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KRASG12D is the most frequent KRAS mutation in human cancer with particularly high frequencies in pancreatic and colorectal cancer. Informed by the structure of the KRASG12C inhibitor adagrasib, Hallin et al. have now, through multiple rounds of structure-based drug design, identified and validated a potent, selective, and noncovalent KRASG12D inhibitor, MRTX1133. This study demonstrated that MRTX1133 inhibited both the inactive and active state of KRASG12D and showed potent antitumor activity in several preclinical models of pancreatic and colorectal cancer, especially when combined with cetuximab, a monoclonal antibody against the EGFR, or BYL-719, a potent PI3Kα inhibitor., This work was supported by grants from the European Research Council (ERC-2015-AdG/695566, THERACAN), the CRIS Cancer Foundation and the CIBERONC to M.B. M.B. is a recipient of an Endowed Chair from the AXA Research Fund.

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.

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.

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.

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.

For decades, KRAS mutant lung adenocarcinomas (LUAD) have been refractory to therapeutic strategies based on personalized medicine owing to the complexity of designing inhibitors to selectively target KRAS and downstream targets with acceptable toxicities. The recent development of selective KRASG12C inhibitors represents a landmark after 40 years of intense research efforts since the identification of KRAS as a human oncogene. Here, we discuss the mechanisms responsible for the rapid development of resistance to these inhibitors, as well as potential strategies to overcome this limitation. Other therapeutic strategies aimed at inhibiting KRAS oncogenic signaling by targeting either upstream activators or downstream effectors are also reviewed. Finally, we discuss the effect of targeting the mitogen-activated protein kinase (MAPK) pathway, both based on the failure of MEK and ERK inhibitors in clinical trials, as well as on the recent identification of RAF1 as a potential target due to its MAPK-independent activity. These new developments, taken together, are likely to open new avenues to effectively treat KRAS mutant LUAD. 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, RTI2018-094664-BI00) the Autonomous Community of Madrid (B2017/BMD-3884 iLUNG-CM) and the CRIS Cancer Foundation (to MB) as well as the Spanish Ministry of Science and Innovation (PID2020-116705RB-100) (to MD). MB is a recipient of an Endowed Chair from the AXA Research Fund. Sí

The kinase suppressor of rat sarcoma (RAS) proteins (KSR1 and KSR2) have long been considered as scaffolding proteins required for optimal mitogen-activated protein kinase (MAPK) pathway signalling. However, recent evidence suggests that they play a more complex role within this pathway. Here, we demonstrate that ectopic expression of KSR1 or KSR2 is sufficient to activate the MAPK pathway and to induce cell proliferation in the absence of RAS proteins. In contrast, the ectopic expression of KSR proteins is not sufficient to induce cell proliferation in the absence of either rapidly accelerated fibrosarcoma (RAF) or MAPK-ERK kinase proteins, indicating that they act upstream of RAF. Indeed, KSR1 requires dimerization with at least one member of the RAF family to stimulate proliferation, an event that results in the translocation of the heterodimerized RAF protein to the cell membrane. Mutations in the conserved aspartic acid–phenylalanine–glycine motif of KSR1 that affect ATP binding impair the induction of cell proliferation. We also show that increased expression levels of KSR1 decrease the responsiveness to the KRASG12C inhibitor sotorasib in human cancer cell lines, thus suggesting that increased levels of expression of KSR may make tumour cells less dependent on KRAS oncogenic signalling., 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), the CRIS Cancer Foundation and the Asociación Española contra el Cáncer (AECC) (GC166173694BARB). MB is a recipient of an Endowed Chair from the AXA Research Fund. GP has been supported by a fellowship from the Programa de Atracción de Talento of the Autonomous Community of Madrid. SGA is a recipient of a postdoctoral fellowship from the AECC. OB is a recipient of a fellowship from the Formación de Personal Investigador (FPI) program of the Spanish Ministry of Science, Innovation and Universities.

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.

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.

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.

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.

Activating mutations in KRAS and EGFR, the two most frequent oncogenes in human lung adenocarcinoma, are mutually exclusive, a phenotype attributed to functional redundancy implying lack of positive selection. Employing a mouse model expressing EGFRL858R in advanced KrasG12V-driven tumors we show that their mutual exclusivity can be explained by detrimental effects of their co-expression in lung adenocarcinoma. In vivo, expression of EGFRL858R in KrasG12V-driven tumors triggers replicative stress and apoptosis, while the surviving cells enter a transient cytostatic state incompatible with tumor development that is fully reversible upon discontinued EGFRL858R expression. Eventually, sustained expression of both mutants induces attenuation of oncogenic signaling to levels compatible with proliferation and tumor growth resulting in high sensitivity to Mek inhibition. Our results indicate that the mutual exclusivity of KRAS and EGFR mutations occurs as a combination of cellular toxicity and signal adjustment resulting in lack of selective advantage for cells expressing both oncogenes.

KRAS oncogenes are responsible for one fourth of all human tumors, including lung and pancreatic adenocarcinomas, two tumor types with some of the worse prognoses. Unfortunately, development of suitable therapies to treat these tumors has remained elusive for the last 35 years. Although KRAS remains an undruggable target (with the possible exception of the KRASG12C isoform), most of its downstream effectors are druggable kinases. Indeed, pharma and biotech companies have already developed dozens of selective inhibitors against these kinases. Yet none of them has been approved by the FDA, mostly due to unacceptable toxicities at antitumor effective doses. Hence, patients are still treated with old chemotherapy drugs. To address this important health issue, we have used a new generation of genetically engineered mouse (GEM) tumor models of KRas/Tp53 driven lung and pancreatic tumors to deconstruct oncogenic KRas signaling to identify those effectors whose systemic ablation/inactivation in tumor-bearing mice will result in therapeutic responses without inducing unacceptable toxicities. We have used this experimental strategy to interrogate the effect of ablating/inactivating each of the members of the MAPK cascade, including the Raf, Mek, and Erk kinases, as well as key effectors of the PI3K pathway, including PI3K p110alpha and mTOR. We have also evaluated additional downstream signaling elements such as the cell cycle Cdks. This systematic approach has revealed that most of the KRas signaling effectors are not suitable therapeutic targets due to either lack of therapeutic activity, such as Cdk2, Cdk6 A-Raf, or B-Raf, or to the induction of unacceptable toxicities such as the Mek1/2 and Erk1/2 kinases, PI3K p110alpha, mTOR, and Cdk1. Only Raf1 and Cdk4 turned out to be suitable therapeutic targets, based not only on their antitumor properties but also on the well-tolerated toxicities. Surprisingly, EGFR, an upstream effect of KRas, also has suitable therapeutic activity in pancreatic but not in lung tumors. We are now combining these targets to define more efficacious therapeutic strategies that could be eventually translated to the clinic. Moreover, we are using these experimental GEM tumor models to identify potential resistance mechanisms to these therapies. Citation Format: Mariano Barbacid. The use of GEM tumor models to identify effective therapies against KRAS mutant tumors [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr IA11.

The presence of K-RAS oncogenes in human tumors has been known for over 35 years. Indeed, extensive analysis of cancer genomes has revealed that they are present in at least a quarter of all solid tumors. Moreover, there is overwhelming evidence that they represent the initiating event in lung and pancreatic adenocarcinomas, two cancer types with some of the worse prognoses. Recent studies in genetically engineered mouse (GEM) tumor models have shown that inhibition of K-RAS oncogene expression results in rapid tumor regression. Yet in spite of recent progress, K-RAS oncogenes remain for the most part undruggable. Ironically, the main K-RAS downstream effectors are druggable kinases. Selective inhibitors against members of the MAPK and PI3K signaling pathways have been developed over the last two decades. Yet none of them has yielded suitable results in clinical trials to merit FDA approval against K-RAS mutant malignancies. In an effort to identify K-RAS vulnerabilities that may have been missed using biochemical or pharmacologic approaches, we decided a decade ago to deconstruct K-Ras oncogenic signaling in GEM tumor models by interrogating the effect of ablating or inactivating its downstream kinases during tumor initiation. In addition, we examined the consequences of systemically eliminating or inhibiting these targets during adult homeostasis, in order to evaluate the potential toxic effects that may result from tampering with the MAPK and PI3K signaling pathways. These studies revealed three classes of downstream targets: those whose ablation had no consequences on tumor development, those that prevented tumor development but were unacceptably toxic when ablated systemically, and a limited number (c-Raf and Cdk4) that prevented or thwarted tumor development without causing significant toxicities upon systemic elimination. In addition to these downstream targets, we were also able to identify the EGFR as a potential target in pancreatic but not in lung tumors. More recently, we have established a new generation of “therapeutic” GEM tumor models in which we can separate, both spatially and temporally, tumor development from target ablation/inactivation by using two independent recombinase systems. In these models, whereas K-Ras or K-Ras/TP53-driven tumor development is mediated by the Flp(o)/frt system, we use the inducible CreERT2/loxP system for systemic target ablation/inactivation. These “therapeutic” models have allowed us to explore the therapeutic potential of these previously validated targets in tumor-bearing mice. Our results have revealed that c-Raf ablation induces significant levels of tumor regression in K-Ras/TP53-driven lung tumors, a therapeutic effect that is further potentiated by the concomitant expression of a kinase dead isoform of Cdk4. In the corresponding GEM pancreatic tumor model, ablation or inactivation of c-Raf, Cdk4, or EGFR failed to prevent tumor regression, although tampering with Cdk4 or EGFR resulted in significant tumor delays. Importantly, concomitant ablation of c-Raf and EGFR in K-Ras/TP53-driven advanced pancreatic tumors resulted in complete tumor regression in half of the mice. Preliminary comparison of these “responder” tumors with those that did not respond to c-Raf/EGFR ablation is revealing some vulnerabilities that may provide additional targets against these advanced tumors. We hope that these results may serve to pave the way to define more efficacious therapeutic strategies against K-RAS mutant tumors that might eventually be translated to the clinic. Citation Format: Mariano Barbacid. Deconstructing K-RAS oncogenic signaling in lung and pancreatic tumors [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA01.

E-type cyclins E1 (CcnE1) and E2 (CcnE2) are regulatory subunits of cyclin-dependent kinase 2 (Cdk2) and thought to control the transition of quiescent cells into the cell cycle. Initial findings indicated that CcnE1 and CcnE2 have largely overlapping functions for cancer development in several tumor entities including hepatocellular carcinoma (HCC). In the present study, we dissected the differential contributions of CcnE1, CcnE2, and Cdk2 for initiation and progression of HCC in mice and patients. To this end, we tested the HCC susceptibility in mice with constitutive deficiency for CcnE1 or CcnE2 as well as in mice lacking Cdk2 in hepatocytes. Genetic inactivation of CcnE1 largely prevented development of liver cancer in mice in two established HCC models, while ablation of CcnE2 had no effect on hepatocarcinogenesis. Importantly, CcnE1-driven HCC initiation was dependent on Cdk2. However, isolated primary hepatoma cells typically acquired independence on CcnE1 and Cdk2 with increasing progression in vitro, which was associated with a gene signature involving secondary induction of CcnE2 and up-regulation of cell cycle and DNA repair pathways. Importantly, a similar expression profile was also found in HCC patients with elevated CcnE2 expression and poor survival. In general, overall survival in HCC patients was synergistically affected by expression of CcnE1 and CcnE2, but not through Cdk2. Our study suggests that HCC initiation specifically depends on CcnE1 and Cdk2, while HCC progression requires expression of any E-cyclin, but no Cdk2.

K-RAS signaling has been intensely studied for over 40 years. Yet, as of today, no drugs have been approved to treat K-RAS mutant cancers. Since the turn of the century, scientists have used genetically engineered mouse (GEM) models to reproduce K-RAS mutant cancers in a laboratory setting to elucidate those molecular events responsible for the onset and progression of these tumors and to identify suitable therapies. In this review, we outline a brief description of available GEM models for two tumor types known to be driven by K-RAS mutations: lung adenocarcinoma and pancreatic ductal adenocarcinoma. In addition, we summarize a series of studies that have used these GEM tumor models to validate, either by genetic or pharmacological approaches, the therapeutic potential of a variety of targets, with the ultimate goal of translating these results to the clinical setting. This work was supported by grants from the European Research Council (ERC-ADG/695566-THERACAN), the Spanish Ministry of Economy and Competitiveness (SAF2014-59864-R) and the Spanish Association against Cancer (AECC, GC16173694BARB). Sí

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the presence of abundant desmoplastic stroma primarily composed of cancer-associated fibroblasts (CAFs). It is generally accepted that CAFs stimulate tumor progression and might be implicated in drug resistance and immunosuppression. Here, we have compared the transcriptional profile of PDGFRα + CAFs isolated from genetically engineered mouse PDAC tumors with that of normal pancreatic fibroblasts to identify genes potentially implicated in their protumorigenic properties. We report that the most differentially expressed gene, Saa3, a member of the serum amyloid A (SAA) apolipoprotein family, is a key mediator of the protumorigenic activity of PDGFRα + CAFs. Whereas Saa3-competent CAFs stimulate the growth of tumor cells in an orthotopic model, Saa3-null CAFs inhibit tumor growth. Saa3 also plays a role in the cross talk between CAFs and tumor cells. Ablation of Saa3 in pancreatic tumor cells makes them insensitive to the inhibitory effect of Saa3-null CAFs. As a consequence, germline ablation of Saa3 does not prevent PDAC development in mice. The protumorigenic activity of Saa3 in CAFs is mediated by Mpp6, a member of the palmitoylated membrane protein subfamily of the peripheral membrane-associated guanylate kinases (MAGUK). Finally, we interrogated whether these observations could be translated to a human scenario. Indeed, SAA1, the ortholog of murine Saa3, is overexpressed in human CAFs. Moreover, high levels of SAA1 in the stromal component correlate with worse survival. These findings support the concept that selective inhibition of SAA1 in CAFs may provide potential therapeutic benefit to PDAC patients., This work was supported by European Research Council Grants ERC-AG/250297-RAS AHEAD and ERC-AG/695566-THERACAN, Spanish Ministry of Economy and Competitiveness Grant SAF2014-59864-R, and Asociación Española contra el Cáncer Grant GC16173694BARB (to M. Barbacid). M.D. was supported by a fellowship from La Caixa International Fellowship Program. M. Barbacid is the recipient of an Endowed Chair from the AXA Research Fund

Telomeres are considered anti-cancer targets, as telomere maintenance above a minimum length is necessary for cancer growth. Telomerase abrogation in cancer-prone mouse models, however, only decreased tumor growth after several mouse generations when telomeres reach a critically short length, and this effect was lost upon p53 mutation. Here, we address whether induction of telomere uncapping by inhibition of the TRF1 shelterin protein can effectively block cancer growth independently of telomere length. We show that genetic Trf1 ablation impairs the growth of p53-null K-Ras(G12V)-induced lung carcinomas and increases mouse survival independently of telomere length. This is accompanied by induction of telomeric DNA damage, apoptosis, decreased proliferation, and G2 arrest. Long-term whole-body Trf1 deletion in adult mice did not impact on mouse survival and viability, although some mice showed a moderately decreased cellularity in bone marrow and blood. Importantly, inhibition of TRF1 binding to telomeres by small molecules blocks the growth of already established lung carcinomas without affecting mouse survival or tissue function. Thus, induction of acute telomere uncapping emerges as a potential new therapeutic target for lung cancer.

On the basis of clinical trials using first-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), it became a doctrine that V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-RAS) mutations drive resistance to EGFR inhibition in non-small cell lung cancer (NSCLC). Conversely, we provide evidence that EGFR signaling is engaged in K-RAS-driven lung tumorigenesis in humans and in mice. Specifically, genetic mouse models revealed that deletion of Egfr quenches mutant K-RAS activity and transiently reduces tumor growth. However, EGFR inhibition initiates a rapid resistance mechanism involving non-EGFR ERBB family members. This tumor escape mechanism clarifies the disappointing outcome of first-generation TKIs and suggests high therapeutic potential of pan-ERBB inhibitors. On the basis of various experimental models including genetically engineered mouse models, patient-derived and cell line-derived xenografts, and in vitro experiments, we demonstrate that the U.S. Food and Drug Administration-approved pan-ERBB inhibitor afatinib effectively impairs K-RAS-driven lung tumorigenesis. Our data support reconsidering the use of pan-ERBB inhibition in clinical trials to treat K-RAS-mutated NSCLC.

HMG-box proteins, including Sox/SRY (Sox) and TCF/LEF1 (TCF) family members, bind DNA via their HMG-box. This binding, however, is relatively weak and both Sox and TCF factors employ distinct mechanisms for enhancing their affinity and specificity for DNA. Here we report that Capicua (CIC), an HMG-box transcriptional repressor involved in Ras/MAPK signaling and cancer progression, employs an additional distinct mode of DNA binding that enables selective recognition of its targets. We find that, contrary to previous assumptions, the HMG-box of CIC does not bind DNA alone but instead requires a distant motif (referred to as C1) present at the C-terminus of all CIC proteins. The HMG-box and C1 domains are both necessary for binding specific TGAATGAA-like sites, do not function via dimerization, and are active in the absence of cofactors, suggesting that they form a bipartite structure for sequence-specific binding to DNA. We demonstrate that this binding mechanism operates throughout Drosophila development and in human cells, ensuring specific regulation of multiple CIC targets. It thus appears that HMG-box proteins generally depend on auxiliary DNA binding mechanisms for regulating their appropriate genomic targets, but that each sub-family has evolved unique strategies for this purpose. Finally, the key role of C1 in DNA binding also explains the fact that this domain is a hotspot for inactivating mutations in oligodendroglioma and other tumors, while being preserved in oncogenic CIC-DUX4 fusion chimeras associated to Ewing-like sarcomas, This work was funded by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (BFU2011-23611 and BFU2014-52863-P) to GJ; the European Research Council (ERC-AG/ 250297-RAS AHEAD), the EU-Framework Programme (LSHG-CT-2007-037665/CHEMORES, HEALTH-F2-2010-259770/LUNGTARGET and HEALTH-2010-260791/EUROCANPLATFORM), the MINECO (SAF2011-30173 and SAF2014-59864-R), the Autonomous Community of Madrid (S2011/BDM-2470/ONCOCYCLE) to MB; and the Fundació La Marató de TV3 (20131730/1). LSC was supported by a fellowship from the Programa de Formación de Personal Investigador (FPI, MINECO). MB is a recipient of an Endowed Chair from the AXA Research Fund. GJ is an ICREA investigator.

Cancer evolution is a process that is still poorly understood because of the lack of versatile in vivo longitudinal studies. By generating murine non–small cell lung cancer (NSCLC) orthoallobanks and paired primary cell lines, we provide a detailed description of an in vivo, time-dependent cancer malignization process. We identify the acquisition of metastatic dissemination potential, the selection of co-driver mutations, and the appearance of naturally occurring intratumor heterogeneity, thus recapitulating the stochastic nature of human cancer development. This approach combines the robustness of genetically engineered cancer models with the flexibility of allograft methodology. We have applied this tool for the preclinical evaluation of therapeutic approaches. This system can be implemented to improve the design of future treatments for patients with NSCLC. Cancer Res; 74(21); 5978–88. ©2014 AACR.

Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. NS also is associated with a risk for developing myeloproliferative disorders (MPD), including juvenile myelomonocytic leukemia (JMML). Mutations responsible for NS occur in at least 11 different loci including KRAS. Here we describe a mouse model for NS induced by K-Ras(V14I), a recurrent KRAS mutation in NS patients. K-Ras(V14I)-mutant mice displayed multiple NS-associated developmental defects such as growth delay, craniofacial dysmorphia, cardiac defects, and hematologic abnormalities including a severe form of MPD that resembles human JMML. Homozygous animals had perinatal lethality whose penetrance varied with genetic background. Exposure of pregnant mothers to a MEK inhibitor rescued perinatal lethality and prevented craniofacial dysmorphia and cardiac defects. However, Mek inhibition was not sufficient to correct these defects when mice were treated after weaning. Interestingly, Mek inhibition did not correct the neoplastic MPD characteristic of these mutant mice, regardless of the timing at which the mice were treated, thus suggesting that MPD is driven by additional signaling pathways. These genetically engineered K-Ras(V14I)-mutant mice offer an experimental tool for studying the molecular mechanisms underlying the clinical manifestations of NS. Perhaps more importantly, they should be useful as a preclinical model to test new therapies aimed at preventing or ameliorating those deficits associated with this syndrome.

In this implementation phase of the European Cancer Patient's Bill of Rights (BoR), we confirm the following three patient-centred principles that underpin this initiative: The right of every European citizen to receive the most accurate information and to be proactively involved in his/her care. The right of every European citizen to optimal and timely access to a diagnosis and to appropriate specialised care, underpinned by research and innovation. The right of every European citizen to receive care in health systems that ensure the best possible cancer prevention, the earliest possible diagnosis of their cancer, improved outcomes, patient rehabilitation, best quality of life and affordable health care. The key aspects of working towards implementing the BoR are: Agree our high-level goal. The vision of 70% long-term survival for patients with cancer in 2035, promoting cancer prevention and cancer control and the associated progress in ensuring good patient experience and quality of life. Establish the major mechanisms to underpin its delivery. (1) The systematic and rigorous sharing of best practice between and across European cancer healthcare systems and (2) the active promotion of Research and Innovation focused on improving outcomes; (3) Improving access to new and established cancer care by sharing best practice in the development, approval, procurement and reimbursement of cancer diagnostic tests and treatments. Work with other organisations to bring into being a Europe based centre that will (1) systematically identify, evaluate and validate and disseminate best practice in cancer management for the different countries and regions and (2) promote Research and Innovation and its translation to maximise its impact to improve outcomes., SCOPUS: re.j, info:eu-repo/semantics/published

Background: Nab-paclitaxel and gemcitabine have demonstrated a survival benefit over gemcitabine alone in advanced pancreatic cancer (PDA). This study aimed to investigate the clinical, biological, and imaging effects of the regimen in patients with operable PDA. Methods: Patients with operable PDA received two cycles of nab-paclitaxel and gemcitabine before surgical resection. FDG-PET and CA19.9 tumour marker levels were used to measure clinical activity. Effects on tumour stroma were determined by endoscopic ultrasound (EUS) elastography. The collagen content and architecture as well as density of cancer-associated fibroblasts (CAFs) were determined in the resected surgical specimen and compared with a group of untreated and treated with conventional chemoradiation therapy controls. A co-clinical study in a mouse model of PDA was conducted to differentiate between the effects of nab-paclitaxel and gemcitabine. Results: A total of 16 patients were enrolled. Treatment resulted in significant antitumour effects with 50% of patients achieving a >75% decrease in circulating CA19.9 tumour marker and a response by FDG-PET. There was also a significant decrement in tumour stiffness as measured by EUS elastography. Seven of 12 patients who completed treatment and were operated had major pathological regressions. Analysis of residual tumours showed a marked disorganised collagen with a very low density of CAF, which was not observed in the untreated or conventionally treated control groups. The preclinical co-clinical study showed that these effects were specific of nab-paclitaxel and not gemcitabine. Conclusion: These data suggest that nab-paclitaxel and gemcitabine decreases CAF content inducing a marked alteration in cancer stroma that results in tumour softening. This regimen should be studied in patients with operable PDA.

Proliferation in the epidermis is a tightly controlled process. During skin development, epidermis formation and hair follicle morphogenesis crucially depend on the regulated balance between proliferation and differentiation. Here we deleted all three Ras loci (H-Ras, N-Ras and K-Ras) from keratinocytes in vitro as well as specifically from the epidermis in mice using a K5Cre strain. Upon Ras elimination, keratinocytes ceased proliferation and entered into senescence without any signs of apoptosis induction. Constitutive activation of the mitogen-activated protein kinase pathway was able to partially rescue the proliferative defects. In mice, Ras signaling was essential for proper development of the epidermis and hair follicles. Deletion of the three Ras loci during epidermis formation in mouse embryos caused a dramatic decrease in proliferation, resulting in a substantially thinner epidermis and delayed appearance of differentiation markers. We could not detect apoptotic or senescent cells in these embryos suggesting that loss of Ras protein expression only leads to severe hypoproliferation. These observations provide genetic evidence for an essential role of Ras proteins in the control of keratinocyte and epidermal proliferation.

Understanding the early evolution of cancer heterogeneity during the initial steps of tumorigenesis can uncover vulnerabilities of cancer cells that may be masked at later stages. We describe a comprehensive approach employing gene expression analysis in early lesions to identify novel therapeutic targets and the use of mouse models to test synthetic lethal drug combinations to treat human Kirsten rat sarcoma viral oncogene homologue (KRAS)-driven lung adenocarcinoma.

Hypoactive BRAF mutants bind more tightly than wild type to the upstream regulator RAS, thus amplifying to amplify ERK signalling; tumours expressing these mutants require coexistent mechanisms for RAS activation to grow and are sensitive to their inhibition. Mutant alleles of the kinase BRAF have been identified in human tumours, some of which have activated catalytic functions, whereas others have impaired or absent kinase activity. Here Neal Rosen and colleagues have characterized the less-understood hypoactive BRAF mutants. They find that these mutants bind more tightly to the upstream regulator RAS in order to amplify signalling and require coexistent mechanisms for RAS activation in the tumour in order to function. The mechanistic action of these hypoactive BRAF mutants is different to that of the activated mutants and dictates their sensitivity to therapeutic inhibitors of the pathway. Elsewhere in this issue, David Santamaria and colleagues show that kinase-inactive BRAF mutants can initiate the development of lung adenocarcinoma in mice. Approximately 200 BRAF mutant alleles have been identified in human tumours. Activating BRAF mutants cause feedback inhibition of GTP-bound RAS, are RAS-independent and signal either as active monomers (class 1) or constitutively active dimers (class 2)1. Here we characterize a third class of BRAF mutants—those that have impaired kinase activity or are kinase-dead. These mutants are sensitive to ERK-mediated feedback and their activation of signalling is RAS-dependent. The mutants bind more tightly than wild-type BRAF to RAS–GTP, and their binding to and activation of wild-type CRAF is enhanced, leading to increased ERK signalling. The model suggests that dysregulation of signalling by these mutants in tumours requires coexistent mechanisms for maintaining RAS activation despite ERK-dependent feedback. Consistent with this hypothesis, melanomas with these class 3 BRAF mutations also harbour RAS mutations or NF1 deletions. By contrast, in lung and colorectal cancers with class 3 BRAF mutants, RAS is typically activated by receptor tyrosine kinase signalling. These tumours are sensitive to the inhibition of RAS activation by inhibitors of receptor tyrosine kinases. We have thus defined three distinct functional classes of BRAF mutants in human tumours. The mutants activate ERK signalling by different mechanisms that dictate their sensitivity to therapeutic inhibitors of the pathway.

SummaryClinical evidence indicates that mutation/activation of EGF receptors (EGFRs) is mutually exclusive with the presence of K-RAS oncogenes in lung and colon tumors. We have validated these observations using genetically engineered mouse models. However, development of pancreatic ductal adenocarcinomas driven by K-Ras oncogenes are totally dependent on EGFR signaling. Similar results were obtained using human pancreatic tumor cell lines. EGFRs were also essential even in the context of pancreatic injury and absence of p16Ink4a/p19Arf. Only loss of p53 made pancreatic tumors independent of EGFR signaling. Additional inhibition of PI3K and STAT3 effectively prevented proliferation of explants derived from these p53-defective pancreatic tumors. These findings may provide the bases for more rational approaches to treat pancreatic tumors in the clinic.

Oncogene-induced replicative stress activates an Atr- and Chk1-dependent response, which has been proposed to be widespread in tumors. We explored whether the presence of replicative stress could be exploited for the selective elimination of cancer cells. To this end, we evaluated the impact of targeting the replicative stress-response on cancer development. In mice (Mus musculus), the reduced levels of Atr found on a mouse model of the Atr-Seckel syndrome completely prevented the development of Myc-induced lymphomas or pancreatic tumors, both of which showed abundant levels of replicative stress. Moreover, Chk1 inhibitors were highly effective in killing Myc-driven lymphomas. By contrast, pancreatic adenocarcinomas initiated by K-Ras(G12V) showed no detectable evidence of replicative stress and were nonresponsive to this therapy. Besides its impact on cancer, Myc overexpression aggravated the phenotypes of Atr-Seckel mice, revealing that oncogenes can modulate the severity of replicative stress-associated diseases.

SummaryPancreatic acinar cells of adult mice (≥P60) are resistant to transformation by some of the most robust oncogenic insults including expression of K-Ras oncogenes and loss of p16Ink4a/p19Arf or Trp53 tumor suppressors. Yet, these acinar cells yield pancreatic intraepithelial neoplasias (mPanIN) and ductal adenocarcinomas (mPDAC) if exposed to limited bouts of non-acute pancreatitis, providing they harbor K-Ras oncogenes. Pancreatitis contributes to tumor progression by abrogating the senescence barrier characteristic of low-grade mPanINs. Attenuation of pancreatitis-induced inflammation also accelerates tissue repair and thwarts mPanIN expansion. Patients with chronic pancreatitis display senescent PanINs, providing they have received antiinflammatory drugs. These results support the concept that antiinflammatory treatment of people diagnosed with pancreatitis may reduce their risk of developing PDAC.

Keratinocytes contribute to melanocyte transformation by affecting their microenvironment, in part, through the secretion of paracrine factors. Here we report a loss of expression of nuclear receptor RXRα in epidermal keratinocytes during human melanoma progression. We show that absence of keratinocytic RXRα in combination with mutant Cdk4 generated cutaneous melanoma that metastasized to lymph nodes in a bigenic mouse model. Expression of several keratinocyte-derived mitogenic growth factors (Et-1, Hgf, Scf, α-MSH and Fgf2) were elevated in skin of bigenic mice, while Fas, E-cadherin and Pten, implicated in apoptosis, cellular invasion and melanomagenesis, respectively, were downregulated within the microdissected melanocytic tumors. We demonstrated that RXRα is recruited on the proximal promoter of both Et-1 and Hgf, possibly directly regulating their transcription in keratinocytes. These studies demonstrate the contribution of keratinocytic paracrine signaling during the cellular transformation and malignant conversion of melanocytes. Significance Malignant melanoma continues to evade modern curative efforts as a result of the complex and elusive nature of metastatic tumors. We demonstrated that RXRα expression is lost in epidermal keratinocytes during melanoma progression in humans. We have presented evidence for the association of keratinocytic nuclear receptor RXRα-mediated paracrine/juxtacrine signaling and the malignant transformation of melanocytic tumors. The present work highlights cooperative effects of aberrant keratinocytic signaling and activated Cdk4 in melanoma metastasis. This mouse model will allow greater understanding of the mechanisms responsible for transition towards a malignant phenotype. Manipulation of these receptor-mediated signaling pathways could hold therapeutic value when combating metastatic disease.

Activated oncogenes induce compensatory tumour-suppressive responses, such as cellular senescence or apoptosis, but the signals determining the main outcome remain to be fully understood. Here, we uncover a role for Cdk2 (cyclin-dependent kinase 2) in suppressing Myc-induced senescence. Short-term activation of Myc promoted cell-cycle progression in either wild-type or Cdk2 knockout mouse embryo fibroblasts (MEFs). In the knockout MEFs, however, the initial hyper-proliferative response was followed by cellular senescence. Loss of Cdk2 also caused sensitization to Myc-induced senescence in pancreatic beta-cells or splenic B-cells in vivo, correlating with delayed lymphoma onset in the latter. Cdk2-/- MEFs also senesced upon ectopic Wnt signalling or, without an oncogene, upon oxygen-induced culture shock. Myc also causes senescence in cells lacking the DNA repair protein Wrn. However, unlike loss of Wrn, loss of Cdk2 did not enhance Myc-induced replication stress, implying that these proteins suppress senescence through different routes. In MEFs, Myc-induced senescence was genetically dependent on the ARF-p53-p21Cip1 and p16INK4a-pRb pathways, p21Cip1 and p16INK4a being selectively induced in Cdk2-/- cells. Thus, although redundant for cell-cycle progression and development, Cdk2 has a unique role in suppressing oncogene- and/or stress-induced senescence. Pharmacological inhibition of Cdk2 induced Myc-dependent senescence in various cell types, including a p53-null human cancer cell line. Our data warrant re-assessment of Cdk2 as a therapeutic target in Myc- or Wnt-driven tumours.

Mutational activation of RAS proteins occurs in nearly 30% of all human tumors. To date direct pharmacological inhibition of RAS oncoproteins has not been possible. As a consequence, current strategies are focusing on the development of inhibitors that target those kinases acting downstream of RAS proteins, including those of the RAF/MEK/ERK and PI3K/AKT pathways. Most of these inhibitors have undesired off-target effects that mask the potential therapeutic effect of blocking their targeted kinases. To facilitate the screening of selective inhibitors, we have generated lines of mouse embryonic fibroblasts that lack endogenous Ras proteins. These cells proliferate due to ectopic expression of either Ras oncoproteins that selectively activate the Raf/Mek/Erk pathway such as H-Ras(G12V/D38E) or constitutively active kinases such as B-Raf and Mek1. These cell lines were exposed to inhibitors against the RAF, MEK, and AKT kinases as well as inhibitors of other kinases known to crosstalk with RAS signaling such as JNK and p38. Amongst all compounds tested, only the MEK inhibitors U0126 and PD0325901, showed the expected specificity pattern. Yet, PD0325901, but not U0126, was able to inhibit a cell line lacking Ras proteins that owed its proliferative properties to loss of p53. Thus, suggesting unexpected off-target activities for this compound. The use of cell lines whose proliferative properties exclusively depend on selective targets provide a novel strategy to analyze the specificity of selective inhibitors designed against molecular targets implicated in human cancer.

Lung cancer is the leading cause of cancer-related deaths worldwide, and even today, the 5-year survival rate is still below 15%. Lung adenocarcinoma is the most frequent subtype, and approximately 25% of the cases harbor activating mutations in the KRAS gene. To date, there is no effective treatment for patients carrying KRAS mutations due, at least in part, to the challenge posed by direct targeting of the KRAS oncoprotein. During the last decade, scientists have developed genetically engineered mouse models that faithfully recapitulate the natural history of the human tumors. These models have been used as a preclinical platform to validate a number of relevant downstream effectors of KRAS signaling. Targets displaying synthetic lethality with the KRAS oncoprotein have also been validated in these models. Here, we review these studies and discuss their potential value in the clinical setting. We also provide an outlook as of how to improve the significance of target validation studies in preclinical platforms.

Oncogenic mutations in the K- ras gene occur in ≈50% of human colorectal cancers. However, the precise role that K- ras oncogenes play in tumor formation is still unclear. To address this issue, we have conditionally expressed an oncogenic K- ras V12 allele in the small intestine of adult mice either alone or in the context of Apc deficiency. We found that expression of K- ras V12 does not affect normal intestinal homeostasis or the immediate phenotypes associated with Apc deficiency. Mechanistically we failed to find activation of the Raf/MEK/ERK pathway, which may be a consequence of the up-regulation of a number of negative feedback loops. However, K- ras V12 expression accelerates intestinal tumorigenesis and confers invasive properties after Apc loss over the long term. In renal epithelium, expression of the oncogenic K- ras V12 allele in the absence of Apc induces the rapid development of renal carcinoma. These tumors, unlike those of intestinal origin, display activation of the Raf/MEK/ERK and Akt signaling pathways. Taken together, these data indicate that normal intestinal and kidney epithelium are resistant to malignant transformation by an endogenous K- ras oncogene. However, activation of K- ras V12 after Apc loss results in increased tumorigenesis with distinct kinetics. Whereas the effect of K- ras oncogenes in the intestine can been observed only after long latencies, they result in rapid carcinogenesis in the kidney epithelium. These data imply a window of opportunity for anti-K-ras therapies after tumor initiation in preventing tumor growth and invasion.

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)

We evaluated the efficacy of a candidate melanoma vaccine approach in mice genetically prone to develop melanoma due to the introduction of an oncogenic mutation (R24C) in the germline sequence of the cyclin-dependent kinase 4 (cdk4), a protein critically involved in cell cycle regulation. Melanomas were induced in cdk4-mutant mice by chemical carcinogenesis and UVB irradiation. A genetic prime-boost strategy targeting the clinically relevant differentiation antigen tyrosinase-related protein 2 (TRP2) was performed which was able to stimulate a melanocyte-specific cellular immune response associated with localized autoimmune vitiligo-like depigmentation. However, significant destruction of carcinogen-induced autochthonous melanocytic neoplasms in the skin was not observed following immunization. We provide evidence that autochthonous melanomas expressed TRP2 but not the MHC molecule H2-Kb and are immunologically tolerated in the skin. Our results highlight the importance of assessing melanoma vaccines in genetic mouse models that more adequately represent the expected clinical situation in order to identify strategies, which eventually may be of benefit for melanoma patients. © 2005 Wiley-Liss, Inc.