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Supplemental Data

Mutations at multiple sites in MEK1 occur in cancer, suggesting that their mechanisms of activation might be different. We analyzed 17 tumor-associated MEK1 mutants and found that they drove ERK signaling autonomously or in a RAS/RAF-dependent manner. The latter are sensitive to feedback inhibition of RAF, which limits their functional output, and often cooccur with RAS or RAF mutations. They act as amplifiers of RAF signaling. In contrast, another class of mutants deletes a hitherto unrecognized negative regulatory segment of MEK1, is RAF- and phosphorylation-independent, is unaffected by feedback inhibition of upstream signaling, and drives high ERK output and transformation in the absence of RAF activity. Moreover, these RAF-independent mutants are insensitive to allosteric MEK inhibitors, which preferentially bind to the inactivated form of MEK1. All the mutants are sensitive to an ATP-competitive MEK inhibitor. Thus, our study comprises a novel therapeutic strategy for tumors driven by RAF-independent MEK1 mutants.Significance: Mutants with which MEK1 mutants coexist and their sensitivity to inhibitors are determined by allele-specific properties. This study shows the importance of functional characterization of mutant alleles in single oncogenes and identifies a new class of MEK1 mutants, insensitive to current MEK1 inhibitors but treatable with a new ATP-competitive inhibitor. Cancer Discov; 8(5); 648–61. ©2018 AACR.See related commentary by Maust et al., p. 534.This article is highlighted in the In This Issue feature, p. 517

Supplemental Table

Document describing important findings related to the main manuscript.

Supplementary Figure S1. Generation of conditional knock-in mice that express the H-RasG12V oncoprotein from the K-Ras locus. Supplementary Figure S2. Flow cytometry analysis of myeloid cell expansion in spleens and BM of H-RasG12V expressing mice. Supplementary Figure S3. Characterization of the T-ALL induced by the H-RasG12V oncoprotein expressed from the K-Ras locus. Supplementary Figure S4. Pancreatic and lung lesions in mice expressing H-RasG12V and K-RasG12V oncoproteins from the K-Ras locus. Supplementary Figure S5. Lack of apoptosis or canonical OIS in lung cells expressing the H-RasG12V oncoprotein from the K-Ras locus. Supplementary Figure S6. H-RasG12V induced senescence-like arrest in lung cells is partially mediated by p53. Supplementary Figure S7. Differential effects of expressing the K-RasG12V and H-RasG12V oncoproteins driven from the K-Ras locus in MEFs.

Document describing important mouse lines, treatment protocols, antibodies and primers used in the present study.

Genetic studies in mice have provided evidence that H-Ras and K-Ras proteins are bioequivalent. However, human tumors display marked differences in the association of RAS oncogenes with tumor type. Thus, to further assess the bioequivalence of oncogenic H-Ras and K-Ras, we replaced the coding region of the murine K-Ras locus with H-RasG12V oncogene sequences. Germline expression of H-RasG12V or K-RasG12V from the K-Ras locus resulted in embryonic lethality. However, expression of these genes in adult mice led to different tumor phenotypes. Whereas H-RasG12V elicited papillomas and hematopoietic tumors, K-RasG12V induced lung tumors and gastric lesions. Pulmonary expression of H-RasG12V created a senescence-like state caused by excessive MAPK signaling. Likewise, H-RasG12V but not K-RasG12V induced senescence in mouse embryonic fibroblasts. Label-free quantitative analysis revealed that minor differences in H-RasG12V expression levels led to drastically different biological outputs, suggesting that subtle differences in MAPK signaling confer nonequivalent functions that influence tumor spectra induced by RAS oncoproteins. Cancer Res; 77(3); 707–18. ©2016 AACR.

Supplementary Methods and Supplementary Figure Legends

Pancreatic acinar cells are a cell type of origin for pancreatic cancer that become progressively less sensitive to tumorigenesis induced by oncogenic Kras mutations after birth. This sensitivity is increased when Kras mutations are combined with pancreatitis. Molecular mechanisms underlying these observations are still largely unknown. To identify these mechanisms, we generated the first CRISPR-edited mouse models that enable detection of wild-type and mutant KRAS proteins in vivo. Analysis of these mouse models revealed that more than 75% of adult acinar cells are devoid of detectable KRAS protein. In the 25% of acinar cells expressing KRAS protein, transcriptomic analysis highlighted a slight upregulation of the RAS and MAPK pathways. However, at the protein level, only marginal pancreatic expression of essential KRAS effectors, including C-RAF, was observed. The expression of KRAS and its effectors gradually decreased after birth. The low sensitivity of adult acinar cells to Kras mutations resulted from low expression of KRAS and its effectors and the subsequent lack of activation of RAS/MAPK pathways. Pancreatitis triggered expression of KRAS and its effectors as well as subsequent activation of downstream signaling; this induction required the activity of EGFR. Finally, expression of C-RAF in adult pancreas was required for pancreatic tumorigenesis. In conclusion, our study reveals that control of the expression of KRAS and its effectors regulates the sensitivity of acinar cells to transformation by oncogenic Kras mutations.Significance:This study generates new mouse models to study regulation of KRAS during pancreatic tumorigenesis and highlights a novel mechanism through which pancreatitis sensitizes acinar cells to Kras mutations.

Supplementary Tables S1-S7. K-RasG12V tumors serially transplanted in Crl:NU-Foxn1nu mice and paired derived cell lines (S1); Semiquantitative assessment of the percentages of histologic components in passage #1 engrafted tumors (S2); EML4-ALK tumors orthotopically implanted in Crl:NU-Foxn1nu mice (S3); Histologic progression of orthoallografts upon sequential mice-to-mice passages (S4); Presence of distal metastasis in animals implanted with the indicated orthoallografts (S5); Summary of primary cell lines obtained from the indicated orthoallograft implants (S6); Histopathological characterization of EML4-ALK implants after crizotinib treatment (S7).

Deregulation of the G1-S transition of the cell cycle is a common feature of human cancer. Tumor-associated alterations in this process frequently affect cyclin-dependent kinases (Cdk), their regulators (cyclins, INK4 inhibitors, or p27Kip1), and their substrates (retinoblastoma protein). Although these proteins are generally thought to act in a linear pathway, mutations in different components frequently cooperate in tumor development. Using gene-targeted mouse models, we report in this article that Cdk4 resistance to INK4 inhibitors, due to the Cdk4 R24C mutation, strongly cooperates with p27Kip1 deficiency in tumor development. No such cooperation is observed between Cdk4 R24C and p18INK4c absence, suggesting that the only function of p18INK4c is inhibiting Cdk4 in this model. Cdk4R/R knock in mice, which express the Cdk4 R24C mutant protein, develop pituitary tumors with complete penetrance and short latency in a p27Kip1−/− or p27Kip1+/− background. We have investigated whether this tumor model could be useful to assess the therapeutic activity of cell cycle inhibitors. We show here that exposure to flavopiridol, a wide-spectrum Cdk inhibitor, significantly delays tumor progression and leads to tumor-free survival in a significant percentage of treated mice. These data suggest that genetically engineered tumor models involving key cell cycle regulators are a valuable tool to evaluate drugs with potential therapeutic benefit in human cancer.

Supplementary Figure Legends. Legend for Supplementary Figures S1-S4.

Supplementary Figures S1-S4. Common histological subtypes of NSCLC orthoallografts (S1); In vitro characterization of primary cell lines derived from NSCLC orthoallografts (S2); Lung colonization and tumor formation upon reintroduction of mKLC cell lines (S3); Inoculation of mKLC single cell clones gives rise to NSCLC heterogeneity in vivo (S4).

Supplementary Figures 1-2, Table 1 from Spontaneous and UV Radiation–Induced Multiple Metastatic Melanomas in Cdk4R24C/R24C/TPras Mice

Supplementary Table 1 from Cooperation between Cdk4 and p27kip1 in Tumor Development: A Preclinical Model to Evaluate Cell Cycle Inhibitors with Therapeutic Activity

Human melanoma susceptibility is often characterized by germ-line inactivating CDKN2A (INK4A/ARF) mutations, or mutations that activate CDK4 by preventing its binding to and inhibition by INK4A. We have previously shown that a single neonatal UV radiation (UVR) dose delivered to mice that carry melanocyte-specific activation of Hras (TPras) increases melanoma penetrance from 0% to 57%. Here, we report that activated Cdk4 cooperates with activated Hras to enhance susceptibility to melanoma in mice. Whereas UVR treatment failed to induce melanomas in Cdk4R24C/R24C mice, it greatly increased the penetrance and decreased the age of onset of melanoma development in Cdk4R24C/R24C/TPras animals compared with TPras alone. This increased penetrance was dependent on the threshold of Cdk4 activation as Cdk4R24C/+/TPras animals did not show an increase in UVR-induced melanoma penetrance compared with TPras alone. In addition, Cdk4R24C/R24C/TPras mice invariably developed multiple lesions, which occurred rarely in TPras mice. These results indicate that germ-line defects abrogating the pRb pathway may enhance UVR-induced melanoma. TPras and Cdk4R24C/R24C/TPras tumors were comparable histopathologically but the latter were larger and more aggressive and cultured cells derived from such melanomas were also larger and had higher levels of nuclear atypia. Moreover, the melanomas in Cdk4R24C/R24C/TPras mice, but not in TPras mice, readily metastasized to regional lymph nodes. Thus, it seems that in the mouse, Hras activation initiates UVR-induced melanoma development whereas the cell cycle defect introduced by mutant Cdk4 contributes to tumor progression, producing more aggressive, metastatic tumors. (Cancer Res 2006; 66(6): 2946-52)

Mutant KRAS is a major driver of oncogenesis in a multitude of cancers but remains a challenging target for classical small molecule drugs, motivating the exploration of alternative approaches. Here, we show that aggregation-prone regions (APRs) in the primary sequence of the oncoprotein constitute intrinsic vulnerabilities that can be exploited to misfold KRAS into protein aggregates. Conveniently, this propensity that is present in wild-type KRAS is increased in the common oncogenic mutations at positions 12 and 13. We show that synthetic peptides (Pept-ins™) derived from two distinct KRAS APRs could induce the misfolding and subsequent loss of function of oncogenic KRAS, both of recombinantly produced protein in solution, during cell-free translation and in cancer cells. The Pept-ins exerted antiproliferative activity against a range of mutant KRAS cell lines and abrogated tumor growth in a syngeneic lung adenocarcinoma mouse model driven by mutant KRAS G12V. These findings provide proof-of-concept that the intrinsic misfolding propensity of the KRAS oncoprotein can be exploited to cause its functional inactivation.

Accumulating evidence suggests that both the nature of oncogenic lesions and the cell-of-origin can strongly influence cancer histopathology, tumor aggressiveness and response to therapy. Although oncogenic Kras expression and loss of Trp53 tumor suppressor gene function have been demonstrated to initiate murine lung adenocarcinomas (LUADs) in alveolar type II (AT2) cells, clear evidence that Club cells, representing the second major subset of lung epithelial cells, can also act as cells-of-origin for LUAD is lacking. Equally, the exact anatomic location of Club cells that are susceptible to Kras transformation and the resulting tumor histotype remains to be established. Here, we provide definitive evidence for Club cells as progenitors for LUAD. Using in vivo lineage tracing, we find that a subset of Kras12V -expressing and Trp53-deficient Club cells act as precursors for LUAD and we define the stepwise trajectory of Club cell-initiated tumors leading to lineage marker conversion and aggressive LUAD. Our results establish Club cells as cells-of-origin for LUAD and demonstrate that Club cell-initiated tumors have the potential to develop aggressive LUAD.

Platinum-based chemotherapy is commonly used for non-small cell lung cancer (NSCLC) treatment, yet clinical outcomes remain poor. Cellular senescence and its associated secretory phenotype (SASP) can have multiple tumour-promoting activities, although these are largely unexplored in lung cancer. Here we show that cisplatin-derived SASP enhances the malignant phenotype of lung cancer cells. Using xenograft, orthotopic and KrasG12V-driven murine NSCLC models, we demonstrate that cisplatin-induced senescent cells strongly promote tumour progression. Mechanistically, we find that a TGF-β-enriched SASP drives pro-proliferative effects through TGFβR1 and Akt/mTOR pathway activation. We validate the translational relevance of chemotherapy-induced SASP using clinical NSCLC samples from patients who received neoadjuvant platinum-based chemotherapy. Importantly, TGFβR1 inhibition with galunisertib or senolytic treatment significantly reduces tumour promotion driven by cisplatin-induced senescence. Finally, we demonstrate, using distinct murine NSCLC models, that addition of TGFBR1 inhibitors to platinum-based chemotherapy reduces tumour burden and improves survival, providing pre-clinical proof-of-concept for future trial designs.

KRAS is the most frequently mutated RAS family member. Within non-small cell lung cancer (NSCLC), G12C is the most prevalent KRAS mutation (~13%) and the KRAS G12C inhibitors (G12Ci) sotorasib and adagrasib are now FDA approved. However, it has been shown that simultaneous targeting of multiple nodes in the MAPK pathway may be optimal for deeper and more durable responses. Furthermore, acquired mutations in the MAPK pathway occur clinically upon progression on sotorasib or adagrasib, altogether supporting the need for clinical combinations with G12Ci. Based on the clinical success of G12Ci, several KRAS G12D inhibitors (G12Di) are now being developed as G12D is the most prevalent KRAS mutation across human cancers including pancreatic (~28%) and colorectal (~11%) cancers. Avutometinib (VS-6766; avuto) is a RAF/MEK clamp that potently inhibits MEK kinase activity and induces a dominant negative RAF-MEK complex preventing phosphorylation of MEK by ARAF, BRAF and CRAF. This unique mechanism allows avuto to block MEK signaling without the compensatory re-activation of MEK that appears to limit the efficacy of MEK-only inhibitors. Here, we tested whether addition of avuto to G12Ci or G12Di could improve MAPK pathway blockade and anti-tumor efficacy. Avuto was synergistic with sotorasib and adagrasib in reducing viability of a panel of KRAS G12C cancer cell lines in 3D culture, and the combination of avuto + G12Ci showed improved depth and duration of MAPK pathway inhibition, stronger inhibition of cell cycle/proliferation markers and stronger activation of pro-apoptotic markers relative to G12Ci alone. In mouse models, avuto enhanced efficacy of G12Ci in KRAS G12C NSCLC xenograft models. Furthermore, avuto potentiated G12Ci efficacy in a KRAS(+/G12C);p53null NSCLC GEMM model with avuto + G12Ci combination inducing complete response in 25% of the mice. To evaluate efficacy of avuto against KRAS mutations that have been observed clinically upon acquired resistance to G12Ci, proliferation of Ba/F3 cells expressing these mutations was assessed. While avuto potently inhibited (IC50 < 15 nM) proliferation of cells bearing KRAS G12C/R68S, G12C/H95D or G12C/Y96C mutations, adagrasib showed diminished potency (IC50 ≥ 85 nM) against all 3 of these mutations and sotorasib showed diminished potency (IC50 ≥ 150 nM) against G12C/R68S and G12C/Y96C. Accordingly, avuto strongly inhibited tumor growth of an in vivo model expressing KRAS G12C/Y96D, while sotorasib was ineffective. Similarly, avuto was found to be synergistic with the G12Di MRTX1133 in reducing viability of a panel of KRAS G12D cancer cell lines, and avuto enhanced anti-tumor efficacy of MRTX1133 in KRAS G12D colorectal (CR3300) and pancreatic (PA1252) cancer PDX models. These results support ongoing clinical studies of avutometinib in combination with sotorasib (NCT05074810) and adagrasib (NCT05375994) for patients with KRAS G12C NSCLC. Additionally, these preclinical data support combination of avutometinib with a G12D inhibitor for patients with KRAS G12D colorectal or pancreatic cancers. Citation Format: Silvia Coma, Monica Musteanu, Cristina Caffarra, Alessia Mira, Enrico Patrucco, Julien Dilly, Andrew J. Aguirre, Mariano Barbacid, Chiara Ambrogio, Jonathan A. Pachter. The RAF/MEK clamp avutometinib (VS-6766) enhances antitumor efficacy of KRAS G12C and G12D inhibitors through vertical inhibition of RAS, RAF and MEK. [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B025.

KRAS, an oncogene responsible for at least one fourth of all human cancers, was discovered four decades ago. Yet, the first KRAS selective inhibitor could not be approved until 2021. In spite of this long-awaited breakthrough, approved KRAS inhibitors only block one of the multiple oncogenic isoforms, KRASG12C, an isoform primarily present in lung adenocarcinomas. Moreover, tumor resistance to these KRAS inhibitors is becoming a significant issue in the clinic (Awad et al., N Engl J Med, 2021; Zhao et al., Nature 2021). To provide experimental information that might help to improve current KRAS targeting strategies, we have generated two experimental mouse tumor models of Kras-driven lung adenocarcinoma to compare the therapeutic consequences of eliminating, instead of inhibiting, KRAS oncoproteins. In addition, we have interrogated the molecular mechanisms responsible for the induction of resistance in sotorasib treated KrasG12C/p53 mouse lung adenocarcinomas. We have also investigated the potential role of tampering with KRAS signaling pathways to develop alternative therapeutic strategies against KRAS mutant lung and pancreatic cancers. Previous studies have shown that ablation of RAF1, either by itself, or in combination with the expression of a CDK4 kinase dead isoform, induce significant levels of tumor regression in Kras/p53 driven-lung adenocarcinomas without inducing significant toxicities (Sanclemente et al. Cancer Cell, 2018; Esteban-Burgos et al., PNAS 2020). However, Kras/p53 driven-pancreatic ductal adenocarcinomas (PDAC) are completely resistant to RAF1 ablation, unless RAF1 is deleted in combination with EGFR, an upstream KRAS effector (Blasco et al, Cancer Cell 2019). Yet, ablation of RAF1 and EGFR only resulted in the complete regression of a fraction of PDAC tumors. We have now identified activation of STAT3 as the main mechanism responsible for the resistance of PDAC tumors to RAF1/EGFR ablation. Indeed, combined ablation of RAF1/EGFR/STAT3 completely blocked proliferation of all tested mouse tumor cell lines and organoids. Moreover, all tumors tested in orthotopic allograft models regressed completely with no evidence of tumor regression for at least two months of TMX exposure. Finally, I will discuss potential strategies to pharmacologically validate this triple therapeutic strategy as well as plans to combine it with current KRAS inhibitors. Citation Format: Mariano Barbacid. Targeting KRAS: Light at the end of the tunnel [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA01.

CDK2 is a core cell-cycle enzyme that phosphorylates a variety of substrates to drive progression through the cell cycle. CDK2 is hyperactivated in multiple cancers and is therefore an attractive therapeutic target. Here, application of a novel small molecule CDK2/4/6 inhibitor, PF-06873600, enabled in-depth interrogation of CDK2 substrate phosphorylation, cell-cycle progression, and drug adaptation in preclinical models. CDK2-specific inhibition leads to rapid loss of substrate phosphorylation, but unexpectedly, CDK2 substrate phosphorylation rebounds within several hours. Whereas CDK1 is known to compensate for loss of CDK2 in Cdk2-/- mice, here we demonstrate an early dependence on CDK4/6 activity. CDK2 inhibition reveals latent CDK4/6 activity throughout S phase that backstops the proliferative program by maintaining Rb1 hyperphosphorylation, active E2F transcription, and Cyclin A2 expression, enabling re-activation of CDK2. Our results augment our understanding of CDK plasticity and indicate that co-inhibition of CDK2 and CDK4/6 is required to suppress adaptation to CDK2 inhibitors. Citation Format: Mansi Arora, Justin Moser, Timothy E. Hoffman, Lotte P. Watts, Mingwei Min, Monica Musteanu, Yao Rong, Jordan Schneider, C Ryland Ill, Varuna Nangia, Manuel Sanclemente, John Lapek, Lisa Nguyen, Sherry Niessen, Stephen Dann, Todd van Arsdale, Mariano Barbacid, Nichol Miller, Sabrina L. Spencer. Cells rapidly adapt to CDK2 inhibitors via plasticity of the CDK2/4/6-Rb-E2F axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5992.

Pancreatic cancer evades most of the current therapies and there is an urgent need for new treatments that could efficiently eliminate this aggressive tumor, such as the blocking of routes driving cell proliferation. In this work, we propose the use of small interfering RNA (siRNA) to inhibit the combined expression of FOSL-1 and YAP, two signaling proteins related with tumor cell proliferation and survival. To improve the efficacy of cell transfection, DODAB:MO (1:2) liposomes were used as siRNA nanocarriers, forming a complex denominated siRNA-lipoplexes. Liposomes and lipoplexes (carrying two siRNA for each targeted protein, or the combination of four siRNAs) were physico-chemically and biologically characterized. They showed very good biocompatibility and stability. The efficient targeting of FOSL-1 and YAP expression at both mRNA and protein levels was first proved in vitro using mouse pancreatic tumoral cell lines (KRASG12V and p53 knockout), followed by in vivo studies using subcutaneous allografts on mice. The peri-tumoral injection of lipoplexes lead to a significant decrease in the tumor growth in both Athymic Nude-Foxn1nu and C57BL/6 mice, mainly in those receiving the combination of four siRNAs, targeting both YAP and FOSL-1. These results open a new perspective to overcome the fast tumor progression in pancreatic cancer., government of Spain (ref. BIO2017-84974-R) and the Xunta de Galicia (Grupo de Referencia Competitiva [ED431C 2020/02]) and Centro singular de investigacion de Galicia and the European Regional Development Fund (ERDF)-[ED431G2019/06]. This work was also supported by grants from the CRIS Cancer Foundation and the Spanish Ministry of Science, Innovation and Universities (RTI2018-094664-B-I00) to M.B. M.B. is a recipient of an Endowed Chair from the AXA Research Fund.

RAF kinases are RAS-activated enzymes that initiate signalling through the MAPK cascade to control cellular proliferation, differentiation, and survival. Here, we describe the structure of the full-length RAF1 protein in complex with HSP90 and CDC37 obtained by cryo-electron microscopy. The reconstruction reveals a RAF1 kinase with an unfolded N-lobe separated from its C-lobe. The hydrophobic core of the N-lobe is trapped in the HSP90 dimer while CDC37 wraps around the chaperone and interacts with the N- and C-lobes of the kinase. The structure indicates how CDC37 can discriminate between the different members of the RAF family. Our structural analysis also reveals that the folded RAF1 assembles with 14-3-3 dimers, suggesting that after folding follows a similar activation as B-RAF. Finally, disruption of the interaction between CDC37 and the DFG segment of RAF1 unveils potential vulnerabilities to attempt the pharmacological degradation of RAF1 for therapeutic purposes.

KRAS G12C inhibitors (G12Ci), sotorasib and adagrasib, have demonstrated antitumor activity in patients with KRAS G12C mutant (mt) non-small cell lung cancer (NSCLC), and sotorasib has recently received FDA approval. It has been shown that simultaneous targeting of multiple nodes in the RAS/RAF/MEK/ERK (MAPK) pathway may be optimal for durable response. Furthermore, acquired mutations in the MAPK pathway occur clinically upon progression on adagrasib. Therefore, clinical combinations with G12Ci are needed. VS-6766 is a unique dual RAF/MEK inhibitor. In contrast to MEK-only inhibitors (MEKi), VS-6766 is a potent allosteric inhibitor of MEK kinase activity and induces a dominant negative RAF/MEK complex preventing phosphorylation of MEK by BRAF and CRAF. The combination of VS-6766 with the focal adhesion kinase (FAK) inhibitor defactinib with an intermittent schedule has shown clinical activity for patients with KRAS G12V and KRAS G12C mt NSCLC with a manageable safety profile relative to MEKi. Here, we tested whether combination of G12Ci with VS-6766 for vertical blockade of RAS, RAF and MEK might yield superior pathway blockade and antitumor efficacy. In 3D proliferation assays, VS-6766 was synergistic with both sotorasib and adagrasib in reducing viability of a panel of KRAS G12C mt cancer cell lines. Accordingly, VS-6766 effectively suppressed MAPK pathway signaling (pMEK, pERK) as a single agent, and the combination of VS-6766 + G12Ci showed improved depth and duration of MAPK pathway inhibition relative to G12Ci alone in KRAS G12C mt NSCLC models in vitro and in vivo. Reverse phase protein array (RPPA) analysis showed stronger inhibition of cell cycle/proliferation markers (e.g. aurora A/B, cyclin B1, pRb, pCDK1, pS6) with the combination than with VS-6766 or G12Ci alone. Additionally, the combination showed stronger activation of pro-apoptotic markers (e.g. cleaved caspase 7) and potential resistance markers (e.g. pFAK) than either agent alone. We are currently testing VS-6766 against a panel of cell lines expressing mutations identified in patients progressing on adagrasib. In KRAS G12C mt NSCLC models in vivo, VS-6766 combination augmented tumor growth inhibition by sotorasib, whereas trametinib combination was much less effective in augmenting sotorasib efficacy. In the H358 KRAS G12C mt NSCLC model, sotorasib monotherapy or sotorasib + trametinib failed to induce substantial tumor regression, whereas sotorasib + VS-6766 induced >25% tumor regression in 10/10 mice. Similarly, in the H2122 KRAS G12C mt NSCLC model, sotorasib + VS-6766 induced >20% tumor regression in 5/10 mice while sotorasib or sotorasib + trametinib were relatively ineffective. These results support the imminent clinical evaluation of VS-6766 in combination with a G12C inhibitor for treatment of KRAS G12C mt NSCLC in both G12Ci naïve patients and patients progressing on G12Ci treatment. Citation Format: Silvia Coma, Sanjib Chowdhury, Julien Dilly, Monica Musteanu, Mariano Barbacid, Andrew J. Aguirre, Jonathan A. Pachter. Dual RAF/MEK inhibitor VS-6766 enhances antitumor efficacy of KRAS G12C inhibitors through vertical inhibition of RAS, RAF and MEK [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 402.

RAS proteins are key players in multiple cellular processes. To study the role of RAS proteins individually or in combination, we have developed MEFs that can be rendered RASless, i.e., devoid of all endogenous RAS isoforms. These cells have significantly contributed to our understanding of the requirements for RAS functions in cell proliferation as well as their implications in diverse cellular processes. Here, we describe methods using RASless MEFs to study RAS-dependent cellular activities with special emphasis on proliferation. We provide the details to identify inducers of RAS-independent proliferation in colony assays. We recommend following these stringent guidelines to avoid false-positive results. Moreover, this protocol can be adapted to generate RASless MEFs ectopically expressing RAS variants to interrogate their function in the absence of endogenous RAS isoforms or to perform experiments in the absence of RAS. Finally, we also describe protocols to generate and use RASless MEFs for cell cycle analyses using the FUCCI cell cycle indicator.

La Lección Cajal es una conferencia anual dictada desde 2019 en la Universidad de Zaragoza por una figura académica relevante en su campo del saber, impulsada por el Vicerrectorado de Cultura y Proyección Social para conmemorar el 150 aniversario de la entrada de Santiago Ramón y Cajal en esta universidad, su «venerada alma mater». MARIANO BARBACID estudió Ciencias Químicas en la Universidad Complutense y se doctoró en 1974. Entre 1974 y 1977 completó su formación postdoctoral en el Instituto del Cáncer (NCI) de Estados Unidos. En 1978 formó su propio grupo de investigación en el NCI, donde trabajó hasta 1988. Durante la siguiente década (1988-1998) fue vicepresidente de Oncología Preclínica de la multinacional Bristol-Myers Squibb. En 1998 regresó a España para fundar y diri- gir el Centro Nacional de Investigaciones Oncológicas (CNIO). El Dr. Barbacid es miembro extranjero de la Academia de Ciencias de EE. UU., un honor que tan solo ostentan otros siete investigado- res españoles. En 2014 fue nombrado Fellow de la Academia de la Asociación Americana de Investigación en Cáncer (AACR), el primer español en recibir esta distinción. Es doctor honoris causa por la Universidad Internacional Menéndez Pelayo (1995), la Universidad de Cantabria (2011) y la Universidad de Barcelona (2014). En 2011 reci- bió la Gran Cruz del Dos de Mayo, la más alta distinción que otorga la Comunidad de Madrid. Entre los premios internacionales destacan la Medalla Burkitt (Irlanda, 2017), la Medalla de Honor de la Agencia Internacional del Cáncer de la Organización Mundial de la Salud (Francia, 2007) y el Premio Charles Rodolphe Brupbacher (Suiza, 2005). En la actualidad, el «Índice h» (Hirsch Index) del Dr. Barbacid es de 121, el más alto de España en las áreas de Bioquímica y Biología Molecular y el segundo más alto en Oncología.

RAS proteins are key players in multiple cellular processes. To study the role of RAS proteins individually or in combination, we have developed MEFs that can be rendered RASless, i.e., devoid of all endogenous RAS isoforms. These cells have significantly contributed to our understanding of the requirements for RAS functions in cell proliferation as well as their implications in diverse cellular processes. Here, we describe methods using RASless MEFs to study RAS-dependent cellular activities with special emphasis on proliferation. We provide the details to identify inducers of RAS-independent proliferation in colony assays. We recommend following these stringent guidelines to avoid false-positive results. Moreover, this protocol can be adapted to generate RASless MEFs ectopically expressing RAS variants to interrogate their function in the absence of endogenous RAS isoforms or to perform experiments in the absence of RAS. Finally, we also describe protocols to generate and use RASless MEFs for cell cycle analyses using the FUCCI cell cycle indicator.

In mammals, the KRAS locus encodes two protein isoforms, KRAS4A and KRAS4B, which differ only in their C terminus via alternative splicing of distinct fourth exons. Previous studies have shown that whereas KRAS expression is essential for mouse development, the KRAS4A isoform is expendable. Here, we have generated a mouse strain that carries a terminator codon in exon 4B that leads to the expression of an unstable KRAS4B154 truncated polypeptide, hence resulting in a bona fide Kras4B-null allele. In contrast, this terminator codon leaves expression of the KRAS4A isoform unaffected. Mice selectively lacking KRAS4B expression developed to term but died perinatally because of hypertrabeculation of the ventricular wall, a defect reminiscent of that observed in embryos lacking the Kras locus. Mouse embryonic fibroblasts (MEFs) obtained from Kras4B-/- embryos proliferated less than did wild-type MEFs, because of limited expression of KRAS4A, a defect that can be compensated for by ectopic expression of this isoform. Introduction of the same terminator codon into a Kras FSFG12V allele allowed expression of an endogenous KRAS4AG12V oncogenic isoform in the absence of KRAS4B. Exposure of Kras +/FSF4AG12V4B- mice to Adeno-FLPo particles induced lung tumors with complete penetrance, albeit with increased latencies as compared with control Kras +/FSFG12V animals. Moreover, a significant percentage of these mice developed proximal metastasis, a feature seldom observed in mice expressing both mutant isoforms. These results illustrate that expression of the KRAS4AG12V mutant isoform is sufficient to induce lung tumors, thus suggesting that selective targeting of the KRAS4BG12V oncoprotein may not have significant therapeutic consequences. We thank Marta San Roman, Raquel Villar, and Nuria Cabrera for excellent technical assistance; Mayte Lamparero and Isabel Blanco (Animal Facility) for mouse work; the Histopathology Unit for processing of mouse tissues; Lola Martinez (Flow Cytometry Unit) for her help with flow cytometry analyses; Diego Megias and Manuel Perez (Confocal Microscopy Unit) for assistance with confocal microscopy; and the Mouse Genome Editing Unit for support with the generation of the mouse strains described here. We also thank Ignacio Perez de Castro (Instituto de Salud Carlos III, Madrid, Spain) for sharing the EGFP-KRAS4B plasmid and Orlando Dominguez (Genomics Unit) and Pedro P. Lopez-Casas (Clinical Research Program) for their advice on exome sequencing. This work was supported by grants from the European Research Council (ERC-2015-AdG/695566, THERACAN), the Spanish Ministry of Science, Innovation and Universities (RTC-2017-6576-1), and the Autonomous Community of Madrid (B2017/BMD-3884 iLUNG-CM); a grant from the CRIS Cancer Foundation (to M.B.); and a grant from the Spanish Ministry of Science, Innovation and Universities (RTI2018-094664-B-I00, to M.B. and M.M.). M.B. is a recipient of an Endowed Chair from the AXA Research Fund. M.S. was supported by predoctoral contract "Severo Ochoa" (BES-2016-079096) from the SpanishMinistry of Science, Innovation and Universities. G.P. was a recipient of a "Young Ph.D." grant from the Government of the Community of Madrid. F.F.-G. was supported by a formacion de profesorado universitario (FPU) fellowship from the Spanish Ministry of Science, Innovation and Universities. Sí

Pancreatic acinar cells are a cell type of origin for pancreatic cancer that become progressively less sensitive to tumorigenesis induced by oncogenic Kras mutations after birth. This sensitivity is increased when Kras mutations are combined with pancreatitis. Molecular mechanisms underlying these observations are still largely unknown. To identify these mechanisms, we generated the first CRISPR-edited mouse models that enable detection of wild-type and mutant KRAS proteins in vivo. Analysis of these mouse models revealed that more than 75% of adult acinar cells are devoid of detectable KRAS protein. In the 25% of acinar cells expressing KRAS protein, transcriptomic analysis highlighted a slight upregulation of the RAS and MAPK pathways. However, at the protein level, only marginal pancreatic expression of essential KRAS effectors, including C-RAF, was observed. The expression of KRAS and its effectors gradually decreased after birth. The low sensitivity of adult acinar cells to Kras mutations resulted from low expression of KRAS and its effectors and the subsequent lack of activation of RAS/MAPK pathways. Pancreatitis triggered expression of KRAS and its effectors as well as subsequent activation of downstream signaling; this induction required the activity of EGFR. Finally, expression of C-RAF in adult pancreas was required for pancreatic tumorigenesis. In conclusion, our study reveals that control of the expression of KRAS and its effectors regulates the sensitivity of acinar cells to transformation by oncogenic Kras mutations. Significance: This study generates new mouse models to study regulation of KRAS during pancreatic tumorigenesis and highlights a novel mechanism through which pancreatitis sensitizes acinar cells to Kras mutations.

International audience; Platinum-based chemotherapy in combination with immune-checkpoint inhibitors is the current standard of care for patients with advanced lung adenocarcinoma (LUAD). However, tumor progression evolves in most cases. Therefore, predictive biomarkers are needed for better patient stratification and for the identification of new therapeutic strategies, including enhancing the efficacy of chemotoxic agents. Here, we hypothesized that discoidin domain receptor 1 (DDR1) may be both a predictive factor for chemoresistance in patients with LUAD and a potential target positively selected in resistant cells. By using biopsies from patients with LUAD, KRAS-mutant LUAD cell lines, and in vivo genetically engineered KRAS-driven mouse models, we evaluated the role of DDR1 in the context of chemotherapy treatment. We found that DDR1 is upregulated during chemotherapy both in vitro and in vivo. Moreover, analysis of a cohort of patients with LUAD suggested that high DDR1 levels in pretreatment biopsies correlated with poor response to chemotherapy. Additionally, we showed that combining DDR1 inhibition with chemotherapy prompted a synergistic therapeutic effect and enhanced cell death of KRAS-mutant tumors in vivo. Collectively, this study suggests a potential role for DDR1 as both a predictive and prognostic biomarker, potentially improving the chemotherapy response of patients with LUAD.

Malignant transformation entails important changes in the control of cell proliferation through the rewiring of selected signaling pathways. Cancer cells then become very dependent on the proper function of those pathways, and their inhibition offers therapeutic opportunities. Here we identify the stress kinase p38α as a nononcogenic signaling molecule that enables the progression of KrasG12V-driven lung cancer. We demonstrate in vivo that, despite acting as a tumor suppressor in healthy alveolar progenitor cells, p38α contributes to the proliferation and malignization of lung cancer epithelial cells. We show that high expression levels of p38α correlate with poor survival in lung adenocarcinoma patients, and that genetic or chemical inhibition of p38α halts tumor growth in lung cancer mouse models. Moreover, we reveal a lung cancer epithelial cell-autonomous function for p38α promoting the expression of TIMP-1, which in turn stimulates cell proliferation in an autocrine manner. Altogether, our results suggest that epithelial p38α promotes KrasG12V-driven lung cancer progression via maintenance of cellular self-growth stimulatory signals. Fil: Vitos Faleato, Jessica. Instituto de Investigación Biomédica de Barcelona.; España Fil: Real Varela, Sebastián Martín. Instituto de Investigación Biomédica de Barcelona.; España Fil: Gutiérrez Prat, Nuria. Instituto de Investigación Biomédica de Barcelona.; España Fil: Villanueva, Alberto. Instituto Catalán de Oncología; España Fil: Llonch llongueras, Elisabet. Instituto de Investigación Biomédica de Barcelona.; España Fil: Drosten, Matthias. Centro Nacional de Investigaciones Oncologicas; España Fil: Barbacid, Mariano. Centro Nacional de Investigaciones Oncologicas; España Fil: Nebreda, Ángel R.. Instituto de Investigación Biomédica de Barcelona.; España

KRAS mutations occur in a quarter of all of human cancers, yet no selective drug has been approved to treat these tumors. Despite the recent development of drugs that block KRASG12C, the majority of KRAS oncoproteins remain undruggable. Here, we review recent efforts to validate individual components of the mitogen-activated protein kinase (MAPK) pathway as targets to treat KRAS-mutant cancers by comparing genetic information derived from experimental mouse models of KRAS-driven lung and pancreatic tumors with the outcome of selective MAPK inhibitors in clinical trials. We also review the potential of RAF1 as a key target to block KRAS-mutant cancers. This work was supported by grants from the European Research Council (ERC-AG/695566, THERACAN), the Spanish Ministry of Science, Innovation and Universities (RTI2018-094664-B-I00 and RTC2017-6576-1), the Autonomous Community of Madrid (B2017/BMD-3884 iLUNG-CM), and the Asociacion Espanola contra el Cancer (GC166173694BARB). M.B. is a recipient of an Endowed Chair from the AXA Research Fund. Sí

Myocardial uptake can hamper visualization of lung tumors, atherosclerotic plaques, and inflammatory diseases in 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) studies because it leads to spillover in adjacent structures. Several preparatory pre-imaging protocols (including dietary restrictions and drugs) have been proposed to decrease physiological [18F]FDG uptake by the heart, although their effect on tumor glucose metabolism remains largely unknown. The objective of this study was to assess the effects of a ketogenic diet (as an alternative protocol to fasting) on tumor glucose metabolism assessed by [18F]FDG positron emission tomography (PET) in a mouse model of lung cancer. PET scans were performed 60 min after injection of 18.5 MBq of [18F]FDG. PET data were collected for 45 min, and an x-ray computed tomograph (CT) image was acquired after the PET scan. A PET/CT study was obtained for each mouse after fasting and after the ketogenic diet. Quantitative data were obtained from regions of interest in the left ventricular myocardium and lung tumor. Three days on a ketogenic diet decreased mean standard uptake value (SUVmean) in the myocardium (SUVmean 0.95 ± 0.36) more than one night of fasting (SUVmean 1.64 ± 0.93). Tumor uptake did not change under either dietary condition. These results show that 3 days on high-fat diets prior to [18F]FDG-PET imaging does not change tumor glucose metabolism compared with one night of fasting, although high-fat diets suppress myocardial [18F]FDG uptake better than fasting.

Mutations at multiple sites in MEK1 occur in cancer, suggesting that their mechanisms of activation might be different. We analyzed 17 tumor-associated MEK1 mutants and found that they drove ERK signaling autonomously or in a RAS/RAF-dependent manner. The latter are sensitive to feedback inhibition of RAF, which limits their functional output, and often cooccur with RAS or RAF mutations. They act as amplifiers of RAF signaling. In contrast, another class of mutants deletes a hitherto unrecognized negative regulatory segment of MEK1, is RAF- and phosphorylation-independent, is unaffected by feedback inhibition of upstream signaling, and drives high ERK output and transformation in the absence of RAF activity. Moreover, these RAF-independent mutants are insensitive to allosteric MEK inhibitors, which preferentially bind to the inactivated form of MEK1. All the mutants are sensitive to an ATP-competitive MEK inhibitor. Thus, our study comprises a novel therapeutic strategy for tumors driven by RAF-independent MEK1 mutants. Significance: Mutants with which MEK1 mutants coexist and their sensitivity to inhibitors are determined by allele-specific properties. This study shows the importance of functional characterization of mutant alleles in single oncogenes and identifies a new class of MEK1 mutants, insensitive to current MEK1 inhibitors but treatable with a new ATP-competitive inhibitor. Cancer Discov; 8(5); 648–61. ©2018 AACR. See related commentary by Maust et al., p. 534. This article is highlighted in the In This Issue feature, p. 517

The RAS/RAF/MEK/ERK (MAPK) pathway shows frequent mutations in cancer which often confer worse prognosis. Whereas KRAS mutations (mt) are prevalent in pancreatic cancer (PDAC; 98%), colorectal cancer (CRC; 45%) and non-small cell lung cancer (NSCLC; 31%), melanoma shows frequent mutations in BRAF (60%) and NRAS (28%). Although several BRAF and MEK inhibitors (MEKi) are FDA approved, there is still a need for agents with improved response rate, duration of response, and tolerability. VS-6766 is a unique dual RAF/MEK inhibitor. In contrast to MEKi, VS-6766 is a potent allosteric inhibitor of MEK kinase activity, which promotes a dominant negative RAF/MEK complex preventing phosphorylation of MEK by wildtype BRAF, BRAF V600E and CRAF. This unique mechanism allows VS-6766 to block MEK signaling without the compensatory activation of MEK that appears to limit the efficacy of MEKi. In 3D proliferation assays in vitro, VS-6766 inhibited cell proliferation across multiple MAPK pathway alterations, including KRAS (G12C, G12D, G12V, G13D and Q61K), BRAF (V600E and class 2), NRAS and NF1 mt. Cell lines with KRAS G12V mt (n = 10; IC50 = 10 - 400 nM, average = 120 nM) were generally more sensitive than cell lines with KRAS G12D mt (n = 9; IC50 = 150 – 12,000 nM; average = 2.4 mM). Cell lines with NRAS mt also appeared to be especially sensitive to VS-6766 (average IC50 = 80 nM). Overall, VS-6766 inhibited proliferation of cell lines representing multiple cancers including NSCLC, PDAC, CRC and melanoma. VS-6766 effectively suppressed RAS pathway signaling (pMEK, pERK, DUSP4, pEphA2) throughout a 48-hour period among a panel of KRAS mt NSCLC cell lines. In a genetically engineered mouse tumor model of KRAS G12V mt/Trp53 KO NSCLC, which has previously been shown to be CRAF-dependent (Sanclemente et al., 2018), VS-6766 (0.1 mg/kg QD) induced regression of all tumors, whereas the MEKi trametinib at the same dose did not induce significant tumor growth inhibition. In KRAS mt NSCLC (H358) and ovarian cancer (TOV21G) xenograft models, VS-6766 significantly inhibited tumor growth (tumor growth inhibition, TGI = 70% and 69%, respectively), whereas the equivalent dose of trametinib was much less effective in inhibiting tumor growth (TGI = 13% and 29%, respectively). Clinically, intermittent dosing of VS-6766 as monotherapy has shown partial responses in patients with KRAS or BRAF mt gynecological malignancies (3/5) and KRAS mt NSCLC (3/10) (Guo, 2020). Interestingly, in NSCLC, responses occurred especially in patients with KRAS G12V mt tumors, which correlates with the greater anti-proliferative potency of VS-6766 observed across KRAS G12V mt NSCLC, CRC and PDAC cell lines. These data support the recent initiation of two registration-directed studies evaluating VS-6766 ± defactinib (FAK inhibitor) for the treatment of recurrent low-grade serous ovarian cancer (NCT04625270) and recurrent NSCLC with KRAS G12V or other KRAS mutation (NCT04620330), and suggest potential utility of VS-6766 for treatment of additional tumor types and oncogenic mutations in the MAPK pathway. Citation Format: Silvia Coma, Sanjib Chowdhury, Monica Musteanu, Mariano Barbacid, Jonathan A. Pachter. Dual RAF/MEK inhibitor VS-6766 for treatment of solid tumors with diverse MAPK pathway alterations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P121.

Insulin is one of the standard components used to culture primary neurospheres. Although it stimulates growth of different types of cells, the effects of insulin on adult neural stem cells (NSCs) have not been well characterized. Here, we reveal that insulin stimulates proliferation, but not survival or self-renewal, of adult NSCs. This effect is mediated by insulin receptor substrate 2 (IRS2) and subsequent activation of the protein kinase B (or Akt), leading to increased activity of the G1-phase cyclin-dependent kinase 4 (Cdk4) and cell cycle progression. Neurospheres isolated from Irs2-deficient mice are reduced in size and fail to expand in culture and this impaired proliferation is rescued by introduction of a constitutively active Cdk4 (Cdk4R24C/R24C). More interestingly, activation of the IRS2/Akt/Cdk4 signaling pathway by insulin is also necessary for the generation in vitro of neurons and oligodendrocytes from NSCs. Furthermore, the IRS2/Cdk4 pathway is also required for neuritogenesis, an aspect of neuronal maturation that has not been previously linked to regulation of the cell cycle. Differentiation of NSCs usually follows exit from the cell cycle due to increased levels of CDK-inhibitors which prevent activation of CDKs. In contrast, our data indicate that IRS2-mediated Cdk4 activity in response to a mitogen such as insulin promotes terminal differentiation of adult NSCs.

The initiating oncogenic event in almost half of human lung adenocarcinomas is still unknown, a fact that complicates the development of selective targeted therapies. Yet these tumours harbour a number of alterations without obvious oncogenic function including BRAF-inactivating mutations. Inactivating BRAF mutants in lung predominate over the activating V600E mutant that is frequently observed in other tumour types. Here we demonstrate that the expression of an endogenous Braf(D631A) kinase-inactive isoform in mice (corresponding to the human BRAF(D594A) mutation) triggers lung adenocarcinoma in vivo, indicating that BRAF-inactivating mutations are initiating events in lung oncogenesis. Moreover, inactivating BRAF mutations have also been identified in a subset of KRAS-driven human lung tumours. Co-expression of Kras(G12V) and Braf(D631A) in mouse lung cells markedly enhances tumour initiation, a phenomenon mediated by Craf kinase activity, and effectively accelerates tumour progression when activated in advanced lung adenocarcinomas. We also report a key role for the wild-type Braf kinase in sustaining Kras(G12V)/Braf(D631A)-driven tumours. Ablation of the wild-type Braf allele prevents the development of lung adenocarcinoma by inducing a further increase in MAPK signalling that results in oncogenic toxicity; this effect can be abolished by pharmacological inhibition of Mek to restore tumour growth. However, the loss of wild-type Braf also induces transdifferentiation of club cells, which leads to the rapid development of lethal intrabronchiolar lesions. These observations indicate that the signal intensity of the MAPK pathway is a critical determinant not only in tumour development, but also in dictating the nature of the cancer-initiating cell and ultimately the resulting tumour phenotype.

Genetic studies in mice have provided evidence that H-Ras and K-Ras proteins are bioequivalent. However, human tumors display marked differences in the association of RAS oncogenes with tumor type. Thus, to further assess the bioequivalence of oncogenic H-Ras and K-Ras, we replaced the coding region of the murine K-Ras locus with H-Ras oncogene sequences. Germline expression of H-Ras or K-Ras from the K-Ras locus resulted in embryonic lethality. However, expression of these genes in adult mice led to different tumor phenotypes. Whereas H-Ras elicited papillomas and hematopoietictumors, K-Ras induced lung tumors and gastric lesions. Pulmonary expression of H-Ras created a senescence-like state caused by excessive MAPK signaling. Likewise, H-Ras but not K-Ras induced senescence in mouse embryonic fibroblasts. Label-free quantitative analysis revealed that minor differences in H-Ras expression levels led to drastically different biological outputs, suggesting that subtle differences in MAPK signaling confer nonequivalent functions that influence tumor spectra induced by RAS oncoproteins., This work was supported by grants from the European Research Council (ERC-AG/ 250297-RAS AHEAD), EU-Framework Programme (LSHG-CT-2007-037665/CHEMORES, HEALTH-F2-2010-259770/LUNGTARGET and HEALTH2010-260791/EUROCANPLATFORM), Spanish Ministry of Economy and Competitiveness (SAF2011-30173 and SAF2014-59864-R) and Autonomous Community of Madrid (S2011/BDM-2470/ONCOCYCLE) to MB, Castilla-León Autonomous Government (CSI101U13, BIO/SA01/15), the Spanish Ministry of Economy and Competitiveness (SAF2012-31371, RD12/0036/0002), Worldwide Cancer Research (14-1248), the Solórzano Foundation, and the Ramón Areces Foundation to XRB. Spanish government-sponsored funding to XRB is partially supported by the European Regional Development Fund. MB is the recipient of an Endowed Chair from the AXA Research Fund.

CIC (also known as Capicua) is a transcriptional repressor negatively regulated by RAS/MAPK signaling. Whereas the functions of Cic have been well characterized in Drosophila, little is known about its role in mammals. CIC is inactivated in a variety of human tumors and has been implicated recently in the promotion of lung metastases. Here, we describe a mouse model in which we inactivated Cic by selectively disabling its DNA-binding activity, a mutation that causes derepression of its target genes. Germline Cic inactivation causes perinatal lethality due to lung differentiation defects. However, its systemic inactivation in adult mice induces T-cell acute lymphoblastic lymphoma (T-ALL), a tumor type known to carry CIC mutations, albeit with low incidence. Cic inactivation in mice induces T-ALL by a mechanism involving derepression of its well-known target, Etv4. Importantly, human T-ALL also relies on ETV4 expression for maintaining its oncogenic phenotype. Moreover, Cic inactivation renders T-ALL insensitive to MEK inhibitors in both mouse and human cell lines. Finally, we show that Ras-induced mouse T-ALL as well as human T-ALL carrying mutations in the RAS/MAPK pathway display a genetic signature indicative of Cic inactivation. These observations illustrate that CIC inactivation plays a key role in this human malignancy, This work was supported by grants from the Fundació La Marató de TV3 (20131730/1) to G.J. and M.B., and the European Research Council (ERC-AG/ 250297-RAS AHEAD), the EU-Framework Programme (HEALTH-F2-2010-259770/LUNGTARGET and HEALTH-2010- 260791/EUROCANPLATFORM), the Spanish Ministry of Economy and Competitiveness (SAF2014-59864-R), the Autonomous Community of Madrid (S2011/BDM-2470/ONCOCYCLE), and the Asociación Española contra el Cáncer (AECC) (GC16173694BARB) to M.B. M.B. is the recipient of an Endowed Chair from the AXA Research Fund. L.S.-C. was supported by a fellowship from the Programa de Formación de Personal Investigator (FPI) of the Spanish Ministry of Economy and Competitiveness.