Activating mutations in KRAS are among the most frequent events in diverse human carcinomas and are particularly prominent in human pancreatic ductal adenocarcinoma (PDAC). An inducible Kras G12D -driven mouse model of PDAC has established a critical role for sustained Kras G12D expression in tumor maintenance, providing a model to determine the potential for and the underlying mechanisms of Kras G12D –independent PDAC recurrence. Here, we show that some tumors undergo spontaneous relapse and are devoid of Kras G12D expression and downstream canonical MAPK signaling and instead acquire amplification and overexpression of the transcriptional coactivator Yap1. Functional studies established the role of Yap1 and the transcriptional factor Tead2 in driving Kras G12D -independent tumor maintenance. The Yap1/Tead2 complex acts cooperatively with E2F transcription factors to activate a cell cycle and DNA replication program. Our studies, along with corroborating evidence from human PDAC models, portend a novel mechanism of escape from oncogenic Kras addiction in PDAC.

Despite its critical role in PDAC biology, we sought to determine whether sustained oncogenic Kras suppression would result in tumor relapse and would illuminate tumor resistance mechanisms. Employing our previously described doxycycline (doxy)-inducible KrasGEM PDAC model, we identified relapse tumors (after Krasextinction induced tumor regression) that lacked transgene expression and instead harbored an activated Yap1/Tead2 transcriptional program enabling Kras-independent tumor cell proliferation that enlists the cooperative actions of the E2F transcription factor. Interestingly, our findings in the mouse model are reinforced by observation in human PDAC showing a prominence of similar transcriptional programs in the quasimesenchymal subset (QM subset) of pancreatic cancers, which are notable for lower dependency on oncogenic KRAS relative to other PDAC subsets ().

Given the essential roles of oncogenic Kras in both PDAC initiation and maintenance, mutant KRAS and its signaling pathways have been a major focus for the development of disease models for human PDAC (). To model anti-Ras therapy, we and others have generated an inducible KrasGEM PDAC model and established that extinction of Krasinduced rapid tumor regression, highlighting the potential clinical utility of targeting oncogenic KRAS in pancreatic cancer ().

Oncogenic Kras is required for both the initiation and maintenance of pancreatic cancer in mice.

Oncogenic Kras is required for both the initiation and maintenance of pancreatic cancer in mice.

The oncogene addiction and tumor maintenance paradigm () has rationalized the striking clinical responses achieved with drugs targeting driver oncogenes (). Despite significant clinical responses to targeted therapies, nearly all tumor remissions are followed by acquired resistance and tumor relapse. Resistance mechanisms vary considerably and include mutations blocking drug-target interaction, genetic alterations sustaining signaling in downstream pathways, or alternate survival pathways (). The pervasive disease recurrence following targeted therapy has motivated the use of inducible driver oncogene GEM models of cancers to proactively illuminate potential mechanisms of resistance employed by human cancers ().

Pancreatic ductal adenocarcinoma (PDAC) remains a largely incurable lethal disease with a median survival of ∼6 months (). The PDAC genome is characterized by a number of signature mutations involving the KRAS oncogene and the CDKN2A, TP53, and SMAD4 tumor suppressor genes and by significant chromosomal aberrations resulted from telomere dysfunction and centrosome abnormalities, among other mechanisms (). Activating mutations in KRAS are present in the majority of human PDAC cases, and genetically engineered mouse (GEM) models have substantiated critical roles of oncogenic Kras in driving tumor initiation and in enabling tumor progression along with deficiencies of P53, Ink4a/Arf, Smad4, and/or Pten tumor suppressors (). The panoply of signaling pathways engaged by oncogenic Kras provides a basis for its diverse tumor biological roles in proliferation, survival, metabolism, and tumor microenvironment remodeling ().

To gain insight into whether YAP1 might have a role in driving growth of human Kras-independent PDAC, we first compared its expression in previously established human Kras-dependent and Kras-independent PDAC cells (). In line with our murine findings, YAP1 expression was significantly higher in the human KRAS-independent PDAC cells ( Figure 6 B). Next, to determine whether YAP1 is required for growth of QM KRAS-independent subtype cells, we performed knockdown of YAP1 in two KRAS-independent QM human PDAC cell lines (Panc1 and PaTu8988T) and one wild-type KRAS cell line BxPC-3 ( Figures 6 C and 6D). shRNA-mediated knockdown of YAP1 strongly suppressed the proliferation of these cells, implying that YAP1 is indeed essential for their growth ( Figure 6 D). Together, our data indicate that the oncogenic Kras-independent relapse tumors tend to resemble the QM subtype of human PDAC and rely on alternative oncogenic mechanisms, including Yap1, for their growth.

Human PDAC was recently defined into three subtypes based on transcriptional profiles: classical, quasimesenchymal, and exocrine-like, which correspond with distinctive clinical outcome and therapeutic responses (). Six out of ten Kras-positive tumors, including the doxy-induced iKras lines and the iKrasrelapse tumors, show expression signatures of classical PDAC, which are highly linked to oncogenic Kras activity. In contrast, five of eight iKraslines clustered with QM subtype of human PDAC, which have been reported to show high expression of mesenchymal genes, lower KRAS expression, and less KRAS dependency ( Figure 6 A;). The fact that we see a statistically significant association between iKrasrelapse tumors and the quasimesenchymal subtype of human PDAC aligns well with the observation that these human tumors tend to be less KRAS dependent (chi-square test, p = 0.01). Interestingly,demonstrated that a YAP/FOS-mediated epithelial-to-mesenchymal transition (EMT) program can also drive KRAS-independent tumor growth, further corroborating the relationship between KRAS independence and Yap1 as well as underscoring the complexity of Yap1 signaling.

As noted, a subset of human PDAC cell lines becomes less dependent on oncogenic KRAS (). To assess the potential clinical relevance of our Kras-independent relapse PDAC tumors, we compared the transcriptomic profiling of the relapse tumors with those reported in primary human PDACs and PDAC cell lines (). As a control, we also performed transcriptomic analysis of doxy-induced iKras PDAC lines. When subjected to unbiased clustering analysis, 5 of 8 iKrasrelapsed tumor profiles clustered closely with each other, and 5 of 8 iKrasrelapse tumors clustered with the doxy-induced primary PDAC lines ( Figure 6 A), reinforcing the view that iKrasrelapse tumors are molecularly distinct from Kras-dependent tumors.

(D) Representative wells (top) of clonogenic growth of Kras-independent QM human PDAC cells (Panc1 and PaTu8988T) and the wild-type KRAS cell line BxPC-3 upon YAP1 knockdown. Quantification (bottom) from a representative experiment is shown on the right. Error bars represent SD of triplicate wells. ∗∗∗ p < 0.001.

(C) Western blots validating the knockdown of YAP1 in the indicated human PDAC cell lines by two independent shRNAs.

(B) Gene expression data reanalyzed fromshowing that YAP1 expression is significantly higher in the Kras-independent lines compared to Kras-dependent human PDAC cells. The y axis indicates gene expression data expressed as logmedian centered intensity. Boxed bars indicate the medians.

(A) Hierarchical clustering of murine PDAC iKras cells and the relapse tumors into different PDAC subtypes using PDAssigner genes (). Subtype analysis found statistically significant association between iKrasrelapse tumors and the QM subtype, whereas the iKrastumors are associated with the classical subtype (chi-square test, p = 0.01;). The subtype identity of the samples (in gray) is not apparent.

Next, to assess the functional importance of E2F binding, we blocked E2F activity utilizing a dominant-negative form of E2F1 (E2F1) that lacks the transactivation domain (). Expression of the dominant-negative E2F1 suppressed proliferation of Yap1-expressing cells ( Figure 5 F), supporting a role for E2F1 activity in enabling Yap1/Tead2-mediated bypass of tumor regression upon Krasextinction.

To assess a potential cooperative role of E2F in Yap1/Tead2-mediated bypass of tumor regression, we first sought to determine co-occurrence of TEAD- and E2F-binding sites among the differentially expressed genes in the Yap1-bypassed tumors. Using the TRANSFAC position frequency matrix, we found significant enrichment for genes containing putative binding sites for both Tead and E2F specifically in the promoters of genes that were upregulated (2-fold upregulated, p < 0.005) in the Yap1-bypassed tumors (19/241; p < 0.05). Using chromatin immunoprecipitation (ChIP), we next validated occupancy of E2F1 along with YAP1 (Flag-tagged)/TEAD2 (V5-tagged) at promoters of several representative genes with predicted E2F- and TEAD-binding sites and showed no occupancy in an intergenic region lacking any putative E2F/TEAD-binding sites ( Figure 5 E). We further validated occupancy of endogenous Yap1 at three representative loci in early passage cultures derived from Yap1 amplicon-positive tumors (E-1 and E-2), but not Yap1 amplicon-negative (E-3) tumor ( Figure S5 ). As a specificity control, a nonspecific IgG antibody failed to immunoprecipitate any of the above promoter fragments ( Figure 5 E). These in silico and ChIP analyses support the view that E2f cooperates with Yap1/Tead2 in coordinating downstream gene expression.

Endogenous ChIP for Yap1 in short term cultures from relapse tumors. Note Yap1 occupancy is seen at three representative loci preferentially in E-1 and E-2 but not in E-3. No occupancy was seen in the control intergenic region. Bars represent enrichment at target regions in the promoter relative to the 3′ region of each gene.

It has been suggested that Yap/Tead-mediated gene regulation may rely on a combinatorial network of transcription factors to drive gene expression thresholds (). To determine whether Yap/Tead cooperates with a particular transcription factor in a coordinated gene expression program, we performed promoter analysis and identified several transcription factor motifs, including E2F, several members of the ATF family (activating transcription factors), and CREB1 (cyclic-AMP response element binding protein 1) enriched in the promoters of differentially expressed genes in Yap1-bypassed tumors ( Figure 5 D and data not shown). We focused further study on the E2F family of transcription factors, as recent studies in Drosophila have emphasized that E2f1 is required for the full activation of specific target genes by fly Yap1 and Tead orthologs Yki and Sd ().

Our findings are in agreement with the known role of Yap1 in regulating normal cell proliferation through a Tead-mediated transcriptional program (). Indeed, several bona fide Yap1 target genes, including Ccnd1 and Birc5, were documented to be upregulated in Yap1-bypassed tumors. Together, these analyses support the view that Yap1 enables tumor growth upon Krasextinction through the coordinate activation of genes governing cell cycle and DNA replication.

Next, transcriptomic analyses were conducted to elucidate the molecular network of Yap1 actions that facilitate Kras-independent tumor growth. We defined the baseline gene expression upon extinction of oncogenic Kras and subsequently compared this expression profile to that in Yap1-bypassed tumors. To surmise molecular pathways associated with Yap1 overexpression, we performed gene set enrichment analysis (GSEA) of the expression profiles using gene sets for the canonical pathways in the Molecular Signature Database (MSigDB) (). Consistent with our tumor biological observations, GSEA indicated that a significant fraction of Kras-dependent gene sets that are rescued in the Yap1-bypassed tumors related to cell proliferation, DNA synthesis, and replication ( Figures 5 A–5C). We validated several of the differentially expressed genes by qRT-PCR, including mitotic kinases (including aurora kinase A [Aurka] and aurora kinase B [Aurkb]), budding uninhibited by benzimidazoles (Bub1), cyclins (including Ccna2, Ccnb1, Ccnb2, and Ccnd1), cell-division-associated proteins (including Cdc2, Cdc20, and Cdc25c), and DNA replication proteins (including minichromosome maintenance complex proteins Mcm5, Mcm6, and Mcm10).

(F) Dominant-negative E2F1 (E2F1 DN ) suppresses proliferation of Yap1 (or Yap1 S127A ) expressing cells. Quantification from a representative experiment is shown on the right. Error bars represent SD of triplicate wells. ∗∗∗ p < 0.001.

(E) ChIP showing YAP1 and TEAD2 occupancy at E2F1-bound promoters of several representative genes. No occupancy was seen in the control intergenic region (lacking any putative E2F/TEAD binding sites). IgG served as a specificity control for the antibody. Bars represent enrichment at target regions in the promoter relative to the 3′ region of each gene. Error bars represent SD of the mean.

(A) Representative heatmaps of the cell cycle and DNA replication genes enriched in Yap1-bypassed tumors compared to control (Gfp, off doxy for 24 hr). Expression levels shown are representative of log2 values of each replicate. Red signal denotes higher expression relative to the mean expression level within the group, and blue signal denotes lower expression relative to the mean expression level within the group.

Second, we examined whether increasing Tead2 levels could mimic the effect of Yap1 overexpression. Overexpression of full-length Tead2 did not promote anchorage-independent cell growth or orthotopic tumor growth, consistent with a lack of intrinsic transactivation activity of Tead2 (data not shown). However, expression of a transcriptionally active form of Tead2, Tead2-VP16 (a fusion protein of the N-terminal region of Tead2 containing the TEA domain and the activation domain of herpes simplex virus VP16;), in two independent iKras cells promoted orthotopic tumor growth ( Figures 4 G and S4 G), in the absence of Krasexpression. Together, these multiple lines of evidence establish a critical role for TEAD2 in mediating Yap1-driven tumor cell growth upon Krasextinction in our model system.

First, shRNA-mediated knockdown of Tead2 reduced the proliferation of early passage cultures derived from orthotopic Yap1-bypassed tumors, whereas Tead2 knockdown had no effect on the Kras-expressing iKras lines ( Figures 4 E and S4 E). Furthermore, overexpression of a previously characterized dominant-negative Tead2 mutant (Tead2), which harbors a deletion of the C-terminal Yap1-interacting domain while retaining the ability to bind DNA, strongly blocked tumor cell proliferation of Yap1-expressing cells in clonogenic assays ( Figures 4 F and S4 F). In this case as well, growth-suppressive activity of Tead2is specific to Yap1-expressing cells, as overexpression of Tead2did not suppress cell proliferation induced by oncogenic Kras ( Figure 4 F).

We thus performed a series of experiments to test whether Yap1 exerted its growth effects through Tead2. Mutation in Tead-binding domain (Tead-binding-defective Yap1and Yap1 Figures S4 B and S4C) completely abolished the ability of Yap1 to drive proliferation and substitute for oncogenic Kras both in vitro ( Figure 4 C) and in vivo ( Figure 4 D). Similar results were obtained using the Yap1double mutant ( Figures 4 D and S4 D). These results strongly suggest that Tead2 is a critical partner of Yap1 in promoting tumor cell growth in the absence of oncogenic Kras. Next, we sought corroborating evidence of the importance of Tead2 in mediating Yap1 function by directly blocking its activity using two complementary approaches.

Because Yap1 is a transcriptional coactivator and does not bind directly to DNA, we sought to determine whether transcription factors known to interact with Yap1 signaling might mediate its activity. Smad1, p73, Runx2, and Tead transcription factors are all known to mediate the effects of Yap1 in different contexts (). Interestingly, only Tead2 levels were significantly higher in iKrasrelapse tumors compared to the iKrasones ( Figure 4 A), and Tead2 physically interacted with endogenous Yap1 in early passage cultures from primary relapse tumors and in cells derived from Yap1-bypassed tumors ( Figures S4 A and Figure 4 B), implicating Tead2 as a candidate transcription factor mediating Yap1’s activity in our system. In line with this hypothesis, the TEAD family of transcription factors are genetically and biochemically validated mediators of YAP1’s proliferation-inducing function (), and this established activity is consistent with the observed robust tumor cell proliferation profile in recurrent tumors ( Figure 3 C).

(E) Expression of Tead2 in iKras or Yap1- or Yap1 S127 -expressing cells upon knockdown by two independent Tead2 shRNAs and the control shRNA (sh_Scr). Expression levels are relative to levels in control shRNA (normalized to 1). Error bars represent SD of the mean.

(D) Sustained expression of Yap1 S127A but not TEAD binding defective Yap1 mutant, Yap S94A/S127A can promote anchorage independent growth of iKras cells in the absence of doxy. For each condition, five random fields were counted. Error bars represent SD of the mean, ∗∗∗ p < 0.001.

(C) Gal4-fused TEAD2 was cotransfected with a 5 × UAS-Luciferase reporter and the indicated Yap1 construct to monitor the ability of Yap1 to transactivate the reporter. Data were normalized using Renilla luciferase. Experiments were performed in triplicates. Error bars represent SD of the mean.

(G) The transcriptionally active form of Tead2 (Tead2-VP16) can substitute oncogenic Kras for in vivo tumor growth. Representative images are shown at 6 weeks off doxy (n = 5 per group).

(F) Dominant-negative Tead2 (Tead2 DN ) selectively suppresses proliferation of Yap1 (or Yap1 S127A ) expressing cells, but not the Kras G12D -expressing iKras cells. Quantification of cell growth is shown below. Error bars represent SD of triplicate wells. ∗∗∗ p < 0.001.

(E) Representative wells (top) of the clonogenic growth assay upon knockdown of Tead2 by two independent shRNAs in Yap1 (or Yap1) expressing cells (described in Figure 3 C). Quantification of cell growth is shown below. Error bars represent SD of triplicate wells.p < 0.001.

(D) Mutation in Tead-binding domain of Yap1 (S94A) dramatically decreases the ability of Yap1 or Yap1 S127A to substitute for oncogenic Kras in vivo. Representative images are shown at 6 weeks off doxy (n = 5 per group).

(C) Sustained expression of wild-type Yap1, but not TEAD-binding-defective Yap1 mutants (Yap S94A and Yap1 Δ60–89 ), can promote anchorage-independent growth of iKras cells off doxy. For each condition, five random fields were counted. Error bars represent SD of the mean. ∗∗∗ p < 0.001.

(B) Tead2 interacts with Yap1 in Yap1 (Flag-tagged) expressing cells (described in Figure 3 C). Input (25%) is used as a reference.

(A) qRT-PCR for expression of Tead family of transcription factors (Tead1–4) in iKras − and iKras + relapse tumors. Error bars represent SD of the mean. ∗∗∗ p < 0.001.

These cell-culture-based findings were further substantiated in vivo by the ability of enforced Yap1 expression to substitute for Krasactivity in tumor maintenance. Specifically, Yap1-, Yap1-, and Kras-expressing iKras tumor cells grown orthotopically ( Figure 3 C) or subcutaneously ( Figure S3 A) in nude mice were able to resist tumor regression upon extinction of oncogenic Kras and were able to promote tumor growth and proliferation (as measured by Ki-67 staining, Figure 3 D), whereas GFP-expressing control iKras tumor cells fully regressed upon doxy withdrawal ( Figures 3 C and S3 A). Additionally, Yap1- or Yap1-expressing iKras cells (using two independently derived lines) showed Kras-independent tumor growth when injected into nude mice ( Figures S3 C and S3D). Furthermore, shRNA-mediated knockdown of Yap1 or Yapdramatically suppressed proliferation of short-term cultures from these Yap1-expressing orthotopic tumors (described in Figure 3 C), confirming that the growth of bypassed tumors was indeed Yap1 dependent ( Figures S3 E–S3G). Notably, Yap1-bypassed tumors and early passage derivative cell lines showed lower MAPK activity when compared to Kras-bypassed tumors. ( Figures 3 E, 3F, and S3 A). Together, these results indicate that enforced Yap1 expression can substitute for oncogenic Kras-driven tumor maintenance and associated tumor cell proliferation.

(F and G) Representative wells (F) of the clonogenic growth assay demonstrating reduced proliferation upon knockdown of Yap1 in Yap1 expressing cells. Quantification of cell growth is shown in (G). Error bars represent SD of duplicate wells.

(E) Expression of Yap1 in cultures from the Yap1- or Yap1 S127 -expressing orthotopic tumors (described in 3C) upon knockdown by two independent Yap1 shRNAs and the control shRNA (sh_Scr). Expression levels are relative to levels in control shRNA (normalized to 1). Error bars represent SD of the mean.

(C and D) Kras-independent tumor growth in two independent iKras cells expressing either Yap1 or Yap1 S127A . Gfp expressing cells are used as control and do not show growth. Tumor volumes (mm 3 ) were measured at the days indicated (n = 5 per group). Error bars represent SD of the mean; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

(B) Protein levels of YAP1 and KRAS in iKras cells engineered to ectopically express Yap1, Yap1 S127A or Kras G12V . Vinculin is used as a loading control.

(A) Yap1 mediated rescue of tumor regression (upon inactivation of Kras G12D ) in vivo in subcutanous xenografts generated from iKras cells expressing either Yap1 or constitutive Kras G12V . Mice (n = 5 per group) were kept on dox (Kras G12D on) for 7 days postimplantation and then switched to off dox. Tumor growth off dox was visualized by bioluminescent imaging and representative images (i) are shown at two weeks off dox, except for Kras G12V for which mice were imaged two weeks after switching to off dox and subsequently tumors harvested. Gfp expressing cells served as negative control and regressed upon Kras G12D inactivation while constitutive Kras G12V and Yap1 expressing cells continued to grow. Xenografts were stained with antibodies against proliferation marker Ki-67 (ii), pErk (iii) and Yap1 (iv). Gfp expressing tumor 48 hr after dox withdrawal was used as a negative control while Kras G12V expressing tumors grown off dox served as a positive control for the staining. Histology of representative tumors is shown in (v).

To evaluate whether Yap1 gain of function would provide a mechanism for Kras-independent PDAC growth, we engineered three independently derived Kras-dependent iKras tumor lines with wild-type Yap1 or constitutively active Yap1mutant (S127A mutation prevents Yap1 cytoplasmic sequestration by Lats;) constructs and assayed them for anchorage-independent growth in the absence of doxy ( Figures 3 A, 3B, and 3B ). As shown in Figures 3 A and 3B, Yap1 or Yap1expression along with Kras-expressing cells dramatically enhanced anchorage-independent growth, whereas GFP-expressing control cells showed profound impairment of cell growth in the absence of doxy. The ability of Yap to drive Kras-independent cell growth aligns with the ability of enforced Yap1 to complement loss of Kras function in human pancreatic cancer cell lines ().

(F) Signaling status of key RAS effectors in short-term cultures derived from three independent Yap1-expressing orthotopic tumors described in (C).

(D and E) IHC for Ki-67 (D, quantified on the right; error bars represent SD of the mean of five random fields) and pErk (E). Gfp-expressing tumor 72 hr after doxy was used as a negative control. Note the proficient proliferation (as visualized by Ki-67 staining in D) and the lack of MAPK activation (as visualized by low pErk staining in E) in the Yap1-expressing tumors.

(C) Yap1-mediated bypass of tumor regression (upon inactivation of Kras G12D ) in orthotopic xenografts generated from iKras cells used in (A). Mice (n = 10 per group) were kept on doxy for 7 days postimplantation and were then switched to off doxy. (Top) Tumor growth off doxy was visualized by bioluminescent imaging at 4 weeks off doxy except for Kras G12V , for which image is taken 2 weeks after switching animals off doxy. Gfp-expressing cells regressed upon Kras G12D inactivation. (Bottom) H&E staining of representative tumors is shown. Scale bar, 100 μM.

(B) Quantification of anchorage-independent growth assay in three independently derived iKras cells (grown off doxy, Kras G12D off). For each condition, five random fields were counted. Error bars represent SD of the mean. ∗∗∗ p < 0.001.

(A) Representative wells of anchorage-independent growth assay demonstrating the ability of Yap1 or Yap1 S127A to substitute for oncogenic Kras in promoting cell growth of iKras cells. Growth of Gfp-infected cells was impaired in the absence of oncogenic Kras.

To assess the possible role of Yap1, Birc2 and Birc3 overexpression in driving growth of the iKrasrelapse tumors, early passage primary cultures generated from tumors with and without the 9qA1 amplicon were monitored for cell growth following shRNA-mediated knockdown of Yap1, Birc2, or Birc3. Birc2 or Birc3 knockdown had no impact on cell growth relative to control shRNA-expressing cells ( Figures S2 E and S2F). In contrast, two independent shRNAs targeting Yap1 reduced proliferation in clonogenic assays as well as tumor growth and tumor cell proliferation (Ki-67, Figure 2 G) in Yap1-amplified relapse tumors (E-1 and E-2) but exerted no impact on cells without Yap1 amplification (E-9 and E-10) ( Figures 2 D–2F, S2 G, and S2H). Interestingly, persistent Yap1 knockdown in xenografts generated from Yap1 shRNA-expressing cells resulted in resumption of Krastransgene expression, which coincided with a modest increase in MAPK activity and increased tumor cell proliferation, further underscoring the importance of activated Kras in PDAC maintenance (data not shown). Together, these genomic and functional studies strongly support a role for Yap1 amplification-driven overexpression as a mechanism for Kras-independent PDAC recurrence.

Next, we explored the molecular mechanisms underlying spontaneous tumor relapse after Krasextinction. Array-based comparative genomic hybridization (aCGH) revealed that all iKrasrelapse tumors exhibited focal amplification of the Rosa26 locus rtTA allele, providing a likely basis for doxy-independent re-expression of the iKras transgene ( Figure S2 A, chromosome 6q). In iKrasrelapse tumors, the only recurrent genomic alteration was amplification of chromosome 9qA1 region, encompassing 11 genes encoding several metalloproteinases, the transcriptional coactivator Yap1, and the antiapoptotic genes Birc2 (cIap1) and Birc3 (cIap2). Of these, only Yap1, Birc2, and Birc3 showed a copy-number-linked increase in gene expression ( Figures 2 A–2C and S2 D). Further, YAP1 protein was found to be elevated in iKrasrelapse tumors bearing the 9qA1 amplicon (E-1, E-2, and E-5), whereas iKrastumors lacking the amplicon showed low levels of Yap1 ( Figures 2 B, 2C, and S2 C), pointing to additional escape mechanisms (see Discussion and[this issue of Cell]).

(G) Quantification of IHC staining for Ki-67 displayed as a percentage of cells positive for Ki-67 staining. Error bars represent SD of the mean of five random fields. ∗∗∗ p < 0.001.

(E) Xenograft tumor growth of cell cultures derived from E-2 expressing two independent Yap1 shRNA or control shRNA. Tumor volume was measured at the days indicated, and data shown is representative of results from two independent experiments (n = 5 per group). Error bars represent SD of the mean. ∗∗ p < 0.01; ∗∗∗ p < 0.001.

(D) Representative wells of the clonogenic growth assay upon knockdown of Yap1 by two independent shRNAs primary cultures in Yap1 amplicon + tumors (E-1 and E-2) and the iKras + tumors (E-9 and E-10). Nontargeting shRNA (sh_Scr) was used as control. Quantification of cell growth is shown at the bottom. Error bars represent SD of triplicate wells. ∗∗∗ p < 0.001.

(C) qRT-PCR for Yap1 in relapse tumors. Relative expression levels normalized to reference gene. Error bars represent SD of the mean.

(B) IHC for Yap1 in relapse tumors. Note the increased Yap1 expression in 9qA1 amplicon + tumors E-1, E-2, and E-5, but not 9qA1 amplicon − tumor E-3.

(A) aCGH plots of relapse tumors show that the 9qA1 locus containing Yap1 is focally amplified (denoted by red arrow) in E-1, E-2, and E-5. Normalized log 2 ratios for each probe are plotted.

(H) Xenograft growth of primary cell cultures derived from E-1 expressing two independent Yap1 shRNA or control shRNA. Tumor volumes (mm 3 ) were measured at the days indicated (n = 5 per group). Error bars represent SD of the mean; ∗∗∗ p < 0.001.

(G) Protein levels of YAP1 in E-1 and E-2 cells infected with control shRNA and two independent shRNA against Yap1.

(F) Representative wells (left) of the clonogenic growth assay demonstrating no effect on proliferation upon knockdown of Birc2 or Birc3 in E-1 and E-3. sh_Scr infected cells were used as controls. Quantification of cell growth is shown on the right. Error bars represent SD of duplicate wells.

(E) Expression of Birc2 and Birc3 in E-1 and E-3 expressing two independent shRNAs against Birc2 and Birc3 and the control shRNA (sh_Scr). Expression levels are relative to levels in control shRNA (normalized to 1).

(D) qRT-PCR expression of Birc2 and Birc3 in all the relapse tumors. Relative expression levels are indicated. Error bars represent SD of the mean.

(C) Yap1 expression in primary cell cultures derived from the indicated relapse tumors. YAP1 protein levels are higher in Yap1 ampliconrelapse tumors E-1, E-2 and E-5 but not in other relapse tumors. pAKT blot from Figure S1 C was stripped and reprobed with Yap1 antibody. Vinculin is used as a loading control and is duplicated from Figure S1 C.

(A) Minimum common region of 6qE3 showing amplification of Rosa26 locus (denoted by solid line) in the iKras + relapse tumors (E9 to E16; amplification indicated by red bar).

Doxy-independent tumor recurrence in this model could potentially result from doxy-independent activation of the iKras transgene or from Kras-independent mechanisms. Indeed, although doxy withdrawal results in loss of Krasexpression and downstream signaling in all primary tumors ( Figures 1 E and S1 C), approximately half of relapse tumors examined exhibited re-expression of the iKras transgene accompanied with canonical downstream signaling; these tumors were designated hereafter as iKrasrelapse tumors ( Figures 1 E–1G and S1 C, tumors E-9 to E-16). The remaining tumors did not express the iKras transgene or hyperactivated endogenous Kras expression and exhibited diminished canonical downstream signaling; these tumors were designated hereafter as iKrasrelapse tumors ( Figures 1 E–1G, S1 C, and S1 D, tumors E-1 to E-8). While displaying similar aggressive histopathological features, the iKrasversus iKrasrelapse tumors were readily distinguished molecularly on the basis of mitogen-activated protein kinase (MAPK) pathway activity. In contrast to iKrastumors, the majority of iKrastumor lines showed relatively lower phospho-Mek (pMek) and phospho-Erk (pErk) both in vivo and in vitro ( Figures 1 G and S1 C). Furthermore the iKrastumors did not show compensatory hyperactivation of AKT pathway, and levels of phospho-ribosomal protein S6 (pS6) were generally lower relative to iKrastumors ( Figure S1 C). Thus, although oncogenic Kras signaling in the primary tumors tightly controls MAPK pathway activity, recurrence of iKrastumors results from mechanisms that do not utilize oncogenic Kras or hyperactivated MAPK/AKT signaling. Because the wild-type Kras allele remains intact upon Krasextinction in our model system, the contribution of basal signaling activity from wild-type Kras or other Ras family members during tumor relapse remains to be elucidated.

The relapsed tumor lesions were of pancreatic origin, as demonstrated by the presence of Cre-mediated p53 deletion in cells cultured from the relapse tumors ( Figure S1 B). Furthermore, because the iKras PDAC mice do not develop pancreatic tumors in the absence of doxy induction (), we conclude that these doxy-independent pancreatic tumors are bona fide tumor relapses of the original primary tumors rather than tumors formed de novo in the absence of oncogene induction.

Using mice engineered with a doxy-inducible Krastransgene and conditional p53 null alleles (p48Cre; tetO_LSL-Kras; ROSA_rtTA; p53, designated iKras), we and others established that sustained Krassignaling is essential for pancreatic tumor maintenance (). To evaluate the potential for recurrence mechanisms following Krasextinction, we utilized MRI imaging to monitor regression of advanced pancreatic tumors measuring at least 8 mm in diameter at the time of doxy withdrawal. Consistent with previous findings (), Krasextinction resulted in complete regression despite significant tumor burdens in all animals (n = 28), with virtually no gross tumor detected by MRI imaging at 3 weeks following doxy withdrawal ( Figure 1 A). However, 70% of the mice (20/28) escaped from doxy withdrawal with evidence of relapse between 9 and 47 weeks, with a median survival of 36.6 weeks compared to 15.4 weeks for iKras mice maintained on continued doxy treatment (p < 0.0001) ( Figure 1 B). On the morphological level, in contrast to the well-differentiated ductal features and predominant CK19 (ductal marker) positivity of the doxy-induced PDACs ( Figure S1 A available online) (), all recurrent tumors exhibited poorly differentiated or sarcomatoid features. They were devoid of acinar (amylase) or endocrine markers (chromogranin; CHGA) staining, although some tumors partially retained scattered CK19 staining ( Figures 1 C and S1 A). Consistent with the development of more aggressive phenotypes, distal metastases to lung or liver were observed in 75% (15/20) of the animals with recurrent tumors versus 21% (8/38) (p < 0.001) of those carrying primary tumors ( Figures 1 C and 1D).

(C) Signaling status of key RAS effectors in short term cultures derived from parental iKras tumors and relapse tumors. Two independent iKras cells were maintained in the presence (+) or absence (-) of doxy for 24 hr and used as controls for the western. All the relapse tumors were grown in the absence of doxy. Vinculin is used as a loading control.

(B) Genomic DNA PCR confirming the presence of Cre-mediated p53 deletion in cells cultured from the relapse tumors (E-1 to E-16). Matching tail DNA is used as a negative control.

(A) Histopathological characterization of the relapse tumors. Representative primary tumors from the iKras mice and the relapse tumors were stained with antibodies to ductal marker (Cytokeratin 19; CK19), acinar marker (AMYLASE) and endocrine marker (chromogranin; CHGA).

(E and F) (E) qRT-PCR for Kras G12D transgene shows expression in relapse tumors. Data represented as relative normalized expression. (F) Measurement of Ras activity in relapse tumors. Two independent iKras cells were maintained in the presence (+) or absence (−) of doxy for 24 hr and used as controls. Error bars represent SD of duplicate samples.

(C) Histopathological characterization of the relapse tumors showing poorly differentiated (i) or sarcomatoid (ii) relapse tumors, with liver (iii) and lung (iv) metastasis (denoted by arrow).

(B) Kaplan-Meier overall survival analysis for iKras; p53 L/+ mice after doxy withdrawal. (On) Mice were fed with doxy. (Off) Mice with advanced PDAC were switched to doxy-free water 8–15 weeks after on doxy and observed for relapse.

Oncogenic Kras is required for both the initiation and maintenance of pancreatic cancer in mice.

Oncogenic Kras is required for both the initiation and maintenance of pancreatic cancer in mice.

Discussion

Shao et al. (2014) Shao D.D.

Xue W.

Krall E.B.

Bhutkar A.

Piccioni F.

Wang X.

Schinzel A.C.

Sood S.

Rosenbluh J.

Kim J.W.

et al. KRAS and YAP1 converge to regulate EMT and tumor survival. In this study, we investigated potential resistance mechanisms to oncogenic Kras extinction in the context of significant tumor burden. Following oncogenic Kras extinction and complete tumor regression in all animals, approximately one-third of the animals indeed remained tumor free over a period of up to 65 weeks. This observation, together with re-expression of the iKras transgene in approximately half of the mice with tumor recurrence, emphasizes the prominent role of oncogenic Kras in tumor maintenance. Tumor recurrence following complete extinction of oncogenic Kras was not anticipated, given the wide spectrum of critical pathways controlled by Kras in cancer. At the same time, our work and that ofdemonstrate the potential for oncogenic Kras-independent bypass mechanisms involving the Yap1 oncogene, emphasizing that PDAC tumor cells can survive in the absence of oncogenic Kras signaling and can acquire alternative mechanisms to foster their own growth, portending the need for anti-Yap1 therapeutic strategies for some tumors in the setting of agents targeting Kras and its signaling pathways.

Our results have important ramifications in anticipating clinical responses in drugs designed directly to target oncogenic Kras. Based on this study and resistance mechanisms discovered in response to other targeted therapies, at least three distinct resistance mechanisms against Kras extinction are possible. First, genomic alterations can act on target itself, driving relapse by circumventing target blockade. This is supported by our observation that, in approximately half of the relapse tumors, the iKras transgene is amplified. Second, augmentation of key growth signaling pathways through activation of compensatory pathways may induce tumor relapse. In agreement with such a notion, Shao et al. showed that expression of receptor tyrosine kinases bypass the dependency on oncogenic Kras. Our preliminary gene expression data raise the intriguing possibility of activation of multiple RTKs and/or their ligands in Yap1 amplicon-negative relapse tumors (E-3, E-4 and E6-E8; data not shown). Third, and most importantly, we and Shao et al., have uncovered a novel mechanism of resistance to Kras inhibition through a Yap1-mediated transcriptional program. Amplification of Yap1 in our study is reminiscent of classic second-site suppression events that substitute for critical functions of oncogenic Kras, particularly tumor cell proliferation, thus allowing Kras-dependent tumors to escape dependency on oncogenic Kras.

Cao et al., 2008 Cao X.

Pfaff S.L.

Gage F.H. YAP regulates neural progenitor cell number via the TEA domain transcription factor. Tamm et al., 2011 Tamm C.

Böwer N.

Annerén C. Regulation of mouse embryonic stem cell self-renewal by a Yes-YAP-TEAD2 signaling pathway downstream of LIF. G12D extinction (A.K., A.V., et al., unpublished data). It is not clear, however, whether YAP1 amplification is already present in these rare oncogene-independent cells before KrasG12D ablation or whether it is acquired after oncogene extinction. Further work using clonal tracking methodologies would be needed to define the relationship between these surviving cells and the relapse tumors seen in our model. In our study, Tead2 is required for Yap1-mediated tumor relapse. Interestingly, Tead2 has been shown to play an important role in stem cell maintenance and in self-renewal (), and thus we speculate that residual surviving tumor cells or tumor stem cells following Kras extinction maintain their viability in a Tead2-dependent manner. This rare subpopulation of PDAC cells may be enriched for tumor-initiating activity and may be capable of surviving genetic or pharmacological inactivation of Kras and its surrogates. Such surviving cells could provide a reservoir of relapsed tumor cells to enable acquisition of Kras-independent tumor maintenance events. In support of this hypothesis, we have generated preliminary data showing Tead2 is highly expressed and is important for survival of a subpopulation of tumor cells that survive Krasextinction (A.K., A.V., et al., unpublished data). It is not clear, however, whether YAP1 amplification is already present in these rare oncogene-independent cells before Krasablation or whether it is acquired after oncogene extinction. Further work using clonal tracking methodologies would be needed to define the relationship between these surviving cells and the relapse tumors seen in our model.

Shao et al. (2014) Shao D.D.

Xue W.

Krall E.B.

Bhutkar A.

Piccioni F.

Wang X.

Schinzel A.C.

Sood S.

Rosenbluh J.

Kim J.W.

et al. KRAS and YAP1 converge to regulate EMT and tumor survival. Shao et al., 2014 Shao D.D.

Xue W.

Krall E.B.

Bhutkar A.

Piccioni F.

Wang X.

Schinzel A.C.

Sood S.

Rosenbluh J.

Kim J.W.

et al. KRAS and YAP1 converge to regulate EMT and tumor survival. Pobbati and Hong, 2013 Pobbati A.V.

Hong W. Emerging roles of TEAD transcription factors and its coactivators in cancers. Zhao et al., 2011 Zhao B.

Tumaneng K.

Guan K.L. The Hippo pathway in organ size control, tissue regeneration and stem cell self-renewal. The findings that Yap1 can substitute for oncogenic Kras in advanced PDAC raise the possibility that Yap1 can similarly substitute for activated RAS in other malignancies or in other cellular contexts. In agreement with this supposition,identified Yap1 in a gain-of-function screen to identify genes that can substitute for Ras signaling in KRAS-dependent human cancer cells. Consistent with the pleiotropic effects of Yap1, both studies converge on overlapping networks, such as ATF and E2Fs, and diverge on distinct transcriptional programs, such as Tead2 (this study) and Fos (). Our convergent and contrasting findings are consistent with the established fact that the Yap1-mediated gene expression program is largely dictated by the cellular context and its interacting transcription factors ().

− relapse tumors display features similar to the QM subtype of human PDACs associated with poor prognosis ( Collisson et al., 2011 Collisson E.A.

Sadanandam A.

Olson P.

Gibb W.J.

Truitt M.

Gu S.

Cooc J.

Weinkle J.

Kim G.E.

Jakkula L.

et al. Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy. Singh et al., 2009 Singh A.

Greninger P.

Rhodes D.

Koopman L.

Violette S.

Bardeesy N.

Settleman J. A gene expression signature associated with “K-Ras addiction” reveals regulators of EMT and tumor cell survival. Interestingly, the iKrasrelapse tumors display features similar to the QM subtype of human PDACs associated with poor prognosis (). Although YAP1-amplified tumors clearly fall into the nonclassical subtype, the limited number of tumors prevents us from definitely subgrouping the tumors with YAP1 amplification. However, the significant correlation between iKras status and classical/QM subtypes aligns with previously published results showing that Kras dependency is strongly linked to epithelial differentiation status and that, upon EMT, Kras dependency is reduced in human cancer cells (). Furthermore, the observation by Shao et al. that YAP1 can replace oncogenic Kras in part by regulating an EMT-like program further supports the link between EMT status, Kras independence, and Yap1 expression and also underscores the complexity of Yap1 signaling.