-vation cannot be simply ascribed to mere reversal of the events originally triggered by Myc activation. For example, Myc deactivation not only terminated further desmoplastic deposition but triggered its rapid degradation, quickly restor-ing blood vessel patency and local tissue oxygenation (Fig. 6C; Supplementary Fig. S9). Furthermore, Myc deactivation not






Figure 5. Continuous Myc activity is required for PDAC maintenance (related to Supplementary Fig. S8). A, Representative H&E (top row), Ki-67 IHC for proliferation (second row), Gomori Trichrome staining for collagen (third row) and α-SMA IHC for myofibroblasts (bottom row) in sections of pancre-ata from 12-week-old KMpdx1 mice in which Myc was continuously activated for 3 weeks (left column) or activated for 3 weeks and then deactivated for 3 weeks (middle column). For comparison, age-matched control pancreata in which Myc was never activated (treated with oil for 3 weeks, then untreated for 3 weeks) are shown (right column). Quantitation of tumor burden in individual mice from each cohort is shown to the right, as is quantitation of Ki-67, collagen, and α–SMA. n = 5–6 per group. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Quantitation after 3 weeks of sustained Myc activation is from the same animals as shown in Fig. 1. B, Representative H&E (left) and Gomori Trichome–stained (right) sections of pancreata from 10-week-old Kpdx1; Trp53ERTAM KI (KPO) mice without (left) or with (right) Omomyc expression for 4 weeks. Quantitation of tumor burden and collagen is shown below. n = 6 per group. ****, P < 0.0001. FOV, field of view.

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Figure 6. Myc deactivation triggers immediate onset of regression of KMp48 tumor stroma (related to Supplementary Fig. S9 and S10). Analysis of pancreata from 12-week-old KMp48 mice treated with tamoxifen (Myc ON) for 3 weeks (Myc OFF 0 days) and 3 days after by tamoxifen withdrawal (Myc OFF 3 days). A, Left, analysis of proliferation (Ki-67 IHC and quantitation). Right, analysis of apoptosis (TUNEL IF and quantitation). Scale bars apply across each column. B, Staining for macrophages (F4/80 IF), neutrophils (Ly-6B IF), T cells (CD3 IHC), and IF labeling of PD-L1 (IF) and B cells (B220 IHC). Broken black line delineates tumor border. T, tumor. Scale bars apply across each row. Relevant quantitation is shown below each panel. C, IHC for α-SMA, Gomori Trichrome staining of collagen, IF for CD31 (endothelial cells), fluorescein isothiocyanate–conjugated Lycopersicon esculentum lectin (visualiza-tion of vascular patency) and IF for HIF1α (hypoxia). Scale bars apply across all columns. Relevant quantitation is shown below each panel. Data were analyzed using unpaired t test. n = 5–7 for each treatment group. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. FOV, field of view.

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only induced proliferative arrest and redifferentiation of pan-creatic tumor cells but also triggered an abrupt wave of tumor cell death (Fig. 6A; Supplementary Fig. S9). To investigate whether such cell death was a consequence of the rapid PD-L1 downregulation–mediated influx of T cells that Myc deactivation induces, we systemically ablated CD4+ and CD8+ T cells. This efficiently abrogated all detectable circulating and splenic T cells (data not shown) and completely blocked repopulation of pancreatic tumors by CD3+ T cells, yet had no inhibitory impact on tumor cell death (Fig. 7A). In con-trast, B-cell behavior was unexpected. Whereas CD20+B220+ B cells rapidly migrated to the PanIN peripheries upon Myc activation (Fig. 2), Myc deactivation triggered their immediate migration into each tumor mass (Fig. 6B). This influx of B cells was accompanied by concurrent influx of natural killer (NK) p46+ NK-like cells, and systemic depletion of either B cells or NKp46+ NK-like cells profoundly suppressed the abrupt wave of tumor cell death induced by Myc deactivation (Fig. 7B and C). Unexpectedly, B-cell ablation also blocked the Myc deactivation–induced influx of NKp46+ NK-like cells (Fig. 7D), suggesting that B cells play a critical role in initiating PDAC regression by facilitating recruitment of innate NK cells.

DISCUSSIONThe low, quasi-physiologic levels of MYC used in our stud-

ies indicates that the mere deregulation of Myc, without any overt amplification or overexpression, is alone sufficient to drive progression of indolent KrasG12D-driven PanIN lesions to aggressive PDAC. There is, apparently, no need for Myc to be overexpressed for any of its oncogenic activities to be manifest. An important implication of this is that MYC levels, specifi-cally elevated MYC levels, cannot be used as surrogate indica-tors of a causal role for MYC in PDAC (or, indeed, any other cancer type). Rather, it is the aberrant persistence of MYC, not its elevation, that is its underlying oncogenic agency.

The tumor–stromal phenotype of the pancreatic adeno-carcinoma lesions induced by activation of Myc in the PanIN epithelial compartment is marked by several notable features— its complexity, its immediacy, its tissue specificity, and, upon subsequent Myc deactivation, its rapid and complete revers-ibility. The complexity of the MYC-induced tumor phenotype is evident in the diversity of stromal cell types involved and in the complicated and highly organized choreography in which they participate. Myc activation within the PanIN epithelium triggers rapid influx of F4/80+CD206+ macrophages, Ly-6B+ neutrophils and/or granulocytic myeloid-derived suppressor cells (MDSC), and B220+ B cells; rapid efflux of CD3+ T cells; myofibrillar activation of neighboring fibroblastic cells, and consequent desmoplastic deposition, blood vessel compres-sion, vascular occlusion, and hypoxia. Many of these stromal features are clearly evident within 24 hours of Myc activation in the epithelial compartment and are presaged by release of a dis-tinct set of instructional paracrine signals, evident as early as 6 hours post Myc activation. Hence, although the adoption of the canonical phenotype of established PDAC tumors takes some longer time to establish, essentially all of the instructive signals and processes that culminate in the signature phenotype of established PDAC appear to be engaged almost immediately following Myc activation. These include potent chemoattrac-

tion of macrophages (67), Ly-6B+ neutrophils and/or granu-locytic MDSCs, and B220+ B cells (68–71); expulsion of CD3+ T cells (67); activation and proliferation of fibroblastic cells; and extensive desmoplastic deposition with concomitant blood vessel compression, vascular involution, and hypoxia (5, 72, 73). The profound tissue specificity of this pancreatic MYC phenotype is evident from comparison with the adeno-carcinomas induced in the lung by the exact same combina-tion of KrasG12D and switchable Myc (ref. 74; Supplementary Table S1). As in pancreas, the MYC-induced adenocarcinoma phenotype in the lung arises immediately following Myc activation, and involves rapid onset of tumor cell prolifera-tion and invasion, influx of macrophages, and T-cell exclu-sion. However, although some stromagenic mechanisms appear broadly similar in both organs, for example, CCL9-dependent MYC-driven influx of macrophages, the MYC-driven adenocarcinoma transitions in pancreas and lung differ dramatically in several key respects, both with regard to the phenotypes induced and the MYC-induced signals that instruct them. Thus, the dramatic and rapid MYC-driven influx of granulocytic and B lymphoid cells into nascent pancreatic tumors is entirely absent in the lung. Indeed, Myc activation has precisely opposite impacts on B cells in lung versus pancreas, driving their expulsion in the former and influx to the tumor periphery in the latter. Such differences are reflected in the underlying paracrine signals responsible in each case. IL23 is responsible for MYC-driven exclusion of B (and T and NK) cells in the lung (74), yet it is not detectably induced in the pancreas: Conversely, the key GAS6–pAXL path-way required for neutrophil/granulocyte and B-cell chemoat-traction in the pancreas appears absent from MYC-driven lung adenocarcinomas. Indeed, even when a common mechanism is ostensibly shared across both tissues, for example, the obligate role of PD-L1 in MYC-driven exclusion of CD3+ T cells, the mechanism of pathway deployment is quite different in the two tissues. In the pancreas, PD-L1 is expressed cell-autonomously in MYC-expressing PanIN epithelial cells, apparently through direct MYC-dependent transcription. In contrast, PD-L1 is never appreciably induced in MYC-expressing lung adenoma epithe-lial cells themselves and is, instead, ported into incipient tumors on incoming alveolar macrophages. Perhaps the most overtly different consequences of MYC action between the two tissues is the rapid activation and proliferation of local fibroblastic and stellate cells in the pancreas, triggering progressive deposition of desmoplastic stroma, leading to rapid blood vessel compression and hypoxia. In contrast, MYC activation in the lung elicits no appreciable activation of local fibroblastic cells or desmoplasia and instead engages a potent proangiogenic switch, fueled by macrophage-derived VEGF, which rapidly restores normoxia to the otherwise indolent, hypoxic adenomas (74).

A most remarkable feature of the complex, multicell lin-eage tumor–stroma phenotypes that MYC instructs in the pancreas and lung is that each closely matches the distinctive tumor–stromal phenotype of spontaneous adenocarcinomas arising from each of those same tissues. The inescapable inference is that adenocarcinoma phenotypes are principally specified by their host tissue of origin rather than by the pecu-liar ensemble of oncogenic mutations that drive them. Thus, RAS and MYC serve as common, tumor-agnostic downstream conduits of the multitude of different oncogenic mutations






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that lie upstream and, rather than specifying each tumor phe-notype, KrasG12D and Myc instead unlock a preconfigured tissue- resident program. The likely source of this tissue-resident program is suggested by the remarkable phenotypic overlap between PDAC (spontaneous and MYC-driven) and that of regenerating pancreas following injury. Both exhibit marked acinar–ductal metaplasia, epithelial proliferation, rapid infil-tration of macrophages and neutrophils, exclusion of T cells, activation of stellate cells, and an extreme fibroinflamma-tory reaction (75–78), attributes so overtly similar as to fre-quently confound differential diagnosis between pancreatitis and early PDAC. To summarize: oncogenic MYC “hacks” the endogenous pancreatic regenerative program (79).

Our data show that Myc activation in KMp48 or KMpdx1 PanIN epithelial cells triggers release of a diverse set of para-crine signals that quickly and synchronously engage highly reproducible and coordinated changes across all stromal com-partments. Macrophage influx, initiated within only a few hours of Myc activation, is long associated with especially poor PDAC prognosis. Pancreatic macrophages, which have diverse origins and functions (80, 81), are, in PDAC, implicated in promoting epithelial-to-mesenchymal transition and tumor cell migration, suppressing immunity, driving myofibroblas-tic transdifferentiation of stellate cells and cancer-associated fibroblasts (CAF) through release of multiple secondary signals including PDGF and TGFβ, and deploying various proteases that actively remodel stroma (81–85). Heavy infiltration with Ly-6B+ neutrophils and their G-MDSC kin is also associated with the most undifferentiated, aggressive, and metastatic forms of PDAC with the poorest prognosis (10, 55, 86, 87) and is thought to be a consequence of their secretion of multiple stromal remodeling proteases and diverse secondary signal-ing molecules including IL8, GM-CSF, CCL3, CXCL10, IL2, TNFα, and oncostatin-M (55), and through suppression of T-cell surveillance (88). CXCL5 is a well-characterized neu-trophil attractant in PDAC (84, 86, 88) that is induced within 6 hours of MYC activation. However, prompted by the previously reported role of GAS6–AXL signaling in neutrophil dynamics (89, 90) and the rapid MYC-dependent induction of GAS6, a member of the Protein S family of vitamin K–dependent plasma glycoproteins, we uncovered an additional signaling pathway necessary for MYC-driven neutrophil influx. GAS6 is a ligand for the TAM receptor tyrosine kinase family (TYRO3,

AXL, and MER) and its induction by MYC temporally corre-lated with appearance of pAXL in a proportion of PanIN epi-thelial cells. Blockade of either GAS6 with low-dose warfarin (59, 90) or AXL kinase with the selective AXL kinase inhibitor TP-0903 (58, 91, 92) blocked MYC-induced neutrophil influx and markedly suppressed proliferation in both tumor cell and stromal compartments, most especially stellate cells and CAFs, while exerting only a modest inhibitory impact on macrophage infiltration (Fig. 4). Unexpectedly, GAS6–AXL blockade also completely blocked the rapid influx of B220+ CD20+ B cells to the peritumoral stroma that MYC trig-gers. A pivotal role for B lymphocytes has lately emerged in PDAC progression and aggression, most notably innate B1b cells, hitherto implicated in physiologic processes such as microbial defense and removal of debris after tissue damage (93, 94). B cells are attracted to incipient PDAC via CXCL13 and, through their release of IL35, are thought to suppress T-cell immunity (95–97), promote PDAC cell proliferation and invasiveness, and inhibit apoptosis (98, 99). Diverse studies have demonstrated GAS6–AXL pathway activation in both tumor and stromal compartments of various solid tumors, including PDAC (57), and shown that GAS6–AXL pathway inhibition impedes growth of many cancers includ-ing PDAC (100) and promotes responses to chemotherapy (60, 91, 92, 100, 101). However, although GAS–AXL signaling is broadly implicated in regulating aspects of immunity (89, 92, 102–105) and tumor–stromal signaling (57), there is no previously described involvement of GAS6–AXL signaling in either neutrophil or B-cell dynamics in PDAC. Currently, it remains unclear what relative contribution of inhibition each of the individual target cell populations of GAS6–AXL blockade—neutrophils, B lymphocytes, or stellate cells—makes to MYC-driven PDAC progression, or whether each of these targets is affected independently or some are contingent upon another. Although our current analysis of signaling mol ecules is less comprehensive than recent proteome-based studies (57), our kinetic data provide unique causal infor-mation as to both the sources and sequence of signals and responses that assemble the PDAC tumor–stromal complex.

Concomitantly with the influx of macrophages, neutro-phils, and B cells, Myc activation induced immediate expul-sion of CD3+ T lymphocytes from PanINs, and this coincided temporally with induction of surface PD-L1 expression on

Figure 7. Depletion of CD20+ B cells or NKp46+ cells, but not CD4+ and CD8+ T cells, significantly retards Myc deactivation–induced tumor cell death in KMp48 PDAC. A, T cells. Tamoxifen was administered to 12-week-old KMp48 mice (Myc ON) for 3 weeks to induce pancreatic tumors. Starting 4 days prior to tamoxifen withdrawal (Myc OFF), mice were injected every other day (injections at −4, −2, 0, +2 days relative to tamoxifen withdrawal at day 0) with either IgG control or with neutralizing antibody against CD8 and twice four days apart (−4 and 0 days) with neutralizing antibody against CD4. Mice were eutha-nized three days after Myc deactivation (+3 days). Sections of treated pancreata were stained for T cells (CD3 IHC, left and middle) and cell death (TUNEL IF). Boxed regions shown at higher magnification (right), and red arrows indicate CD3+ cells. Scale bars apply across each column. Respective quantification is presented to the right of the relevant micrographs. n = 4 per group. B, B cells. Tamoxifen was administered to 12-week-old KMp48 mice (Myc ON) for 3 weeks to induce pancreatic tumors. Two days before cessation of tamoxifen diet (−2 days), mice were injected with either IgG control or a B lymphocyte– specific neutralizing antibody against CD20. Mice were then euthanized three days after Myc deactivation (+3 days). Sections of treated pancreata were then stained for B cells (B220 IF, left and middle) and apoptosis (TUNEL IF). Scale bars apply across each column. Insets show higher magnification. Respective quantification is presented to the right of the relevant micrographs. n = 6 per group. C, NK cells. Tamoxifen was administered to 12-week-old KMp48 mice (Myc ON) for 3 weeks to induce pancreatic tumors. Starting 4 days prior to withdrawal of tamoxifen, mice were then injected every other day (at −4, −2, 0, +2 days) with either IgG control or α-asialo-GM1 blocking antibody and euthanized 3 days after Myc deactivation (+3 days). Sections of pan-creata from treated mice were stained for NK and NK-like cells (anti-NKp46+ IF, left and middle) and cell death (TUNEL IF, right). Scale bars apply across each column. Insets show higher magnification. Respective quantification is presented to the right of the relevant micrographs. n = 6 per group. D, Systemic B-cell ablation blocks NK-cell influx: representative NKp46 IF staining of sections from pancreata from KMp48 mice before Myc deactivation (left), IgG control–treated 3 days after Myc deactivation (middle), and α-CD20-B-cell–ablated mice 3 days after Myc deactivation (right), as described in B above. Regions identified in insets (top row) are shown at higher magnification below. Scale bars apply across each row. T, tumor; broken yellow lines delineate tumor borders. ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant. Data were analyzed using unpaired Student t test. FOV, field of view.






PanIN epithelial cells in vivo. Moreover, ChIP analysis located MYC on the CD274 (PD-L1) promoter, implicating CD274 as a direct MYC target in the pancreas, as suggested in some other tissues (106). Although T-cell expulsion is not currently established as a mechanism of PD-L1 action, the very same physical location dependency of T cells on absence of PD-L1 has also been observed in lung adenocarcinoma progression. Moreover, the rapidity, immediacy, and reversibility of PD-L1 expression and T-cell exclusion suggest that there is a tight causal relationship between the two. Nonetheless, although systemic blockade of PD-L1 abrogated MYC-induced T-cell efflux, indicating that PD-L1 is causally required for MYC-induced T-cell efflux, the consequent persistence of T cells in the tumor masses had no discernible inhibitory impact on either PDAC maintenance or subsequent growth. These data are consistent with previous studies in both pancreas (107) and lung (74) that indicate that enforced persistence of T cells in tumors is, alone, insufficient to engage significant antitumor immune responses.

Activation (α-SMA) and mitogenesis of local stellate and fibroblastic cells was also evident rapidly following Myc acti-vation in PanIN epithelial cells and was rapidly succeeded by progressive desmoplastic deposition, vascular occlusion and hypoxia. A more detailed future analysis will be needed to ascer-tain whether physical proximity of mesenchymal cells to PDAC epithelial cells (108) influences their MYC-dependent activa-tion. In spontaneous PDAC, activated stellate cells and CAFs are known to be the principal agents of desmoplastic deposition (5, 57, 109–111). Although the role of PDAC desmoplasia in vascular compression and hypoxia is generally accepted (5, 6, 8, 72, 73, 109, 112, 113), its influence on disease progression and response to therapy remains contentious (8, 114–117).

The final notable feature of the MYC-driven PDAC pheno-type is its rapid and complete reversibility upon subsequent Myc deactivation. Nor is such reversibility peculiar only to PDACs specifically driven by oncogenic MYC, because inhibi-tion of endogenous MYC triggers identical regression in spon-taneous KrasG12D-induced (and p53-deficient) mouse PDAC. Such rapid and efficient reversal is notwithstanding the fact that Myc is deactivated only in the tumor epithelial compart-ment. It nonetheless has an immediate impact on the diverse cell types that comprise the dense PDAC desmoplastic stroma, unraveling the complex tumor maintenance signals shuttling between them. Indeed, we see no evidence whatsoever of any aspect of PDAC pathobiology that is self-sustaining and inde-pendent of requirement for continuous MYC activity within the driving epithelial tumor compartment. Just like MYC-driven progression, onset of regression is immediate upon Myc deactivation, with overt evidence of phenotypic reversal evident within only a few hours of Myc deactivation: Efflux of macrophages and neutrophils, as well as downregulation of PD-L1 and reentry of CD3+ T cells, is accompanied by rapid deactivation of local mesenchymal stellate and fibroblastic cells, degradation of desmoplasia and blood vessel dilation, and tissue reoxygenation. Sustained blockade of MYC func-tion induces the quantitative and meticulous dismantling of even the most extensive tumor masses, restoring normal exocrine pancreas microarchitecture, albeit in a macroscopic organ badly disfigured by scars and cysts. In the main, PDAC disassembly reverses the protumorigenic processes that MYC

initially induced, but with some important exceptions. Most notably, Myc deactivation triggers a sharp wave of tumor cell death, which is dependent upon comigration of peritumoral B cells and NK-like cells into the tumor mass. We observed a similar requirement for NK-cell activity in regression of lung adenocarcinomas following Myc deactivation in vivo (74), sug-gesting that innate immune lymphocytes play a general role in the regression process, as recently suggested (74, 118).

As with MYC-driven PDAC genesis, the rapidity, reproduci-bility, efficiency, complexity, and tight choreography of PDAC regression all suggest it is a manifestation of some evolved physiologic process. Clues to the origin of this process lie in multiple studies of the postregenerative resolution phase of pancreas injury by which normal pancreas mass, architecture, and function are restored. Such injury resolution involves ductal-to-acinar transdifferentiation and the involution of fibrosis, and requires both the rapid exclusion of F4/80+ macrophages and neutrophils and the influx of lymphocytes (75, 119, 120). It seems plausible that, just as activating Myc hacks into the endogenous pancreas regenerative program, inhibiting MYC (or upstream oncogenic signals) hacks into a physiologic injury-resolution program that evolved to termi-nate postinjury regeneration, prune excess cells, and reinstate normal tissue architecture. Indeed, it is possible that enforced engagement of this same injury-resolution program is the principal mechanism by which targeted inhibition of onco-genic signaling exerts its curative impact in cancers.

METHODSGeneration and Maintenance of Genetically Engineered Mice

LSL-KrasG12D, p48cre/+, Pdx1-Cre, Trp53ERTAM, TRE-Omomyc, and Rosa26-lsl-MERT2 mice have all been described previously (18, 48, 61, 62, 121, 122). CMVrtTA [Tg(rtTAhCMV)4Bjd/J] mice were purchased from Jackson Laboratories. Mice were maintained on a 12-hour light/dark cycle with continuous access to food and water and in compliance with protocols approved by the UK Home Office guidelines under a project license (to G.I. Evan) at the Univer-sity of Cambridge (Cambridge, United Kingdom). Deregulated Myc activity was engaged in pancreatic epithelia of KMpdx1 and KMp48 mice by daily intraperitoneal administration of tamoxifen (Sigma-Aldrich, TS648) dissolved in sunflower oil at a dose of 1 mg/20 g body mass; sunflower oil carrier was administered to control mice. In vivo, tamoxifen is metabolized by the liver into its active ligand 4-Hydroxytamoxifen (4-OHT). For long-term treatment, mice were placed on tamoxifen diet (Harlan Laboratories UK, TAM400 diet) as described previously (64); control mice were maintained on regular diet. Omomyc expression was systemically induced in KPO (pdx1- Cre;LSL-KrasG12D; Trp53ERTAM KI;TRE-Omomyc;CMVrtTA) mice by addi-tion of doxycycline (2 mg/mL plus 5% sucrose) to their drinking water versus 5% sucrose for control mice.

Tissue Preparation and HistologyMice were euthanized by cervical dislocation and cardiac perfused

with PBS followed by 10% neutral-buffered formalin (Sigma-Aldrich, 501320). Pancreata were removed, fixed overnight in 10% neutral- buffered formalin, stored in 70% ethanol and processed for paraffin embedding. Tissue sections (4 μm) were stained with hematoxylin and eosin (H&E) using standard reagents and protocols or with Gomori Trichrome Stain (Polysciences, 24205), according to the manu facturer’s instructions. For frozen sections, pancreata were embedded in OCT (VWR Chemicals, 361603E), frozen on dry ice, and stored at −80°C.






Lectin StainingOne hundred microliters of fluorescein-conjugated Lycopersicon

esculentum lectin (FL-1171, Vector Laboratories) was administered intravenously three minutes before euthanasia. Pancreata were har-vested, and deparaffinized tissue sections were counterstained with Hoechst dye and imaged by fluorescence microscopy.

IHC and ImmunofluorescenceFor IHC or immunofluorescence (IF) analysis, paraffin-embed-

ded sections were deparaffinized, rehydrated, and either boiled in 10 mmol/L citrate buffer (pH 6.0) or treated with 20 μg/mL Pro-teinase K to retrieve antigens, depending on the primary antibody used. For IF studies, OCT-embedded sections were air-dried and fixed for 30 minutes in 1% paraformaldehyde. Primary antibodies used were as follows: rabbit monoclonal anti–Ki-67 (clone SP6) (Lab Vision, Thermo Fisher Scientific, 12603707), rat anti–Ki-67 (Clone SolA15, Thermo Fisher Scientific, 15237437), rat monoclonal anti-CD45 (clone 30-F11, BD Pharmingen, 553076), rat monoclonal anti-neutrophils (Clone 7/4, Cedarlane, CL8993AP), rat monoclonal F4/80 (clone Cl:A3-1, Bio-Rad, MCA497R); goat polyclonal anti-MMR/CD206 (R&D Systems, AF2535), goat polyclonal anti-NKp46/NCR1 (R&D Systems, AF2225), rabbit monoclonal anti-CD3 (clone SP7, Thermo Fisher Scientific, RM-9107-RQ), rabbit polyclonal anti-CD274/PD-L1 (Abcam, ab58810); rat monoclonal anti-CD45R/B220 (clone RA3-6B2, Thermo Fisher Scientific, MA1-70098), rabbit poly-clonal anti-αSMA (Abcam, ab5694), rabbit polyclonal CD31 (Abcam, 28364), rabbit polyclonal anti-HIF1α (Abcam, ab82832), rabbit poly-clonal anti-pAXL (Y779, AF2228, R&D Systems), mouse monoclonal anti Pan-Keratin (clone C11, NEB, 4545S). Primary antibodies were incubated with sections overnight at 4°C except for anti-CD3, which was applied for 20 minutes at room temperature. For IHC analysis, primary antibodies were detected using Vectastain Elite ABC HRP Kits (Vector Laboratories: peroxidase rabbit IgG PK-6101, peroxidase rat IgG PK-6104, peroxidase goat IgG PK-6105) and DAB substrate (Vector Laboratories, SK-4100); slides were then counterstained with hematoxylin solution (Sigma-Aldrich, GHS232). For IF analysis, primary antibodies were visualized using species-appropriate cross-adsorbed secondary antibody Alexa Fluor 488 or 555 conjugates (Thermo Fisher Scientific); slides were counterstained with Hoechst (Sigma-Aldrich, B2883) and mounted in Prolong Gold Antifade Mountant (Thermo Fisher Scientific; P36934). TUNEL staining for apoptosis was performed using the Apoptag Fluorescein in situ Apop-tosis Detection Kit (Millipore; S7110) according to the manufac-turer’s instructions. Images were collected with a Zeiss Axio Imager M2 microscope equipped with Axiovision Rel 4.8 software.

Western Immunoblotting Analysis of Pancreas LysatesPancreatic tissues were ground into powder in liquid nitrogen and

proteins extracted using standard protocols. Total protein lysates were electrophoresed on an SDS-PAGE gel and blotted onto Immobilon-P membrane (Millipore). Membranes were blocked with 5% nonfat milk and primary antibodies incubated overnight at 4°C. Secondary anti-bodies were applied for 1 hour followed by chemiluminescence visuali-zation. The following primary antibodies were used: MYC (ab32072; Abcam, 1:2,000 dilution) and β-actin (sc-69879, Santa-Cruz Biotech-nology, 1:5,000 dilution). HRP-conjugated secondary antibodies: goat anti-rabbit (sc-2301; Santa-Cruz Biotechnology 1:7,500 dilution) and goat anti-mouse (A4416, Sigma-Aldrich, 1:7,500 dilution).

Mouse Inflammation/Cytokine Antibody Arrays and qRT-PCR

Pancreata were collected at appropriate time points and snap- frozen in liquid nitrogen. Whole pancreas protein extracts were isolated and incubated with mouse inflammation/cytokine antibody arrays (40 targets, Abcam, ab133999; 97 targets, Abcam ab169820), according to

the manufacturer’s instructions. Signal intensity was determined using ImageJ software. Differential expression of cytokine RNA was assessed by qRT-PCR. For this, total pancreas RNA was isolated using a Qiagen RNeasy Plus Isolation Kit followed by cDNA synthesis (High Capacity cDNA RT Kit, Applied Biosystems, 4374966). qRT-PCR was performed using TaqMan Universal Master Mix II (Thermo Fisher Scientific, 4440038), according to the manufacturer’s protocol. Primers used were: Cxcl5 (Thermo Fisher Scientific, Mm00441260_m1), Gas6 (Thermo Fisher Scientific, Mm00441242_m1), Ccl9 (Thermo Fisher Scientific, Mm00436451_g1), Ccl2 (Thermo Fisher Scientific, Mm00490378_m1), Tbp (Thermo Fisher Scientific, Mm00446973_m1), Shh (Thermo Fisher Scientific, Mm00436528_m1). Samples were analyzed in triplicate on an Eppendorf Mastercycler Realpex 2 with accompanying software. Tbp was used as an internal amplification control.

RNA-seq AnalysisTotal RNA was isolated from KMp48 mouse pancreas using a Pure-

Link RNA Mini Kit (Thermo Fisher Scientific) and PureLink DNAse set (Thermo Fisher Scientific). The quality, quantity, and integrity of RNA were assessed by NanoDrop1000 spectrophotometer and 2100 Bioanalyzer (Agilent Technologies). RNA libraries were generated using TruSeq Stranded mRNA Library Prep Kit following the manu-facturer’s instructions (Illumina). RNA Libraries were run on the Ilumina NextSeq 500 using the 75 cycle high-output kit (single-end sequencing). The quality of the sequencing data was analyzed by the bioinformatics team at the Cambridge Genomic Services (CGS) at the University of Cambridge (https://www.cgs.path.cam.ac.uk) using FastQC v0.11.4. In summary, reads were trimmed using Trim-Galore v0.4.1 and those less than 20 bases long were discarded. Reads were mapped using STAR v2.5.2a. The Ensembl Mus. Musculus GRCm38 (release 95) reference genome was used with annotated transcripts from the Ensembl Mus. musculus GRC38.95.gtf file. The number of reads mapping to genomic features was calculated using HTSeq v0.6.0. Differential Gene Expression Analysis using the counted reads employed the R package edgeR v3.16.5 and the paired design model as suggested in the edgeR user’s guide. RNA-seq data have been deposited in the ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-8467.

Isolation, Immunofluorescence, Gene Expression, and ChIP-qPCR Analysis of Primary Mouse Pancreatic Cell Lines

Pancreatic tumors were isolated as described previously (8), but with modifications. Briefly, 12-week-old KMpdx1 mice were placed on tamoxifen diet for 3 weeks. Pancreatic adenocarcinomas were then harvested, minced, and digested at 37°C in Hank’s Balanced Salt Solution containing 2 mg/mL type V collagenase (Sigma-Aldrich, C9263) for 30 minutes under constant agitation. Digested tumors were centrifuged and the pellet further digested with 1× Trypsin-EDTA (0.05% trypsin, 0.02% EDTA; Sigma-Aldrich, 59418C) for 5 minutes. Proteases were inactivated by the addition of DMEM (Thermo Fisher Scientific, 41966-029) containing 10% FBS (Thermo Fisher Scientific, 10270106). Established cell lines were propagated in DMEM containing 10% FBS and maintained with 100 nmol/L of 4-OHT (Sigma-Aldrich H7904); they were routinely evaluated for Mycoplasma contamination using the VenorGeM Classic Kit (Myco-plasma Detection Kit for Conventional PCR) according to the manu-facturer’s protocol (Minerva Biolabs, 11-1050). Cells stored at early passage were thawed and cultured for up to 7 weeks in total. For immunofluorescence analysis, cells were fixed in 4% paraformaldehyde and permeabilized with 0.4% Triton X-100. Primary antibodies used were: mouse monoclonal anti-Amylase G-10 (Clone G-10, Santa Cruz Biotechnology, sc-46657), mouse monoclonal anti–pan-cytokeratin (clone AE1/AE3, Abcam, ab27988). Primary antibodies were incubated with fixed cells overnight at 4°C, and were visualized using species-appropriate cross-adsorbed secondary antibody Alexa Fluor 488 or






555 conjugates (Thermo Fisher Scientific) as described above. For gene expression analysis, total RNA was isolated, reverse transcribed, and amplified by RT-PCR as described above. Primers used were for Cd274/pdl1 (Thermo Fisher Scientific, Mm00452054_m1), Tbp (Thermo Fisher Scientific, Mm00446973_m1). Tbp was used as normalization control.

ChIP was performed as described in ref. 123, with modifications. Briefly, 107 cells cultured with 100 nmol/L 4-OHT or ethanol control for 6 or 24 hours were incubated with 1% formaldehyde (Sigma-Aldrich, F8775) at room temperature for 10 minutes. Fixation was quenched by addition of glycine to a final concentration of 125 mmol/L for 5 min-utes at room temperature. Cells were then washed with ice-cold PBS, pelleted, and resuspended in lysis buffer (10 mmol/L Tris-HCl pH 8.0, 85 mmol/L KCl, 0.5% NP40, 1 mmol/L DTT) with protease inhibitors (Sigma-Aldrich, 11836153001). Cross-linked lysates were sonicated to shear DNA to an average fragment size of 100 to 500 bp. The follow-ing antibodies were then used for immunoprecipitation: rabbit poly-clonal anti–c-MYC (Cell Signaling Technology, 9402), rabbit polyclonal anti-ERα (clone HC20; Santa Cruz Biotechnology, sc-543), normal rabbit IgG as a background control (Cell Signaling Technology, 2729). Chromatin regions enriched by ChIP were then identified by qPCR in triplicate using SYBR Green Reaction Mix (Thermo Fisher Scientific, 4385616) according to the manufacturer’s protocol. Pdl1 primers used for amplification were 5′-GTTTCACAGACAGCGGAGGT-3′ (forward) and 5′-CTTTAAAGTGCCCTGCAAGC-3′ (reverse) with NCBI refer-ence sequence NM_021893.3 and described in ref. 124. Calculations of average cycle threshold (Ct) and SD for triplicate reactions were performed and each DNA fraction normalized to its input to account for chromatin sample preparation differences.

Blocking Antibodies, Drug Preparation, and Drug Study Treatment Groups

For PD-L1 blocking studies, KMp48 mice were injected intraperito-neally with 160 μg of LEAF-purified anti-mouse CD274/PD-L1 (clone 10F.9G2, BioLegend, 124309) or rat IgG2b isotype control (clone RTK4530, BioLegend, 400644) every 2 days for 2 weeks, starting 1 day prior to Myc activation. Mice were euthanized after 2 weeks and pancreata harvested for histology. For CD4/CD8 blocking studies, KMp48 mice were administered tamoxifen diet for 3 weeks. Commenc-ing four days prior to returning the mice to normal (tamoxifen-free) diet they were injected twice intraperitoneally with 200 μg of rat anti-mouse CD4 (Clone GK1.5, BioXCell, BE003-1) four days apart, and four times with 200 μg rat anti-mouse CD8 (Clone 2.43, BioXCell, BE0061) every other day. Control mice were injected with an equiva-lent amount of rat IgG2b isotype control (Clone LTF-2, BioXCell, BE0090). Mice were euthanized 3 days post Myc inactivation and pan-creata harvested for histologic analysis. CD20-expressing B cells were blocked with 250 μg of Ultra-LEAF purified anti-mouse CD20 anti-body (clone SA271G2, BioLegend, 152104) or Ultra-LEAF purified Rat IgG2b isotype control (clone RTK4530, BioLegend, 400644). Two days before cessation of tamoxifen administration, mice were injected intravenously with 250 μg of antibody or isotype control. They were then euthanized 3 days post Myc inactivation and pancreata isolated for histologic analysis. To block NKp46+ cells, KMp48 mice were given tamoxifen diet for 3 weeks. Starting four days prior to returning the mice to normal (tamoxifen-free) diet, mice were intravenously injected every other day (total of 4 injections) with 120 μL of Ultra-LEAF Puri-fied anti-Asialo-GM1 Antibody (Clone Poly21460, BioLegend, 14602) or 120 μg of rabbit IgG isotope control (Biotechne, AB-105-C). Mice were euthanized one day after the last antibody injection coincident with third day post tamoxifen diet cessation.

For warfarin blockade of GAS6, KMp48 mice were randomized to receive either normal drinking water or water supplemented with 1 mg/L warfarin (Sigma-Aldrich, A2250) starting 1 day prior to Myc activation. Warfarin-containing water was replenished every 2 days. Mice were euthanized 3 days following Myc activation and pancreata harvested for histology. For AXL receptor tyrosine kinase inhibition,

the specific inhibitor TP-0903.tartrate (Tolero Pharmaceuticals, Inc.) was dissolved in 5% (w/v) Vitamin E TPGS + 1% (v/v) Tween 80 in water and 55 mg/kg administered by oral gavage to KMp48 mice every 2 days, starting 2 hours before initial Myc activation. Mice were eutha-nized 5 days later and pancreata harvested for histologic analysis.

In Vivo Ultrasound ImagingKMp48 mice were anesthetized by continuous infusion of 3% iso-

fluorane using a veterinary anesthesia system (VetEquip, California). The ventral, lateral, and dorsal abdominal surfaces were shaved and mice then injected intraperitoneally with 2 mL of sterile saline. Tumors were imaged using a Vevo 2100 imaging system (Visual Son-ics). Serial images were collected at 0.2-mm intervals throughout the entire tumor. Vevo2100 v1.6.0 software was used to assess tumor size at each time point from 3-D scans, identifying in the Z plane and calculating maximal width and length.

Quantification and Statistical Analysis of Tumor BurdenFor quantification of tumor burden, H&E sections were scanned with

an Aperio AT2 microscope (Leica Biosystems) at 20× magnification (resolution 0.5 μm per pixel) and analyzed with Aperio Software. Sta-tistical significance was assessed by Student t test, with the mean values and the SEM calculated for each group using Prism GraphPad software. Kaplan–Meier survival curves were calculated using the survival time for each mouse from each group, with the log-rank test used to calculate sig-nificant differences between the groups using Prism GraphPad software. Student t test was employed in statistical analyses of ChIP and RT-PCR. IHC and IF staining were quantified using Fiji open source software; statistical significance was determined by Student t test using Prism GraphPad software; data were represented as box-and-whisker plots, which show upper extreme, upper quartile, median, lower quartile, lower extreme. P values (ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001) for each group (the number of animals in each cohort varied between 4 and 7 for each category).

Disclosure of Potential Conflicts of InterestL. Soucek is CEO at Peptomyc S.L. and has ownership interest

(including patents) in the same. G.I. Evan is an advisory board member at AstraZeneca. No potential conflicts of interest were disclosed by the other authors.

Authors’ ContributionsConception and design: N.M. Sodir, L. Soucek, G.I. EvanDevelopment of methodology: N.M. Sodir, R.M. Kortlever, G.I. EvanAcquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): N.M. Sodir, R.M. Kortlever, T. Campos, L. Pellegrinet, S. Kupczak, P. Anastasiou, L. Brown SwigartAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): N.M. Sodir, R.M. Kortlever, V.J.A. Barthet, T. Campos, L. Pellegrinet, M.J. Arends, G.I. EvanWriting, review, and/or revision of the manuscript: N.M. Sodir, R.M. Kortlever, T. Campos, L. Soucek, M.J. Arends, T.D. Littlewood, G.I. EvanAdministrative, technical, or material support (i.e., reporting or organizing data, constructing databases): N.M. Sodir, P. Anastasiou, L. Brown Swigart, G.I. EvanStudy supervision: L. Brown Swigart, T.D. Littlewood, G.I. EvanOther (genotyping of animals, IF staining, assisting with other experiments): P. AnastasiouOther (interpretation of the histopathologic data): M.J. Arends

AcknowledgmentsWe are indebted to the members of the Evan laboratory,

Drs. Daniel Von Hoff (TGEN), Ronald Evans and Michael Downes






(Salk Institute), Aarthi Gopinathan and Christine Feig (Cambridge Cancer Institute), Daniel Masso (Vall d’Hebron Institute), and Fer-nanda Kyle Cezar (King’s College London) for invaluable discussion and advice. We also thank Alessandra Perfetto and Daniel Masso for assistance with animals, Stephanie Whike and Michaela Griffin for assistance with histology, and the Pre-clinical Genome Editing Core at the Cambridge Cancer Research Institute (CI) for assistance with ultrasound scans. We are grateful to Tolero Pharmaceuticals, Inc. for their generous gift of TP-0903. This study was supported by program grants Cancer Research UK C4750/A12077 and C4750/A19013A, the European Research Council (294851), and a Stand Up To Cancer-Cancer Research UK-Lustgarten Foundation Pancreatic Cancer Dream Team Research Grant (grant number: SU2C-AACR-DT20-16; all to G.I. Evan). Stand Up To Cancer (SU2C) is a division of the Entertainment Industry Foundation and the research grant is administered by the American Association for Cancer Research, the Scientific Partner of SU2C.

Received April 17, 2019; revised November 30, 2019; accepted January 10, 2020; published first January 15, 2020.

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2020;10:588-607. Published OnlineFirst January 15, 2020.Cancer Discov Nicole M. Sodir, Roderik M. Kortlever, Valentin J.A. Barthet, et al. PhenotypeMYC Instructs and Maintains Pancreatic Adenocarcinoma

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