Anais Del Curatolo

PTEN: Multiple functions in human malignant tumors

PTEN is the most important negative regulator of the PI3K signaling pathway. In addition to its canonical, PI3K inhibition-dependent functions, PTEN can also function as a tumor suppressor in a PI3K-independent manner. Indeed, the PTEN network regulates a broad spectrum of biological functions, modulating the flow of information from membrane-bound growth factor receptors to nuclear transcription factors, occurring in concert with other tumor suppressors and oncogenic signaling pathways. PTEN acts through its lipid and protein phosphatase activity and other non-enzymatic mechanisms. Studies conducted over the past 10 years have expanded our understanding of the biological role of PTEN, showing that in addition to its ability to regulate proliferation and cell survival, it also plays an intriguing role in regulating genomic stability, cell migration, stem cell self-renewal, and tumor microenvironment. Changes in PTEN protein levels, location, and enzymatic activity through various molecular mechanisms can generate a continuum of functional PTEN levels in inherited syndromes, sporadic cancers, and other diseases. PTEN activity can indeed, be modulated by mutations, epigenetic silencing, transcriptional repression, aberrant protein localization, and post-translational modifications. This review will discuss our current understanding of the biological role of PTEN, how PTEN expression and activity are regulated, and the consequences of PTEN dysregulation in human malignant tumors.

PTEN expression and function in adult cancer stem cells and prospects for therapeutic targeting.

Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a non-redundant lipid phosphatase that restrains and fine tunes the phosphatidylinositol-3-kinase (PI3K) signaling pathway. PTEN is involved in inherited syndromes, which predispose to different types of cancers and is among the most frequently inactivated tumor suppressor genes in sporadic cancers. Indeed, loss of PTEN function occurs in a wide spectrum of human cancers through a variety of mechanisms, including mutations, deletions, transcriptional silencing, or protein instability. PTEN prevents tumorigenesis through multiple mechanisms and regulates a plethora of cellular processes, including survival, proliferation, energy metabolism and cellular architecture. Moreover, recent studies have demonstrated that PTEN is able to exit, exist, and function outside the cell, allowing for inhibition of the PI3K pathway in neighboring cells in a paracrine fashion. Most recently, studies have shown that PTEN is also critical for stem cell maintenance and that PTEN loss can lead to the emergence and proliferation of cancer stem cell (CSC) clones. Depending on the cellular and tissue context of origin, PTEN deletion may result in increased self-renewal capacity or normal stem cell exhaustion and PTEN-defìcient stem and progenitor cells have been reported in prostate, lung, intestinal, and pancreatic tissues before tumor formation; moreover, reversible or irreversible PTEN loss is frequently observed in CSC from a variety of solid and hematologic malignancies, where it may contribute to the functional phenotype of CSC. In this review, we will focus on the role of PTEN expression and function and downstream pathway activation in cancer stem cell biology and regulation of the tumorigenic potential; the emerging role of PTEN in mediating the crosstalk between the PI3K and MAPK pathways will also be discussed, together with prospects for the therapeutic targeting of tumors lacking PTEN expression.

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.

PTEN status is a crucial determinant of the functional outcome of combined MEK and mTOR inhibition in cancer

Combined MAPK/PI3K pathway inhibition represents an attractive, albeit toxic, therapeutic strategy in oncology. Since PTEN lies at the intersection of these two pathways, we investigated whether PTEN status determines the functional response to combined pathway inhibition. PTEN (gene, mRNA, and protein) status was extensively characterized in a panel of cancer cell lines and combined MEK/mTOR inhibition displayed highly synergistic pharmacologic interactions almost exclusively in PTEN-loss models. Genetic manipulation of PTEN status confirmed a mechanistic role for PTEN in determining the functional outcome of combined pathway blockade. Proteomic analysis showed greater phosphoproteomic profile modification(s) in response to combined MEK/mTOR inhibition in PTEN-loss contexts and identified JAK1/STAT3 activation as a potential mediator of synergistic interactions. Overall, our results show that PTEN-loss is a crucial determinant of synergistic interactions between MAPK and PI3K pathway inhibitors, potentially exploitable for the selection of cancer patients at the highest chance of benefit from combined therapeutic strategies.

Emerging Insight into MAPK Inhibitors and Immunotherapy in Colorectal Cancer

Our understanding of the genetic and non-genetic molecular alterations associated with colorectal cancer (CRC) progression and therapy resistance has markedly expanded in the recent years. In addition to their effects on tumor biology, targeted therapies can have effects on host immune responses. However, the mechanisms by which immune cells organize tumor microenvironments to regulate T-cell activity need to be comprehensively defined. There is good evidence in the literature that alterations in different members of the MAPK superfamily (mainly ERKs and p38 MAPKs) modify the inflammatory response and antitumor immunity, enhancing metastatic features of the tumors. In addition, a plethora of alterations that emerge at relapse often converge on the activation of MAPKs, particularly, ERKs, which act in concert with other oncogenic signals to modulate cellular homeostasis and clonal evolution during targeted therapies. Herein, we discuss how this knowledge can be translated into drug development strategies aimed at increasing tumor antigenicity and antitumor immune responses. Insights from these studies could provide a framework for considering additional combinations of targeted therapies and immunotherapies for the treatment of CRC.

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

BackgroundMounting 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.MethodsWe 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.ResultsHere 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.ConclusionsOverall, 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.

Abstract 803: A vertical combination strategy hitting multiple steps along the MAPK cascade: Molecular mechanisms of action and putative genetic determinants of synergism

Preclinical evidence shows that hitting a single point along the RAF/MEK/ERK cascade disrupts intra-pathway negative feedback loop, may cause paradoxical pathway activation and may lead to functional resistance. Thus, the “vertical” combination of agents simultaneously inhibiting RAF and MEK has been proposed as a strategy to synergistically inhibit tumor growth and delay resistance. Experimental procedures: Molecular and functional effects of single and combined MEK (using trametinib, T), BRAF (using dabrafenib, D), and RAF (using the pan-RAF inhibitor, RAF265, R) inhibition were dissected by WB analysis and conservative isobologram analysis to assess functional synergism, using a fixed dose-ratio experimental design. Summary data: We examined the in vitro molecular and functional consequences of D and T, alone or in combination, in a panel of different human BRAFV600E melanoma cell lines; both drugs inhibited cell growth and inactivated the MAPK pathway, but little or no synergistic growth inhibition was observed with their combination (CI: 0.7 - 1.3x106). Conversely, combined D+T suppressed malignant growth with highly synergistic effects in KRAS-mutant lung (2/5 cell lines tested) and pancreatic (4/6 cell lines tested) cancer models (CI: 0.1 - 0.7), in which selective BRAF inhibition induced hyperphosphorylation of MEK, ERK, and p90RSK (paradox effect). At concentrations inhibiting both BRAF and CRAF, R did not induce paradox MAPK activation and did not result in growth inhibitory synergism when combined with T. To define the role of RAS gene status in determining sensitivity/resistance to single and combined RAF and MEK blockade, we analyzed two isogenic tumor cell line models: H1299 expressing individual codon 12 KRAS mutants and isogenic HCT116 clones differing for the presence of a homo or heterozygous G13D KRAS mutation. Conversely, in lung cancer models driven by either EGFR mutations (HCC827, H1650) or HER-2 overexpression (Calu-3), selective BRAF inhibition also induced a paradox MAPK activation, which could be blocked using a reversible EGFR/HER-2 inhibitor (Lapatinib); in this context, combination (D+T) resulted in a non-synergic growth inhibitory effects. Conclusions: Overall, our data indicate that, in appropriate cellular contexts, vertical RAF/MEK inhibition-based combination strategies exert highly synergistic antitumor effects across different cancer models. Citation Format: Anais Del Curatolo, Ursula Cesta Incani, Ludovica Ciuffreda, Italia Falcone, Senji Shirasawa, Massimo Broggini, Isabella Sperduti, Adriana Eramo, Ruggero De Maria, Francesco Cognetti, Michele Milella. A vertical combination strategy hitting multiple steps along the MAPK cascade: Molecular mechanisms of action and putative genetic determinants of synergism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 803. doi:10.1158/1538-7445.AM2014-803

Abstract 2618: PTEN loss as a putative biomarker of synergistic growth inhibitory activity of combined MEK/ERK and PI3K/mTOR pathway blockade

Background. Complex feedback loops and crosstalk between the MEK/ERK and PI3K/mTOR pathways, thus molecular predictors of sensitivity/resistance and synergistic/antagonistic interactions are urgently needed for the effective clinical development of combination strategies. Here we hypothesize that PTEN status critically modulates the growth inhibitory activity of single and combined MEK and PI3K/mTOR inhibition. Methods. Molecular and functional effects of single and combined MEK (trametinib, T) and mTOR (everolimus, E) blockade were assessed in a panel of 29 cancer cell lines with different molecular ‘drivers’. Pharmacologic interactions were analyzed by conservative isobologram analysis. PTEN role was mechanistically assessed by either silencing PTEN expression by shRNA or overexpressing a functional PTEN protein by stable transfection. Specific proteins and their phosphorylation states were analyzed using Kinexus Antibody Microarrays. Results. In the cell line panel analyzed, the presence of a wt-PTEN was the only significant predictor of sensitivity to T (p=0.001), while BRAF mutations were significantly related to E resistance (p=0.015). PTEN silencing slightly increased resistance to T in the wt-PTEN melanoma cell line M14; conversely, wt-PTEN overexpression in the PTEN-del WM115 cell line, dramatically increased sensitivity to T. E-induced growth inhibition was not significantly affected. Combined MEK and mTOR inhibition resulted in a striking growth inhibitory synergism in cells lacking PTEN expression (CI: 0.4-0.0005), but not in those with an intact PTEN (CI: 1.2-107; p=0.001). However, PTEN silencing restored growth inhibitory synergism of combined T and E in M14 cells (CI:0.36); similarly, the slope of the CI/fraction affected curve was dramatically altered in PTEN-overexpressing WM115 cells, as compared to their control-transfected counterpart, again indicating that PTEN expression/function causally influences functional response to combined MEK and mTOR inhibition. Similar results in terms of synergistic/antagonistic pharmacologic interactions were obtained when either double PI3K/mTOR kinase inhibitors (PF-5212384) or AKT allosteric inhibitors (MK-2206) were used to block the PI3K/mTOR pathway in combination with T. Proteomic analysis indicated that a greater modification of protein expression/phosphorylation profiles in response to MEK or combined MEK/mTOR inhibition occurs in cells lacking a functional PTEN, as compared to the wt-PTEN-expressing ones; preliminary analysis suggests that AKT phosphorylation and NF-κB activation maybe be crucial mediators of synergistic growth-inhibitory interactions occurring with combined treatment. Conclusions. PTEN loss may constitute a suitable genetic/molecular marker of synergistic activity interactions between MEK/ERK and PI3K/mTOR pathway inhibitors. Citation Format: Ludovica Ciuffreda, Italia Falcone, Silvia Matteoni, Andrea Sacconi, Federico Malusa, Teresa De Luca, Ursula Cesta Incani, Anais Del Curatolo, Marina Konopleva, Michael Andreeff, Adriana Eramo, Ruggero De Maria, Donatella Del Bufalo, Francesco Cognetti, Michele Milella. PTEN loss as a putative biomarker of synergistic growth inhibitory activity of combined MEK/ERK and PI3K/mTOR pathway blockade. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2618. doi:10.1158/1538-7445.AM2014-2618