Loss of FGFR4 promotes the malignant phenotype of PDAC

Transcriptomic analyses of pancreatic ductal adenocarcinoma (PDAC) have identified two major epithelial subtypes with distinct biology and clinical behaviours. Here, we aimed to clarify the role of FGFR1 and FGFR4 in the definition of aggressive PDAC phenotypes. We found that the expression of FGFR4 is exclusively detected in epithelial cells, significantly elevated in the classical PDAC subtype, and associates with better outcomes. In highly aggressive basal-like/squamous PDAC, reduced FGFR4 expression aligns with hypermethylation of the gene and lower levels of histone marks associated with active transcription in its regulatory regions. Conversely, FGFR1 has more promiscuous expression in both normal and malignant pancreatic tissues and is strongly associated with the EMT phenotype but not with the basal-like cell lineage. Regardless of the genetic background, the increased proliferation of FGFR4-depleted PDAC cells correlates with hyperactivation of the mTORC1 pathway both in vitro and in vivo. Downregulation of FGFR4 in classical cell lines invariably leads to the enrichment of basal-like/squamous gene programs and is associated with either partial or full switch of phenotype. In sum, we show that endogenous levels of FGFR4 limit the malignant phenotype of PDAC cells. Finally, we propose FGFR4 as a valuable marker for the stratification of PDAC patients

Vertical combination strategies endowed with anti-tumor synergism to target MAPK activation in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) ranks as the 4th/5th leading cause of cancer death worldwide. KRAS is mutated (mut) in 90% of human PDAC and results in the constitutive activation of multiple signaling cascades (MAPK, PI3K, NFκB, etc). Hitting a single point along the RAF/MEK/ERK cascade disrupts intra-pathway negative feedback loops, thereby causing paradoxical pathway activation and functional resistance. Thus, combining agents simultaneously inhibiting RAF and MEK represents a potential strategy to synergistically inhibit tumor growth and delay resistance. Molecular and functional effects of single and combined MEK (trametinib, T), BRAF (dabrafenib, D), and RAF (using the pan-RAF inhibitor RAF265, R) inhibition were dissected by WB and conservative isobologram analysis to assess functional synergism. In HPAFII cells (but not in the BRAF-mut M14 melanoma) D and R induced the formation of BRAF/CRAF complexes, as assessed by immunoprecipitation. In a panel of PDAC cell lines selective BRAF inhibition with D induced hyperphosphorylation of MEK, ERK, and p90RSK (paradox effect) and the combination of D+T suppressed cell growth with highly synergistic effects in 6/9 cell lines tested (CI 0.05-0.8); conversely, R did not induce paradox MAPK activation and did not result in growth inhibitory synergism when combined with T. Specific proteins and their phosphorylation states were also analyzed using Kinexus Antibody Microarray and preliminary results show that the different treatments cause upregulation of EGFR family, wnt/ß-catenin and c-KIT signaling, suggesting potential new strategies for both “horizontal” and “vertical” combinations of agents to achieve synergistic PDAC killing. Overall, our data indicate that, in appropriate cellular contexts,vertical RAF/MEK inhibition-based combination strategies exert highly synergistic antitumor effects

Lack of growth inhibitory synergism with combined MAPK/PI3K inhibition in preclinical models of pancreatic cancer

Despite substantial advances in chemotherapy and biology understanding, pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest solid tumors. Attempts at exploiting PDAC biology for therapeutic purposes have failed and the likelihood of approval for new agents that enter phase I testing in this disease is down to a dismal 2.3%[1]. Recently, a combination of MEK (selumetinib) and AKT (MK-2206) inhibitors failed to demonstrate clinical benefit in unselected PDAC patients[2], adding to a long list of targeted agents that have failed clinical testing (EGFR/VEGFR, SMO, and Notch inhibitors, to name a few). We thus asked ourselves whether such failure could have been predicted preclinically. We explored pharmacologic interactions between MEK inhibitors (trametinib) and PI3K pathway inhibitors [gedatolisib (PI3K/mTOR inhibitor) andMK-2206 (AKT inhibitor)] in vitro, using 6 human PDAC cell lines and the “normal pancreatic epithelium” cell line HPDE. Single-agent inhibition of MEK, PI3K/mTOR, or AKT inhibited cell growth to a variable extent in all cell lines examined. However, combined inhibition of MEK and PI3K/mTOR (trametinib/gedatolisib) afforded frankly antagonistic effects in Panc1, MiaPaCa2, T3M4, PaCa44, and HPDE, slightly additive effects in HPAFII, and synergistic effects only in L3.6pl cells (Fig. 1A); similarly, combined MEK/AKT inhibition (trametinib/MK-2206) was antagonistic in all cell lines tested (Fig. 1A-C). Overall, no growth inhibitory synergism in vitro was observed in any of the cell lines tested, with the exception of L3.6pl cells in response to trametinib/gedatolisib combination. Our group has recently shown that combined inhibition of the MAPK and PI3K pathways affords synergistic anti-tumor effects almost exclusively in cancer cells without a functional PTEN gene/protein (PTEN-loss)[3]. We thus examined PTEN expression in the panel of PDAC cell lines examined: no PTEN mutations or bi-allelic loss have been reported for these cells and all displayed detectable levels of PTEN protein (Fig 1A, D), thus falling in the PTEN-competent category according to the definition recently proposed by our group[3]. Consistent results (lack of growth inhibitory synergism) had, indeed, already been obtained in the PTEN-competent cell lines HPAFII and MiaPaCa2, using another combination of MEK and mTOR inhibitors (trametinib and everolimus)[3]. Inactivating PTEN point mutations or LOH rarely occur in human PDAC[4]; thus, based on the preclinical data presented here, the failure of selumetinib/MK-2206 to achieve clinical benefit in unselected PDAC patients would have been largely anticipated. Extensive preclinical modeling and early selection biomarker development are, in our opinion, crucial to successful drug development, in addition to uniform trial eligibility criteria, stringent statistical methods, and detection of robust activity signals in early phase trials. Unfortunately, such rules have been often overlooked in advanced PDAC, resulting in the identification of only 3 clinically meaningful agents or combinations out of 32 phase III trials enrolling >13,000 patients[5]. This line of reasoning especially applies to combined pathway inhibition, which implies increased monetary and toxicity costs, representing a high risk for all stakeholders, should it fail to demonstrate more than additive benefits, and to advanced PDAC, a disease setting in which novel effective therapeutic approaches are urgently needed

Signaling intermediates (MAPK and PI3K) as therapeutic targets in NSCLC

: The RAS/RAF/MEK/ ERK and the PI3K/AKT/mTOR pathways govern fundamental physiological processes, such as cell proliferation, differentiation, metabolism, cytoskeleton reorganization and cell death and survival. Constitutive activation of these signal transduction pathways is a required hallmark of cancer and dysregulation, on either genetic or epigenetic grounds, of these pathways has been implicated in the initiation, progression and metastastic spread of lung cances. Targeting components of the MAPK and PI3K cascades is thus an attractive strategy in the development of novel therapeutic approaches to treat lung cancer, although the use of single pathway inhibitors has met with limited clinical success so far. Indeed, the presence of intra- and inter-pathway compensatory loops that re-activate the very same cascade, either upstream or downstream the point of pharmacological blockade, or activate the alternate pathway following the blockade of one signaling cascade has been demonstrated, potentially driving preclinical (and possibly clinical) resistance. Therefore, the blockade of both pathways with combinations of signaling inhibitors might result in a more efficient anti-tumor effect, and thus potentially overcome and/or delay clinical resistance, as compared with single agent. The current review aims at summarizing the current status of preclinical and clinical research with regard to pathway crosstalks between the MAPK and PI3K cascades in NSCLC and the rationale for combined therapeutic pathway targeting

Therapeutic potential of combined BRAF/MEK blockade in BRAF-wild type preclinical tumor models

Background: Mounting evidence suggests that RAF-mediated MEK activation plays a crucial role in paradox MAPK (re)activation, leading to resistance and therapeutic failure with agents hitting a single step along the MAPK cascade. Methods: We examined the molecular and functional effects of single and combined BRAF (dabrafenib), pan-RAF (RAF265), MEK (trametinib) and EGFR/HER2 (lapatinib) inhibition, using Western Blot and conservative isobologram analysis to assess functional synergism, and explored genetic determinants of synergistic interactions. Immunoprecipitation based assays were used to detect the interaction between BRAF and CRAF. The Mann-Whitney U test was used for comparing quantitative variables. Results: Here we demonstrated that a combination of MEK and BRAF inhibitors overcomes paradoxical MAPK activation (induced by BRAF inhibitors) in BRAF-wt/RAS-mut NSCLC and PDAC in vitro. This results in growth inhibitory synergism, both in vitro and in vivo, in the majority (65%) of the cellular models analyzed, encompassing cell lines and patient-derived cancer stem cells and organoids. However, RAS mutational status is not the sole determinant of functional synergism between RAF and MEK inhibitors, as demonstrated in KRAS isogenic tumor cell line models. Moreover, in EGFR-driven contexts, paradoxical MAPK (re)activation in response to selective BRAF inhibition was dependent on EGFR family signaling and could be offset by simultaneous EGFR/HER-2 blockade. Conclusions: Overall, our data indicate that RAF inhibition-induced paradoxical MAPK activation could be exploited for therapeutic purposes by simultaneously targeting both RAF and MEK (and potentially EGFR family members) in appropriate molecular contexts. KRAS mutation per se does not effectively predict therapeutic synergism and other biomarkers need to be developed to identify patients potentially deriving benefit from combined BRAF/MEK targeting

Advances towards the design and development of personalized non-small-cell lung cancer drug therapy

ntroduction: Non-small-cell lung cancer (NSCLC) subtypes are driven by spe- cific genetic aberrations. For reasons such as this, there is a call for treatment personalization. The ability to instigate NSCLC fragmentation poses new methodological problems, and new \u2018driver\u2019 molecular aberrations are being discovered at an unprecedented pace. Areas covered: This article describes the clinical development of epidermal growth factor-tyrosine kinase inhibitors (EGFR-TKIs) and crizotinib for EGFR-mutant and anaplastic lymphoma kinase (ALK)-rearranged NSCLC. Further, the authors briefly describe the emerging molecular targets in NSCLC, in terms of both rationale for therapeutic targeting and strategies, for clinical development. Expert opinion: Target identification and validation in NSCLC still requires considerable effort, as not all of the molecular alterations are clear \u2018drivers\u2019 nor can they be efficiently targeted with available drugs. However, 50% of the NSCLC cases are without clear-defined molecular aberrations. Clinical trial methodology will need to develop novel paradigms for targeted drug devel- opment, aiming at the validation of an ideal \u2018biology-to-trial\u2019 approach. Despite significant challenges, a truly \u2018personalized\u2019 approach to NSCLC ther- apy appears to be within our reach

Advances towards the design and development of personalized non-small-cell lung cancer drug therapy

Introduction: Non-small-cell lung cancer (NSCLC) subtypes are driven by specific genetic aberrations. For reasons such as this, there is a call for treatment personalization. The ability to instigate NSCLC fragmentation poses new methodological problems, and new 'driver' molecular aberrations are being discovered at an unprecedented pace. Areas covered: This article describes the clinical development of epidermal growth factor-tyrosine kinase inhibitors (EGFR-TKIs) and crizotinib for EGFR-mutant and anaplastic lymphoma kinase (ALK)-rearranged NSCLC. Further, the authors briefly describe the emerging molecular targets in NSCLC, in terms of both rationale for therapeutic targeting and strategies, for clinical development. Expert opinion: Target identification and validation in NSCLC still requires considerable effort, as not all of the molecular alterations are clear 'drivers' nor can they be efficiently targeted with available drugs. However, 50% of the NSCLC cases are without clear-defined molecular aberrations. Clinical trial methodology will need to develop novel paradigms for targeted drug development, aiming at the validation of an ideal 'biology-to-trial' approach. Despite significant challenges, a truly 'personalized' approach to NSCLC therapy appears to be within our reach. \uc2\ua9 2013 Informa UK, Ltd