Luca Mologni

Recurrent SETBP1 Mutations in Atypical Chronic Myeloid Leukemia

Atypical chronic myeloid leukemia (aCML) shares clinical and laboratory features with CML, but it lacks the BCR-ABL1 fusion. We performed exome sequencing of eight aCMLs and identified somatic alterations of SETBP1 (encoding a p.Gly870Ser alteration) in two cases. Targeted resequencing of 70 aCMLs, 574 diverse hematological malignancies and 344 cancer cell lines identified SETBP1 mutations in 24 cases, including 17 of 70 aCMLs (24.3%; 95% confidence interval (CI) = 16–35%). Most mutations (92%) were located between codons 858 and 871 and were identical to changes seen in individuals with Schinzel-Giedion syndrome. Individuals with mutations had higher white blood cell counts (P = 0.008) and worse prognosis (P = 0.01). The p.Gly870Ser alteration abrogated a site for ubiquitination, and cells exogenously expressing this mutant exhibited higher amounts of SETBP1 and SET protein, lower PP2A activity and higher proliferation rates relative to those expressing the wild-type protein. In summary, mutated SETBP1 represents a newly discovered oncogene present in aCML and closely related diseases.

Bcr-Abl stabilizes β-catenin in chronic myeloid leukemia through its tyrosine phosphorylation

Self‐renewal of Bcr‐Abl+ chronic myeloid leukemia (CML) cells is sustained by a nuclear activated serine/threonine‐(S/T) unphosphorylated β‐catenin. Although β‐catenin can be tyrosine (Y)‐phosphorylated, the occurrence and biological relevance of this covalent modification in Bcr‐Abl‐associated leukemogenesis is unknown. Here we show that Bcr‐Abl levels control the degree of β‐catenin protein stabilization by affecting its Y/S/T‐phospho content in CML cells. Bcr‐Abl physically interacts with β‐catenin, and its oncogenic tyrosine kinase activity is required to phosphorylate β‐catenin at Y86 and Y654 residues. This Y‐phospho β‐catenin binds to the TCF4 transcription factor, thus representing a transcriptionally active pool. Imatinib, a Bcr‐Abl antagonist, impairs the β‐catenin/TCF‐related transcription causing a rapid cytosolic retention of Y‐unphosphorylated β‐catenin, which presents an increased binding affinity for the Axin/GSK3β complex. Although Bcr‐Abl does not affect GSK3β autophosphorylation, it prevents, through its effect on β‐catenin Y phosphorylation, Axin/GSK3β binding to β‐catenin and its subsequent S/T phosphorylation. Silencing of β‐catenin by small interfering RNA inhibited proliferation and clonogenicity of Bcr‐Abl+ CML cells, in synergism with Imatinib. These findings indicate the Bcr‐Abl triggered Y phosphorylation of β‐catenin as a new mechanism responsible for its protein stabilization and nuclear signalling activation in CML.

BRAF silencing by short hairpin RNA or chemical blockade by PLX4032 leads to different responses in melanoma and thyroid carcinoma cells

BRAF-activating mutations have been reported in several types of cancer, including melanoma (∼70% of cases), thyroid (30-70%), ovarian (15-30%), and colorectal cancer (5-20%). Mutant BRAF has constitutive kinase activity and causes hyperactivation of the mitogen-activated protein kinase pathway. BRAF silencing induces regression of melanoma xenografts, indicating the essential role of BRAF for cell survival. We set up an inducible short hairpin RNA system to compare the role of oncogenic BRAF in thyroid carcinoma versus melanoma cells. Although BRAF knockdown led to apoptosis in the melanoma cell line A375, the anaplastic thyroid carcinoma cell ARO underwent growth arrest upon silencing, with little or no cell death. Reexpression of the thyroid differentiation marker, sodium iodide symporter, was induced after long-term silencing. The different outcome of BRAF down-regulation in the two cell lines was associated with an opposite regulation of p21CIP1/WAF1 expression levels in response to the block of the BRAF mitogenic signal. These results were confirmed using a specific BRAF small-molecule inhibitor, PLX4032. Restoration of p21CIP1/WAF1 expression rescued melanoma cells from death. Altogether, our data indicate that oncogenic BRAF inhibition can have a different effect on cell fate depending on the cellular type. Furthermore, we suggest that a BRAF-independent mechanism of cell survival exists in anaplastic thyroid cancer cells. (Mol Cancer Res 2008;6(5):751–9)

Crizotinib in Advanced, Chemoresistant Anaplastic Lymphoma Kinase–Positive Lymphoma Patients

Anaplastic lymphoma kinase (ALK)-positive lymphomas respond to chemotherapy, but relapses, which bear a poor prognosis, occur. Crizotinib inhibits ALK in vitro and in vivo and was administered as monotherapy to 11 ALK+ lymphoma patients who were resistant/refractory to cytotoxic therapy. The overall response rate was 10 of 11 (90.9%; 95% confidence interval [CI] = 58.7% to 99.8%). Disease status at the latest follow-up is as follows: four patients are in complete response (CR) (months >21, >30, >35, >40) under continuous crizotinib administration; 4 patients had progression of disease (months 1, 2, 2, 2); 1 patient obtained CR on crizotinib, received an allogeneic bone marrow transplant, and is in CR; 2 patients (treated before and/or after allogeneic bone marrow transplant) obtained and are still in CR but they have stopped crizotinib. Overall and progression-free survival rates at 2 years are 72.7% (95% CI = 39.1% to 94.0%) and 63.7% (95% CI = 30.8% to 89.1%), respectively. ALK mutations conferring resistance to crizotinib in vitro could be identified in relapsed patients. Crizotinib exerted a potent antitumor activity with durable responses in advanced, heavily pretreated ALK+ lymphoma patients, with a benign safety profile.

Sorafenib functions to potently suppress RET tyrosine kinase activity by direct enzymatic inhibition and promoting RET lysosomal degradation independent of proteasomal targeting

Germ line missense mutations in the RET (rearranged during transfection) oncogene are the cause of multiple endocrine neoplasia, type 2 (MEN2), but at present surgery is the only treatment available for MEN2 patients. In this study, the ability of Sorafenib (BAY 43-9006) to act as a RET inhibitor was investigated. Sorafenib inhibited the activity of purified recombinant kinase domain of wild type RET and RETV804M with IC50 values of 5.9 and 7.9 nm, respectively. Interestingly, these values were 6–7-fold lower than the IC50 for the inhibition of B-RAFV600E. In cell-based assays, Sorafenib inhibited the kinase activity and signaling of wild type and oncogenic RET in MEN2 tumor and established cell lines at a concentration between 15 and 150 nm. In contrast, inhibition of oncogenic B-RAF- or epidermal growth factor-induced ERK1/2 phosphorylation required micromolar concentrations of Sorafenib demonstrating the high specificity of this drug in targeting RET. Moreover, prolonged exposure to Sorafenib resulted in inhibition of cell proliferation and RET protein degradation. Using lysosomal and proteasomal inhibitors, we demonstrate that Sorafenib induces RET lysosomal degradation independent of proteasomal targeting. Furthermore, we provide a structural model of the Sorafenib·RET complex in which Sorafenib binds to and induces the DFGout conformation of the RET kinase domain. These results strengthen the argument that Sorafenib may be effective in the treatment of MEN2 patients. In addition, because inhibition of RET is not impaired by mutation of the Val804 gatekeeper residue, MEN2 tumors may be less susceptible to acquired Sorafenib resistance.

Synthesis, Structure-Activity Relationship and Crystallographic Studies of 3-Substituted Indolin-2-One Ret Inhibitors.

The synthesis, structure-activity relationships (SAR) and structural data of a series of indolin-2-one inhibitors of RET tyrosine kinase are described. These compounds were designed to explore the available space around the indolinone scaffold within RET active site. Several substitutions at different positions were tested and biochemical data were used to draw a molecular model of steric and electrostatic interactions, which can be applied to design more potent and selective RET inhibitors. The crystal structures of RET kinase domain in complex with three inhibitors were solved. All three compounds bound in the ATP pocket and formed two hydrogen bonds with the kinase hinge region. Crystallographic analysis confirmed predictions from molecular modelling and helped refine SAR results. These data provide important information for the development of indolinone inhibitors for the treatment of RET-driven cancers.

Ponatinib is a potent inhibitor of wild-type and drug-resistant gatekeeper mutant RET kinase

RET kinase is aberrantly activated in thyroid cancers and in rare cases of lung and colon cancer, and has been validated as a molecular target in these tumors. Vandetanib was recently approved for the treatment of medullary thyroid cancer. However, vandetanib is ineffective in vitro against RET mutants carrying bulky aminoacids at position 804, the gatekeeper residue, similarly to drug-resistant BCR-ABL mutants in chronic myeloid leukemia. Ponatinib is a multi-target kinase inhibitor that was recently approved for treatment-refractory Philadelphia-positive leukemia. We show here potent inhibition of oncogenic RET by ponatinib, including the drug-insensitive V804M/L mutants. Ponatinib inhibited the growth of RET+ and BCR-ABL+ cells with similar potency, while not affecting RET-negative cells. Both in biochemical and in cellular assays ponatinib compared favorably with known RET inhibitors, such as vandetanib, cabozantinib, sorafenib, sunitinib and motesanib, used as reference compounds. We suggest that ponatinib should be considered for the treatment of RET+ tumors, in particular those expressing vandetanib-resistant V804M/L mutations.

Entrectinib, a Pan-TRK, ROS1 and ALK Inhibitor with Activity in Multiple Molecularly Defined Cancer Indications

Activated ALK and ROS1 tyrosine kinases, resulting from chromosomal rearrangements, occur in a subset of non–small cell lung cancers (NSCLC) as well as other tumor types and their oncogenic relevance as actionable targets has been demonstrated by the efficacy of selective kinase inhibitors such as crizotinib, ceritinib, and alectinib. More recently, low-frequency rearrangements of TRK kinases have been described in NSCLC, colorectal carcinoma, glioblastoma, and Spitzoid melanoma. Entrectinib, whose discovery and preclinical characterization are reported herein, is a novel, potent inhibitor of ALK, ROS1, and, importantly, of TRK family kinases, which shows promise for therapy of tumors bearing oncogenic forms of these proteins. Proliferation profiling against over 200 human tumor cell lines revealed that entrectinib is exquisitely potent in vitro against lines that are dependent on the drugs pharmacologic targets. Oral administration of entrectinib to tumor-bearing mice induced regression in relevant human xenograft tumors, including the TRKA-dependent colorectal carcinoma KM12, ROS1-driven tumors, and several ALK-dependent models of different tissue origins, including a model of brain-localized lung cancer metastasis. Entrectinib is currently showing great promise in phase I/II clinical trials, including the first documented objective responses to a TRK inhibitor in colorectal carcinoma and in NSCLC. The drug is, thus, potentially suited to the therapy of several molecularly defined cancer settings, especially that of TRK-dependent tumors, for which no approved drugs are currently available. Mol Cancer Ther; 15(4); 628–39. ©2016 AACR.

Targeted Deletion of the Muscular Dystrophy Gene myotilin Does Not Perturb Muscle Structure or Function in Mice

ABSTRACT Myotilin, palladin, and myopalladin form a novel small subfamily of cytoskeletal proteins that contain immunoglobulin-like domains. Myotilin is a thin filament-associated protein localized at the Z-disk of skeletal and cardiac muscle cells. The direct binding to F-actin, efficient cross-linking of actin filaments, and prevention of induced disassembly of filaments are key roles of myotilin that are thought to be involved in structural maintenance and function of the sarcomere. Missense mutations in the myotilin-encoding gene cause dominant limb girdle muscular dystrophy type 1A and spheroid body myopathy and are the molecular defect that can cause myofibrillar myopathy. Here we describe the generation and analysis of mice that lack myotilin, myo−/− mice. Surprisingly, myo−/− mice maintain normal muscle sarcomeric and sarcolemmal integrity. Also, loss of myotilin does not cause alterations in the heart or other organs of newborn or adult myo−/− mice. The mice develop normally and have a normal life span, and their muscle capacity does not significantly differ from wild-type mice even after prolonged physical stress. The results suggest that either myotilin does not participate in muscle development and basal function maintenance or other proteins serve as structural and functional compensatory molecules when myotilin is absent.

Inhibitors of the anaplastic lymphoma kinase

Introduction: Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase normally expressed in the developing nervous tissue. Genetic alterations of ALK are associated with a number of cancers, including anaplastic large cell lymphoma (ALCL) and a subset of non-small cell lung cancer (NSCLC). Standard therapies for these diseases include surgery plus unspecific cytotoxic agents, with a low therapeutic window and significant treatment-associated systemic toxicity. A few small-molecule inhibitors of ALK kinase activity have been described in the recent years, some of which are currently undergoing clinical evaluation. Areas covered: Literature was searched for all ALK inhibitors that have entered clinical investigation, including published research articles and meeting abstracts. Data on pharmacokinetics, safety and efficacy of crizotinib, as well as preliminary clinical data for second-generation compounds, are reviewed. The issue of drug resistance is discussed. Expert opinion: Understanding the specific genetic aberration that causes cancer development and progression allows major advances in cancer therapy. Along the same way shown by imatinib in chronic myeloid leukemia, compounds that selectively target ALK are bringing a revolution in the treatment of ALK-positive tumors. Crizotinib has just been approved, and new more potent ALK inhibitors will shortly follow. These molecules represent another excellent proof-of-principle for targeted therapy.