Five-year survival for pancreatic ductal adenocarcinoma (PDAC) patients remains below 7% due to the lack of effective treatments. Here, we report that combined ablation of EGFR and c-RAF expression results in complete regression of a significant percentage of PDAC tumors driven by Kras/Trp53 mutations in genetically engineered mice. Moreover, systemic elimination of these targets induces toxicities that are well tolerated. Response to this targeted therapy correlates with transcriptional profiles that resemble those observed in human PDACs. Finally, inhibition of EGFR and c-RAF expression effectively blocked tumor progression in nine independent patient-derived xenografts carrying KRAS and TP53 mutations. These results open the door to the development of targeted therapies for PDAC patients.

To date, no therapeutic strategy has achieved significant regression of human or mouse PDAC. Here, we describe the complete regression of a subset of mouse Kras/Tpr53 mutant tumors upon systemic ablation of EGFR and c-RAF expression. Similar results were obtained upon EGFR and c-RAF knockdown in PDX-derived tumor models. This therapeutic strategy was well tolerated due to the lack of effect on MAPK and PI3K signaling in normal tissues. EGFR inhibitors have already been approved to treat PDAC patients in combination with gemcitabine. Thus, our results should stimulate the identification of selective c-RAF inhibitors that preserve MAPK and PI3K activity. Availability of such inhibitors will make it possible to translate these observations into a clinical scenario.

So far, therapeutic strategies in genetically engineered mouse (GEM) PDAC models have failed to achieve tumor regression. Only tumor models driven by a doxycycline-inducible K-RASundergo tumor regression upon silencing of K-RASexpression (). Genetic studies in mice have illustrated that PDAC development requires EGFR expression due to its essential role in acinar to ductal metaplasia, a natural process thought to be responsible for tumor initiation in the presence of oncogenic K-RAS mutant (). However, in the absence of p53, EGFR expression is no longer essential for tumor development, although tumors appear with considerable delay, thus indicating that EGFR signaling still plays a role in the development of mutant Kras/Trp53-driven PDAC tumors (). Indeed, EGFR inhibitors have shown limited but reproducible responses in PDAC patients, leading to US Food and Drug Administration approval of erlotinib for treating PDAC (). Hence, we reasoned that combining inhibition of EGFR with other effector molecules known to play a role in K-RAS oncogenic signaling might unveil more efficacious therapies to treat PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is the third cause of cancer deaths in the US and is projected to become second after lung cancer by 2030 (). The 5-year survival rate of PDAC patients remains below 7% due to the lack of effective treatments. Gemcitabine, a nucleoside analog approved in 1997, is still the standard of care () and its combination with nab-paclitaxel or erlotinib has shown only modest improvements (). Other therapies such as FOLFIRINOX are very toxic and can only be administered to selected patients (). The main genetic drivers of PDAC have been identified (). Whereas mutations in KRAS appear to be the main initiating event, additional mutations in several tumor suppressors, including TP53, CDKN2A, SMAD4, BRCA2, and TGFΒR2 contribute to tumor progression. Unfortunately, none of these cancer drivers are currently druggable, thus making it difficult to devise effective therapies against PDAC. Only a small percentage of clinically relevant mutations may benefit from available targeted therapies ().

Finally, we determined whether pharmacological inhibition of the kinase activities of EGFR and c-RAF led to similar results. Unfortunately, none of three different c-RAF kinase inhibitors (MLN2480, GW5074, and PLX8394) displayed significant inhibitory activity in various in vitro and in vivo assays (). Thus, we had to inhibit c-RAF by knocking down its expression with specific shRNAs as described above. To block EGFR activity, we used two independent inhibitors, gefitinib and erlotinib (). Exposure of cells derived from the ten independent PDX tumors described above to gefitinib at half maximal inhibitory concentration (IC) determined in short-term cultures ( Table S4 ) along with the two independent c-RAF shRNAS resulted in the complete inhibition of their proliferations, including those cells derived from the PDX-6 tumor model that were partially resistant to EGFR and c-RAF knockdown ( Figure 8 ). Moreover, cells derived from 7 of 10 PDX tumor models underwent cell death, resulting in a reduced number of cells at the end of the 12-day experiment ( Figure 8 ). Similar results were obtained with erlotinib, except for PDX-3- and PDX-6-derived cells, which only displayed partial inhibition ( Figure 8 ). These results, taken together, suggest that pharmacological intervention may also result in significant inhibition of human PDAC tumors in the clinic.

Cell proliferation of the indicated PDX-derived cells infected with scramble shRNA (black), infected with shRNA R1 against c-RAF (red), infected with shRNA R1 against c-RAF and exposed to gefitinib (IC) (green), infected with shRNA R1 against c-RAF and exposed to erlotinib (IC) (blue) (left), and western blot analysis of c-RAF expression in whole-cell extracts obtained from the indicated PDX-derived cells using either a scramble shRNA (−) or a shRNA against c-RAF (R1) (right). Proliferation was determined by CellTiter-Glo and expressed as fold increase in the number of cells determined at each of the indicated days. Error bars indicate means ± SD. GAPDH served as loading control in western blots. See also Table S4

To determine whether combined inhibition of EGFR and c-RAF signaling could provide therapeutic benefit to PDAC patients, we knocked down their expression in cells derived from nine patient-derived xenograft (PDX) tumor models harboring KRAS and TP53 mutations ( Table S3 ). A tenth PDX tumor model (PDX-10) carrying a wild-type TP53 was also included in the study. Individual knockdown of EGFR or c-RAF expression with two independent shRNAs for each locus reduced their proliferative properties to various extents. However, combined knockdown of EGFR and c-RAF expression completely interfered with the proliferative capacity of those cell derived from nine of the ten PDX tumor models ( Figure S4 ). Only those cells derived from PDX-6 were partially inhibited upon EGFR or c-RAF knockdown. Four of the PDX-derived tumor cells that fully responded to EGFR or c-RAF knockdown (PDX-1 to -4) were injected into immunocompromised mice. Again, only the combined knockdown of EGFR and c-RAF effectively inhibited growth of these human PDAC tumor cells in vivo ( Figure 7 ). These observations suggest that combined inhibition of EGFR and c-RAF expression may have significant therapeutic activity in human PDAC tumors.

Western blot analysis of EGFR and c-RAF protein expression in whole-cell extracts obtained from (left), tumor growth curve (center), and quantification of tumor growth at the end of the experiment (right) of the indicated PDX cell line infected with a scramble shRNA (–, black) or with shRNAs against EGFR (E1, blue), c-RAF (R1, red), and EGFR plus c-RAF (E/R, green). GAPDH served as a loading control. Error bars indicate means ± SD. p values were calculated using the unpaired Student's t test.p < 0.05,p < 0.01,p < 0.001. n.s, not significant. See also Table S3 and Figure S4

Gene set enrichment analysis (GSEA) () identified several pathways enriched in RC versus NC cells ( Figure 6 B). The most significantly enriched gene signatures in RC cells included those corresponding to “bile acid, cholesterol, xenobiotic, and fatty acid metabolism,” “apoptosis,” and “p53 pathway” ( Figure 6 B). Significantly enriched gene sets in the NC cells were those corresponding to “E2F targets,” “EMT,” and “MYC targets.” Other enriched pathways included the “PI3K/AKT/mTOR” and “IL6/JAK/STAT3” signaling pathways ( Figure 6 B). Comparison of data obtained by RNA-seq analysis with a transcriptional classification of human PDACs () revealed that NC cells displayed a transcriptional profile most similar to the “squamous subtype.” In contrast, RC cells fit best with the other classifications, “Immunogenic,” “ADEX,” and “Progenitor” () ( Figure 6 C). A list of 57 genes selected from pathways known to play relevant roles in PDAC development and progression is highlighted in Figure 6 D.

Next, we determined the transcriptional profiles by RNA sequencing (RNA-seq) analysis of three RC cell lines and three NC cell lines. As illustrated in Figure 6 A, RC and NC cells displayed distinct transcriptional profiles that included more than 2,000 differentially expressed genes ( Table S2 ).

(D) Genes selected from the 2,000 genes differentially expressed between RC and NC cells based on their involvement in signaling pathways known to participate in the development and/or progression of PDAC. Genes are ordered according to the log2 fold change.

Analysis of known K-RAS effectors in NC and RC cells failed to demonstrate significant differences in the phosphorylation levels of the ERK and AKT kinases as well as in pCOFILIN ( Figure 5 C), suggesting that the MAPK, PI3K, and ROCK1 pathways might not be responsible for the proliferation of NC cells in the absence of EGFR and c-RAF expression. Interestingly, ablation of EGFR and c-RAF expression in NC cells induced increased phosphorylation of STAT3 at the canonical Tyr705 residue ( Figure 5 C). These observations were further substantiated by immunohistochemical (IHC) analysis ( Figures 5 D and 5E). Tumors present in “Non Responder” mice, as well as in mice that became resistant to Egfr and Raf1 ablation, constitutively expressed high levels of nuclear pSTAT3 ( Figures 5 D and 5E). No increase in pSTAT3 expression was observed in control KPeFC tumors or in those few ductal-like cells present in the residual scars of “Regressor” mice ( Figures 5 D and 5E).

To gain insights into the mechanisms responsible for the differential responses of these PDAC tumors to EGFR and c-RAF ablation, we generated tumor cell lines from 15 KPeF;Egfr;Raf1mice that were not enrolled in the preclinical trial because they lacked the UBC-CreERT2 transgene. Elimination of EGFR and c-RAF expression upon infection with AdCre particles led to apoptotic cell death, as determined by cell cycle analysis, in 4 of these 15 cell lines designated as “Regressors” (RC). In contrast, other cell lines (4 of 15) were completely resistant to cell death. They were designated as “Non Responder” cells (NC). The results obtained with 3 RC and 3 NC cell lines are illustrated in Figures 5 A and 5B. The remaining seven cells lines displayed a mixed phenotype with various percentages of cells undergoing cell death upon AdCre infection, thus suggesting the existence of intratumoral heterogeneity in these experimental tumors ().

Moreover, four KPeFC;Egfr;Raf1mice did not respond to the TMX diet. Tumors present in these animals, designated as “Non Responders” (N), progressed similarly to those present in control KPeFC mice ( Figures 4 D and 4E). Histopathological analyses did not reveal significant differences with tumors present in control animals or in KPeFC;Egfr; Raf1mice not exposed to a TMX diet ( Figure 4 F). These tumors did not express EGFR or c-RAF, yet they retained active MAPK and PI3K/AKT signaling pathways ( Figure 4 G). Thus, we hypothesized that these tumors must have undergone additional alterations that made them independent of EGFR/c-RAF signaling. Alternatively, they may have originated from a putative distinct type of acinar cell that does not require these signaling pathways for proliferation.

Tumors present in two mice that initially regressed with kinetics similar to those of the “Regressor” mice started to grow rapidly after 6–8 weeks of TMX exposure, killing them 4–5 weeks later ( Figure 4 A). Western blot analysis of tumor tissue revealed the absence of EGFR and c-RAF, indicating that tumor progression was not due to incomplete recombination of the conditional Egfr and/or Raf1 alleles ( Figure 4 B). Therefore, we have designated these mice as “Resistant” (T). Whether tumor progression was due to the acquisition of new mutations or to the emergence of clones that did not require EGFR and c-RAF signaling remains to be determined. Indeed, the tumor present in the T2 “Resistant” mouse had a distinct sarcomatoid phenotype as illustrated by the lack of expression of CK19 and pERK ( Figure 4 C).

(G) Western blot analysis of EGFR and c-RAF expression in lysates obtained from PDAC present in control KPeFC;Egfr;Raf1mice depicted in Figure 3 A (C1–C3) and in “Non Responder” N1–N3 KPeFC;Egfr;Raf1mice exposed to TMX. Expression levels of pERK1/2, ERK1/2, pAKT, and AKT are also shown. GAPDH served as a loading control.

(F) Representative H&E stained paraffin sections of the pancreata of control C2 KPeFC;Egfr;Raf1mouse (depicted in Figure 3 A) and of “Non Responder” N1 and N2 KPeFC;Egfr;Raf1mice after 4 and 5 weeks of TMX exposure, respectively. Tumors are outlined by dotted lines. Scale bar represents 1,000 μm. Box insets mark areas shown at higher magnification in the adjacent images shown to the right stained for H&E, CK19, pERK, and Ki67. Scale bar represents 50 μm.

(D) Tumor volume visualized by weak ultrasound imaging of KPeFC;Egfr lox/lox ;Raf1 lox/lox mice (n = 4 mice, 4 tumors) exposed to a TMX diet for the indicated time. Each color represents a different mouse. Mice were sacrificed at a humane endpoint due to tumor burden at the last time point indicated in the graph. N1 to N4 identify “Non Responder” mice analyzed in (E) to (F) (see below).

(C) Representative H&E stained paraffin sections of the pancreata of KPeFC;Egfr lox/lox ;Raf1 lox/lox T1 and T2 mice after 10 and 11 weeks of TMX exposure, respectively. Tumors are outlined by a dotted line. Scale bar represents 1,000 μm. Box insets mark areas shown at higher magnification in the adjacent images shown to the right stained for H&E, CK19, pERK, and Ki67. Scale bar represents 50 μm.

(A) Tumor volume visualized by weak ultrasound imaging of KPeFC;Egfr lox/lox ;Raf1 lox/lox mice exposed to TMX. Each color represents a different mouse. Mice were sacrificed at a humane endpoint due to tumor burden at the last time point indicated in the graph. T1 and T2 identify “Resistant” mice analyzed in (B) and (C) (see below).

Finally, the pancreata of these “Regressor” mice contained low-grade PanINs (3–10 per mouse), including the R2 mouse in which the original PDAC had completely disappeared. Most of these lesions expressed EGFR ( Figure S3 C). Whether these PanINs are derived from cells that were not able to progress or represent late events during the course of the study, remains to be determined.

Tumor regression appeared to be mediated by apoptotic cell death. Immunohistochemical analysis of a tumor that regressed around 30% during the first 2 weeks of TMX exposure (“Regressor”) revealed 7% of cleaved caspase-3 expression ( Figure 3 F). In contrast, tumors of two independent mice that continued growing during the same period of time (“Non Responder”) only displayed 0.5% of cleaved caspase-3 expression ( Figure 3 F).

Control KPeFC mice (n = 10) died between 2 and 8 weeks following TMX exposure ( Figure 3 A). To our surprise, 8 of 12 KPeFC;Egfr;Raf1mice displayed a rapid decrease in tumor volume upon TMX exposure. Six mice, designated as “Regressors” (R), became tumor free by micro-ultrasound analysis after 6 weeks of TMX exposure ( Figures 3 B and 3C). Four of these “Regressor” animals (R1–R4) were sacrificed after 6 weeks of TMX exposure, whereas the remaining animals, R5 and R6, were allowed to survive for 10 additional weeks. No tumor reappearance, as evaluated by micro-ultrasound analysis, was observed during this time period ( Figure 3 B). Detailed histological examination of their pancreata revealed normal tissue architecture ( Figure 3 D). One “Regressor” mouse (R2) did not display any lesion at the location where the tumor was formerly located. Yet, the other “Regressor” mice (R1, R3, and R4) exhibited single tiny scars, presumably remnants of their original tumor ( Figure 3 D). These scars appeared as very small fibrotic lesions measuring between 0.05 mmto 0.5 mm, reflecting a reduction in tumor volume over 5,000-fold ( Figure 3 D). They were mostly composed of a dense network of organized collagen fibers, along with a significant content of hyaluronic acid ( Figures S3 A and S3B). We also observed signs of chronic inflammation characterized by the presence of macrophages and T lymphocytes at their edges ( Figures S3 A and S3B). Scars of R1 and R6 mice contained a small percentage of Ki67proliferating cells that expressed significant levels of CK19 and pERK and retained EGFR expression, suggesting that they represent residual unrecombined tumor cells ( Figures 3 D and 3E). Indeed, some of these cells displayed atypia and loss of cellular architecture. Scars present in the remaining “Regressor” mice (R3–R5) also contained CK19epithelial cells organized in ductal-like structures. However, they express low levels of pERK and no EGFR. Although a few of these cells also stained for Ki67, they did not present atypia, suggesting that they may not be neoplastic cells ( Figure 3 E).

(F) Representative IHC staining of cleaved caspase-3 of sections of a pancreatic tumor (“Regressor”) from a KPeFC;Egfr lox/lox ;Raf1 lox/lox mouse that decreased 30% in volume after 2 weeks of TMX exposure and of tumors (“Non Responder”) from two independent KPeFC;Egfr lox/lox ;Raf1 lox/lox mice that continued growing during the same period of time. Scale bar represents 20 μm.

(D) Representative low (left, scale bar represents 1,000 μm) and high (middle and right, scale bar represents 100 μm) magnification images of H&E and anti-cytokeratin19 (CK19) staining of sections of the pancreata of control KPeFC;Egfr +/+ ;Raf1 +/+ (C1) and “Regressor” KPeFC;Egfr lox/lox ;Raf1 lox/lox (R1, R3–R6) mice after TMX exposure. The tumor present in the control C1 mouse is outlined by a dotted line. Box insets mark the areas shown at higher magnification in the right columns.

(C) Representative ultrasound images of the regression of a large tumor present in the “Regressor” R3 mouse after 3 and 6 weeks of TMX exposure. Visible lesions are outlined. Tumor volumes are indicated. ND, not detectable.

(B) Total tumor volume visualized by weak ultrasound imaging of KPeFC;Egfr lox/lox ;Raf1 lox/lox mice (n = 6 mice, 7 tumors) exposed to a TMX diet for the indicated time. Each color represents a different mouse. Mice were sacrificed for histopathological analysis at the last time point indicated in the graph. R1 to R6 identifies the “Regressor” mice analyzed in (D) and (E) (see below).

(A) Total tumor volume visualized by weak ultrasound imaging of KPeFC;Egfr;Raf1control mice (n = 10 mice, 15 tumors) exposed to a TMX diet for the indicated time. Each color represents a different mouse. Mice were sacrificed at a humane endpoint due to tumor burden at the last time point indicated in the graph. C1 identifies the control mouse analyzed in (D) and (E). C2 identifies the control mouse analyzed in Figure 4 F.

Next, we assessed the consequences of systemically ablating EGFR and c-RAF expression in mice carrying advanced Kras/Trp53 mutant PDACs. Tumor-bearing KPeFC (n = 14) and KPeFC;Egfr;Raf1mice (n = 45) carrying lesions ranging from 2 to 50 mmwere exposed to a TMX-containing diet. Unfortunately, 4 of 14 KPeFC and 14 of 45 KPeFC;Egfr;Raflanimals had to be eliminated due to various circumstances, including the appearance of unrelated tumors, mainly sarcomas and papillomas. To determine whether these tumors were a consequence of spurious expression of the FlpO recombinase, we introduced a Rosa26allele in KPeFC mice. These mice displayed FlpO-mediated recombinant activity in skin as well as in other tissues as revealed by the presence of dTomatocells ( Figure S2 A). Moreover, most of these dTomatocells became green upon TMX exposure due to expression of the EGFP marker mediated by the CreERT2 recombinase ( Figure S2 A). In addition, a significant percentage of KPeFC;Egfr;Raf1mice (n = 19) could not be included in the study due to inefficient Cre-mediated recombination ( Figure S2 B). As a consequence, only 10 control KPeFC and 12 KPeFC;Egfr;Raf1mice could be evaluated in the trial.

Previous studies have shown that systemic ablation of the MEK1/2 and ERK1/2 kinases results in the rapid degeneration of the intestinal and colonic crypts, leading to death within 2 weeks of TMX exposure (). Similar results have been observed upon ablation of the three members of the RAF kinase family but not when the systemic targeting was limited to c-RAF (). As illustrated in Figure 2 A, concomitant elimination of Egfr and Raf1 in a variety of tissues did not affect either MAPK or PI3K signaling, two of the main pathways responsible for homeostatic RAS signaling, an observation that may explain the minimal toxic effects observed upon ablation of EGFR and c-RAF expression.

Many therapies fail in the clinic due to unacceptable toxic effects (). Thus, we examined whether concomitant, systemic ablation of EGFR and c-RAF expression could be well tolerated in mice. To this end, we exposed 12-week-old Egfr;Raf1;Tg.UBC-CreERT2 mice to a TMX-containing diet for 15 weeks (n = 5 males, n = 5 females). This treatment resulted in efficient recombination of both floxed alleles ( Figure 2 A). Egfr;Raf1;Tg.UBC-CreERT2 siblings were used as controls. Mice lost weight during the initial treatment, yet they recovered a few weeks later ( Figure 2 B). Egfr;Raf1;Tg.UBC-CreERT2 mice developed skin alterations such as hyperplasia and disorganization of the epidermis, hyperkeratosis, folliculitis, and inflammation with increased numbers of mast cells and significant hair loss ( Figure 2 C). Moreover, these animals occasionally developed ulcers and scabs ( Figure 2 C). These toxic effects were similar to those previously observed in mice lacking EGFR in keratinocytes (). These skin defects are highly reminiscent of the acneiform rash and folliculitis observed in human patients treated with EGFR inhibitors (). We also observed a slight disorganization of the crypts in the small intestine with increased numbers of apoptotic cells, however the overall architecture of the tissue was not affected ( Figure 2 D). No significant toxicities were observed in mice upon ablation of c-RAF expression. Taken together, these observations suggest that combined inhibition of EGFR and c-RAF signaling might be well tolerated by patients.

Target ablation at the time of tumor initiation does not reflect therapeutic intervention in the clinic. Moreover, in most studies, targets are selectively ablated in selected tissues or in those cells that express the oncogenic insult(s) (). These strategies fail to provide information regarding the toxic effects that might occur in the clinic when the targets are inhibited via systemic administration of the corresponding inhibitors. Therefore, we have developed a GEM strain that separates temporally and spatially tumor development from target ablation/inhibition. This strain, Kras;Trp53;Elas-tTA/TetO-FlpO;Tg.UBC-CreERT2, designated as KPeFC, incorporates two distinct recombinases, FlpO and CreERT2. FlpO, responsible for tumor induction, is expressed by the same Tet-Off system used in the KPeC strain. Indeed, KPeFC and KPeC mice develop PanIN lesions and PDACs with complete penetrance and similar kinetics ( Figures S1 A and S1B). Expression of the tamoxifen (TMX)-inducible CreERT2 recombinase is driven by the promoter of the human Ubiquitin C gene (UBC), a locus expressed in all adult tissues (). Thus, exposure of KPeFC mice to a TMX-containing diet allows the systemic recombination of any conditional floxed allele added to this strain.

As illustrated in Figure 1 A, control KPeC mice (n = 20) succumbed to PDAC at the average of 15 weeks of age. Expression of the kinase dead CDK4(n = 14) did not prevent PDAC development, but it increased the median survival of the tumor-bearing mice similar to that observed in the absence of EGFR (). Combined ablation of Egfr and expression of CDK4did not decrease the rate of PDAC development or further extended survival (n = 11) ( Figure 1 A). Surprisingly, in contrast to the results obtained with lung tumors, ablation of Raf1 had no effect on PDAC development, and all animals (n = 13) succumbed to pancreatic tumors with a latency similar to that of KPeC mice ( Figure 1 B). However, concomitant ablation of Egfr and Raf1 completely prevented PDAC development (n = 14), up to 2 years of age ( Figure 1 B). Detailed histological analysis of serial sections of their pancreata failed to identify PanIN lesions or even metaplasias. These mice retained K-RASexpression in their acinar cell compartment as determined by the presence of β-galactosidase, a surrogate marker for K-RASexpression (). Isolation of these cells by laser-capture microdissection confirmed efficient recombination of Raf1and Egfralleles ( Figure 1 C). No such recombination was observed in adjacent acinar cells negative for β-galactosidase expression ( Figure 1 C). Thus, EGFR and c-RAF, but not CDK4, must signal through independent pathways essential for initiation and development of pancreatic tumors. Finally, inhibition of PDAC development requires complete absence of EGFR and c-RAF expression, because different combinations of floxed and wild-type Egfr and Raf1 alleles in KPeC mice delayed, but did not prevent, PDAC development ( Figure 1 B).

(C) PCR analysis of Egfr and Raf1 alleles using DNA extracted from laser captured acinar cells expressing K-RAS G12V (identified by the X-Gal marker). Migration of recombined Egfr − and Raf1 – alleles (lane 1), conditional Egfr lox and Raf1 lox alleles (lane 2), and wild-type Egfr + and Raf1 + alleles (lane 3) used as controls. DNA extracted from X-Gal positive (lane 4) and negative (lane 5) acinar cells of KPeC;Egfr lox/lox ;Raf1 lox/lox mice. Lane 6, blank control. Lane M, DNA size markers. DNA fragment size is indicated.

Among those K-RAS effectors likely to cooperate with EGFR in mediating PDAC development, we selected the CDK4 cell cycle kinase and the c-RAF kinase based on our prior observations that they are essential for the development of K-RASdriven lung tumors (). Moreover, ablation of CDK4 or c-RAF does not induce unacceptable toxic effects such as those observed upon ablation of the MEK1/2 and ERK1/2 kinases (). To interrogate whether tampering with CDK4 activity could cooperate with EGFR ablation in preventing PDAC development, we mutated the endogenous Cdk4 to encode a kinase dead K35M isoform to better recapitulate pharmacological treatments.

We and others have previously reported that Egfr ablation prevented the formation of PanIN lesions in oncogenic Kras-driven GEM PDAC models. Furthermore, the absence of EGFR delayed PDAC development in the absence of p53 (). To identify effector molecules that could cooperate with Egfr ablation in preventing mutant Kras/Trp53-driven PDAC development, we added conditional floxed alleles to the Kras;Trp53;Elas-tTA/TetO-Cre strain. This strain has been designated as KPeC to indicate that the driver mutations are selectively induced by the elastase gene promoter in the acinar cell compartment instead of in all pancreatic cell lineages as in the classic KPC model ().

GEM PDAC models driven by a resident oncogenic Kras mutant and loss or inactivation of Trp53 closely reproduce the natural history and histopathology of human tumors (). Exomic next-generation sequencing of these tumors revealed a number of missense mutations (13.3 mutations/tumor) ( Table S1 ) similar to those reported elsewhere (), with a significant mutational complexity albeit more limited than that of their human counterpart (). Bioinformatic analysis revealed that almost half of these mutated genes could be integrated within the signaling pathways found mutated in human PDACs (). Interestingly, these mouse tumors display a wide heterogeneity since none of the 146 mutated genes identified in our analysis appeared in more than one tumor ( Table S1 ), thus limiting our therapeutic options to those targeting K-RAS signaling pathways.

Detailed analysis of the genomic landscapes of human PDACs beyond the known driver mutations has failed to outline meaningful stratifications that could be correlated with clinical outcome (). Only transcriptional studies have been able to define the existence of distinct PDAC subtypes (). GEM PDAC tumors also exhibit distinct transcriptional signatures. Interestingly, the “Non Responder” tumors displayed a transcriptional pattern similar to the “squamous” signature of human PDACs characterized by worse prognosis (). In contrast, the transcriptional profile of “Regressor” tumors is more similar to the other subtypes (). More importantly, these GEM tumors display drastically different responses to a therapeutic regimen based on Egfr and Raf1 ablation. Recent studies carried out with human PDAC organoids have also described a correlation between their transcriptional profiles and their response to cytotoxic compounds (). Whether such correlation would exist using selected inhibitors against those targets identified here remains to be determined. A deeper understanding of the molecular mechanisms responsible for the lack of response of certain GEM tumors of EGFR and c-RAF inhibition should serve to identify additional therapeutic targets that will increase the limited armamentarium available to fight pancreatic cancer.

Replacement of EGFR ablation by pharmacological inhibition of EGFR kinase activity yielded similar results. Unfortunately, inhibition of c-RAF activity may represent a bigger challenge since currently available panRAF inhibitors will affect the MAPK pathway and hence elicit unacceptable toxicities such as those already observed in the clinic with MEK inhibitors. Moreover, we have been unable to reproduce the results obtained upon c-RAF knockdown with c-RAF kinase inhibitors. Preliminary results using conditional Raf1 kinase alleles in our lung “therapeutic model” suggest that the therapeutic effect observed upon loss of c-RAF expression may not be mediated by its kinase activity (our unpublished data). Therefore, the potential application of our results to a clinical scenario may require sophisticated medicinal chemistry strategies to either block c-RAF kinase independent activities or induce its degradation.

Strategies used to treat PDAC in the clinic have achieved very limited benefit. Likewise, experimental therapeutic strategies in GEM PDAC models have only resulted in modest tumor delays (). Only genetic tampering with oncogenic K-Ras mutant expression in mice has led to significant levels of tumor regression (). In this study, we demonstrate that combined inhibition of EGFR and c-RAF expression is a very effective therapy against PDAC, both in mutant Kras/Trp53-driven GEM tumor models as well as in human PDXs. Of equal relevance for future application of these observations in a clinical scenario is the fact that systemic elimination of these targets results in tolerable toxicities, primarily resulting from the lack of EGFR activity. These observations are likely to be a consequence of the unexpected lack of effect of Egfr and Raf1 ablation on the MAPK cascade, a signaling pathway essential for normal homeostasis (). Human PDACs are genetically more complex than those of GEM tumor models (). Yet, the inhibitory effect of EGFR and c-RAF knockdown in nine of ten independent PDX tumor models illustrates that this therapeutic strategy may also have profound effects in the clinic. It is somewhat surprising that human PDX-derived tumor cells are more sensitive than the corresponding mouse tumors in spite of their more complex mutational profile. Whether the higher sensitivity of the human PDX tumors to EGFR and c-RAF inhibition is a consequence of the lack of desmoplastic tissue and/or their experimental manipulation via passage in immunocompromised mice remains to be determined.

Data are represented as mean ± SD. Significance was calculated with the unpaired Student′s t test using GraphPad Prism software. A p value that was less than 0.05 was considered to be statistically significant for all data sets. Significant differences between experimental groups were: ∗ p< 0.05, ∗∗ p< 0.01 or ∗∗∗ p< 0.001.

RNA sequencing data have been deposited in the Gene Expression Omnibus (GEO) database. The accession number for the RNA sequencing data reported in this paper is GSE112434 . Next generation sequencing data have been deposited in the NCBI’s Sequence Read Archive (SRA). The accession number for the Next generation sequencing data reported in this paper is PRJNA462276.

For pharmacologic studies PDX-derived cells were seeded in 96-well plates at 1,500 and 3,000 cells/well in triplicates, and incubated for 24 hours in DMEM media supplemented with 10% FBS, 2 mM L-glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin (GIBCO-Invitrogen) before adding the IC 50 of the corresponding IC50 concentration of inhibitor in DMSO. The same concentration of DMSO was used as a control. Cells were exposed to the corresponding inhibitor for 12 days, in the presence or absence of a c-RAF shRNA (R1) changing medium and drug every two days. Cell viability was assessed with CellTiter Glo Luminescent Cell Viability.

PDX cell lines were plated at 5,000 cells per well in triplicates in 96-well plates and grown for 24 hours. Cells were treated with a dilution series of Gefitinib (Cymit Química SL), Erlotinib (LC laboratories). Control cells were incubated with media containing DMSO. Cell viability was assessed with CellTiter Glo Luminescent Cell Viability Assay after 72 hours of treatment. Luminescence counts were read in a Victor Instrument (Perkin Elmer) with the recommended settings. To calculate the IC 50 , values were plotted against the inhibitor concentrations and fit to a sigmoid dose–response curve using GraphPad Software.

RNA from PDAC cell explants was extracted with Qiagen RNeasy Mini Kit. 1 μg of total RNA was used for further analysis. PolyAfraction was purified and randomly fragmented, converted to double stranded cDNA and processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters as in Illumina's "TruSeq Stranded mRNA LT Sample Prep Kit". The adapter-ligated library was completed by PCR with Illumina PE primers. The resulting purified cDNA library was applied to an Illumina flow cell for cluster generation and sequenced on an Illumina NovaSeq 6000 instrument by following manufacturer's guidelines. 101 bp single-end reads were analyzed with the Nextpresso pipeline () as follows: sequencing quality was verified with FastQC v0.11.0 ( http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). Reads were aligned to the mouse genome (NCBI37/mm9) with TopHat-2.0.10 () using Bowtie 1.0.0 () and SAMtools 0.1.19 (), allowing 2 mismatches and 20 multihits. Differential expression was tested with DESeq2 () using the Mus musculus NCBI37/mm9 transcript annotations from https://ccb.jhu.edu/software/tophat/igenomes.shtml . GSEAPreranked () was used to perform a gene set enrichment analysis of the described gene signatures on a pre-ranked gene list, setting 1000 gene set permutations. Gene Set Variation Analysis (GSVA) (), was used to estimate the variation of pathway activity over the samples in an unsupervised manner. Heatmaps presented in this study were built with GENE-E software package ( https://software.broadinstitute.org/GENE-E/index.html ).

After 96 hr of infection with AdCre particles cells were harvested by trypsinization and fixed with 70% (v/v) ethanol at 4°C overnight. Fixed cells were incubated in phosphate-buffered saline (PBS) containing 100 μg/ml RNase A for 30 minutes at 37°C, followed by staining with 0.003% of Propidium Iodide for 30 minutes on ice. Thereafter, cells were collected on a nylon mesh filter (pore size, 40 mm), and cell cycle was assayed by flow cytometry (FACSCalibur) at excitation of 488 nm and at emission of 585 nm, and analyzed using a FACSDiva Version 6.1.2 (BD Bioscience).

Tissues were fixed overnight by immersion in 4% paraformaldehyde (PFA) in 0.01 M phosphate-buffered saline (PBS) at 4°C and rinsed in PBS before equilibration in 30% sucrose in PBS for 48 h at 4°C. Samples were thereafter included in O.C.T.™ compound (Sakura) and frozen. Cryosections of the samples were stained with Dapi for nuclei detection (ThermoFisher), mounted with Prolong Gold antifade reagent (ThermoFisher) and visualized with a TCS-SP5 laser scanning confocal microscope (Leica) equipped with AOBS and both 10X/0.4NA and 20X/0.7NA dry objectives. A z-stack was acquired and the maximum projection is shown.

X-Gal staining, laser capture microdissection and Egfr PCR analysis have been previously described (). Raf1 wild-type, floxed and null alleles were identified with forward Raf1 1F (5′-CTGATTGCCCAACTGCCATAA-3′), Raf1 3F (5′-GAGTCAGCAAATGCACTGAAATG-3′) and reverse Raf1 1R (5′-ACTGATCTGGAGCACAGCAAT-3′) primers at 94°C for 1 minute, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds and extension at 72°C for 30s, and finally, followed by a long extension at 72°C for 10 minutes. These primers yielded DNA products of 196 bp, 270 bp and 143 bp for wild-type, floxed and null Raf1 alleles, respectively.

Protein extracts (25 μg) obtained from tumor tissue or cell lines were separated on SDS/PAGE gels (ThermoFisher Scientific), transferred to a nitrocellulose membrane and blotted with antibodies raised against EGFR (Abcam, ab52894), c-RAF (BD Biosciences, 610151), pERK1/2 (Cell Signaling, 9101), ERK1 (BD Biosciences 554100), ERK2 (BD Biosciences, 610103), pAKT (Cell Signaling, 4060), AKT (Cell Signaling, 9272), pSTAT3 (Cell Signaling, 9131), STAT3 (Cell Signaling, 9132), pCOFILIN (Santa Cruz, sc-21867-R) and GAPDH (Sigma, G8795). Primary antibodies were detected against mouse or rabbit IgGs (HRP, Dako and Alexa Fluor 680, Invitrogen) and visualized with ECL Western blot detection solution (GE Healthcare) or Odyssey infrared imaging system (LI-COR Biosciences).

Genomic DNA obtained from 11 paired tumor and tail tissue was enriched in protein-coding sequences using the SureSelect Mouse All Exon kit (Agilent Technologies). The resulting target-enriched pool was amplified and subjected to paired-end sequencing (2 × 100 bp) using HiSeq2000 sequencing instruments at the Beijing Genomics Institute (BGI). Sequencing reads were mapped to the reference genome (mm9) using the Burrows–Wheeler Aligner (BWA) () alignment tool version 0.5.9. Sites that differed from the reference genome (variants) were identified and empirical priors were constructed for the distribution of variant frequencies in each sample independently. High-credibility intervals (posterior probability ≥ 1 – 1e-5) were obtained for the observed frequency of the variants using the statistical algorithm for variant identification (SAVI) algorithm (). Variants were considered absent if their allele frequency was <2% and present if detected with an allele frequency above 15%, corresponding with the sensitivity threshold of direct Sanger sequencing. Variant total depth was also required to be >10x and <300x. Variants were excluded if present in mouse dbSNP database, detected in any of the normal samples, or observed in only one strand. Finally, candidate protein altering somatic variants (nonsense, missense, and small insertions and deletions) were identified when variants were absent in the normal and present in the tumor with at least 1% change in frequency from normal with high posterior probability (≥ 1 – 1e-5).

For routine histological analysis, specimens were fixed in 10% buffered formalin (Sigma) and embedded in paraffin. For histopathological analysis, tissues were serially sectioned (3μm thick) and stained by conventional H&E every ten sections. Antibodies used for immunostaining included those raised against CK19 (CNIO Monoclonal Antibodies Core Unit), cleaved Caspase-3 (Cell Signaling Technology, 9661), CD3 (Santa Cruz Biotechnology, M-20), EGFR (Abcam, ab52894), pERK (Cell Signaling Technology, 9101), F4/80 (ABD Serotec, CI: A3-1), Ki67 (Master Diagnostica, 0003110QD), and pSTAT3 (Cell Signaling Technology; 9145). For detection of HA, biotinylated hyaluronic acid binding protein (HABP) was used (Millipore). Stained slides were scanned using the Mirax scanner (Zeiss). Images were analyzed by Zen2 software and photos were exported using Zen2 software (Zeiss).

Tumors were measured with a micro-ultrasound system (Vevo 770, Visualsonics) with an ultrasound transducer of 40 MHz (RMV704, Visualsonics). To this end, mice were anesthetized with a continuous flow of 1% to 3% isoflurane in 100% oxygen at a rate of 1.5 liter/min. Hypothermia associated with anesthesia was avoided using a bed-heater. Abdominal hair was removed by depilation cream to prepare the examination area. Tumor size was calculated as Length x Width 2 /2. Recombination of the Egfr lox and Raf1 lox conditional alleles was mediated by activation of the inducible CreERT2 recombinase with TMX. To this end, mice were fed with a TMX-containing diet (Teklad CRD TAM400 diet, Harlan) ad libitum. Control mice carrying the corresponding wild-type alleles were also fed with the same diet.

Cells derived from these PDX tumor models were infected with lentiviral supernatants expressing shRNAs against EGFR (E1, TRCN0000121203 and E2, TRCN0000121206), c-RAF (R1, TRCN0000001065 and R2, TRCN0000001068). The E1, E2 and R2 shRNAs were cloned in a plasmid that carries a Puromycin resistant cassette. Instead, the R1 shRNA was cloned in a plasmid that conferred Blasticidine resistance. A scrambled shRNA control vector was used as a negative control. Infected PDX cells were seeded in 96-well plates at a density of 1,500 cells per well and proliferation was assessed using the MTT assay. For in vivo studies, infected cells (0.5x10 6 ) derived from PDAC003T, PDAC013T, Panc-1 and Panc-4 tumor models were injected 1:1 in PBS:Matrigel Matrix (Corning, 354234) into dorsal flanks of immunodeficient mice. Tumor growth was measured every 3 days with a caliper and calculated as Length x Width 2 /2 until humane end point.

PDX tumors models were used include Panc-1, Panc-2, Panc-4, Panc-185, Panc-198, H-PDAC-H-X132, H-PDAC-M-X3 and H-PDAC-M-X7 ( Table S3 ). Panc-1, Panc-2, and Panc-4 were obtained from patients who underwent surgical resection at the Koç University Hospital, Istanbul, Turkey with approval by the Ethical Committee (CEI 60-1057-A068). Panc-185, Panc-198, H-PDAC-H-X132 were obtained from Hospital HM Sanchinaro, Madrid, Spain, with approval by the Ethical Committee (CEIC HM Hospitales, FHM.06.10). H-PDAC-M-X3 and H-PDAC-M-X7 were obtained from the Hospital Virgen de la Arrixaca, Murcia, Spain, with approval by the Ethical Committee (CEIC HCUVA-2013/01). Specific informed consent for PDX model generation was obtained from all patients. PDAC003T and PDAC013T tumor models have been already described ().

To generate mouse PDAC explants, freshly isolated tumors were minced with sterile razor blades, digested with collagenase P (1.5 μg/ml) in Hank’s Balanced Salt Solution (HBSS) for 30 min at 37°C, and cultured in DMEM with 10% of fetal bovine serum (FBS) and 1% Penicillin/Streptomycin. All studies were done on cells maintained in culture for less than ten passages. Their corresponding genotypes were verified by PCR analysis. PDAC cells explants were infected with AdCre particles (multiplicity of infection, 100) and seeded for colony formation assay 5 days after. AdGFP particles were used as negative controls. Cells (5×10 3 ) were seeded and allowed to form colonies for 2 weeks. Plates were fixed with 0.1% glutaraldehyde (Sigma) and stained with 0.5% Crystal Violet (Merck). Colonies were counted and quantified.

Elas-tTA/TetO-Cre;Kras, Trp53, Egfr, Raf1, Kras, Trp53, Tg.UBC-CreERT2 and Rosa26strains have been previously described (). The Cdk4strain was obtained by a Cre-dependent FLEx switch strategy that replaced expression of the wild-type CDK4 protein by a CDK4kinase dead isoform () (). The transgenic TetO-FlpO strain was generated by pronuclear injection of CMV-TetO-FlpO DNA into B6.CBA zygotes (). All mice were maintained in a mixed 129/Sv-C57BL/6 background. Immunodeficient NU-Foxn1nu mice (females, 5-weeks-old) were purchased from Harlan Laboratories. All animal experiments were approved by the Ethical Committees of the Spanish National Cancer Research Centre (CNIO) and they were performed in accordance with the guidelines stated in the International Guiding Principles for Biomedical Research Involving Animal, developed by the Council for International Organizations of Medical Sciences (CIOMS). All strains were genotyped by Transnetyx (Cordova, Tennessee, USA).

Acknowledgments

We thank B. Jiménez, M. San Roman, R. Villar, and S. Jiménez for excellent technical assistance; I. Aragón, A. López, F. Díaz, and I. Blanco (Animal Facility) for mouse work; G. Visdomine, C. Peñalba, and G. Garaulet (Molecular Imaging Unit) for ultrasound studies; P. Vargiu (Transgenic Unit) for help in generating the TetO-FlpO strain; N. Cabrera, A. de Martino (Histopathology Unit) and M. Morente (Tumor Bank) for histopathological analysis, and C. Blanco and A. Cebriá (Experimental Therapeutics) for determining the IC 50 values of gefinitib and erlotinib. Special thanks to J. de la Peña and E. Ortiz (Servicio de Anatomía Patológica HCUVA) and T. Escamez and V. Navarro (Biobanco-IMIM) for their help with the PDX tumor models, and to R. Nieto, J.M. Ligós, and M. Montoya (Cytometry Unit, CNIC) for fluorescence-activated cell sorting analysis of apoptotic cells. This work was supported by grants from the European Research Council (advanced grants ERC-AG/250297-RAS AHEAD and ERC-AG/695566-THERACAN ), from the Spanish Ministry of Economy and Competitiveness ( SAF2014-59864-R ) to M.B. Additional support was also obtained from grants from the Asociación Española contra el Cáncer ( GC16173694BARB ) to M.B. and B.S., from La Ligue Contre le Cancer to J.I., from the European Research Council (advanced grants ERC-2014-ADG ) to M.H., and from the NIH ( U54CA193313 and U54CA209997 ) to R.R. M.T.B was supported by an FPU fellowship from the Spanish Ministry of Education . C.N. was supported by a Juan de la Cierva Award. M. Djurec was partially supported by a pre-doctoral fellowship from La Caixa. J.P.-P. was supported by a Severo Ochoa FPI fellowship from the Spanish Ministry of Economy and Competitiveness. M.B. is the recipient of an Endowed Chair from the AXA Research Fund .

Author Contributions

C.G. and M.B. designed the research; M.T.B. and C.N. performed most of the research and analyzed data; O.G.-C., G.M.-S., J.P.-P., H.K., F.A., and R.R. performed bioinformatic analysis; C.G.L., E.C., E.B.-M., L.M.-C., H.K.C.J., and L.C. performed in vitro studies; L.M.-D., M. Djurec, and J.L. helped with in vivo studies; L.E.-B. contributed to experiments with the Cdk4K35M strain, S.O. generated the TetO-FlpO strain; F.M. carried out ultrasound studies; B.S., N.D., J.I., F.S.-B., and M.H. provided PDX samples; M.M. and M. Drosten provided critical input; M.T.B., C.N., C.G., and M.B. wrote the paper.

Declaration of Interests

M.B. reports a research contract from Pfizer and Eli Lilly and paid consultancy from Amcure. J.I. reports paid consultancy from Dynasio S.A. and Oncomedics, both in France. M.H. reports research contracts and/or paid consultancy with Roche and Astra-Zeneca. None of these relationships are related to the work reported in this manuscript. We declare a patent application related to this work: EP18382555 Barbacid, M., Guerra C., Blasco, M.T., Navas, C. (2018) Combined therapy against cancer.