Marc Billaud

Ponatinib (AP24534) Is a Novel Potent Inhibitor of Oncogenic RET Mutants Associated With Thyroid Cancer

CONTEXTnThe RET tyrosine kinase encoding gene acts as a dominantly transforming oncogene in thyroid carcinoma and other malignancies. Ponatinib (AP24534) is an oral ATP-competitive tyrosine kinase inhibitor that is in advanced clinical experimentation in leukemia.nnnOBJECTIVEnWe tested whether ponatinib inhibited RET kinase and oncogenic activity.nnnMETHODSnPonatinib activity was studied by an in vitro RET immunocomplex kinase assay and immunoblotting. The effects of ponatinib on proliferation of human TT, MZ-CRC-1, and TPC-1 thyroid carcinoma cells, which harbor endogenous oncogenic RET alleles, and of NIH3T3 fibroblasts transfected with oncogenic RET mutants were determined. Ponatinib activity on TT cell xenografted tumors in athymic mice was measured.nnnRESULTSnPonatinib inhibited immunopurified RET kinase at the IC₅₀ of 25.8 nM (95% confidence interval [CI] = 23.15-28.77 nM). It also inhibited (IC₅₀ = 33.9 nM; 95% CI = 26.41-43.58 nM) kinase activity of RET/V804M, a RET mutant displaying resistance to other tyrosine kinase inhibitor. Ponatinib blunted phosphorylation of point-mutant and rearranged RET-derived oncoproteins and inhibited proliferation of RET-transformed fibroblasts and RET mutant thyroid carcinoma cells. Finally, after 3 weeks of treatment with ponatinib (30 mg/kg/d), the volume of TT cell (medullary thyroid carcinoma) xenografts was reduced from 133 mm³ to an unmeasurable size (difference = 133 mm³, 95% CI = -83 to 349 mm³) (P < .001). Ponatinib-treated TT cell tumors displayed a reduction in the mitotic index, RET phosphorylation, and signaling.nnnCONCLUSIONSnPonatinib is a potent inhibitor of RET kinase and has promising preclinical activity in models of RET-driven medullary thyroid carcinoma.

The RNA‐binding E3 ubiquitin ligase MEX‐3C links ubiquitination with MHC‐I mRNA degradation

RNA‐binding E3 ubiquitin ligases were recently identified, though their function remains unclear. While studying the regulation of the MHC class I (MHC‐I) pathway, we here characterize a novel role for ubiquitin in mRNA degradation. MHC‐I molecules provide ligands for both cytotoxic T‐lymphocytes as well as natural killer (NK) cells, and play a central role in innate and adaptive immunity. MHC‐I cell‐surface expression is closely monitored by NK cells, whose killer immunoglobulin‐like receptors encode MHC‐I‐specific activatory and inhibitory receptors, implying that MHC‐I expression needs to be tightly regulated. In a functional siRNA ubiquitome screen we identified MEX‐3C, a novel RNA‐binding ubiquitin E3 ligase, as responsible for the post‐transcriptional, allotype‐specific regulation of MHC‐I. MEX‐3C binds the 3′UTR of HLA‐A2 mRNA, inducing its RING‐dependent degradation. The RING domain of MEX‐3C is not required for HLA‐A2 cell‐surface downregulation, but regulates the degradation of HLA‐A2 mRNA. We have therefore uncovered a novel post‐transcriptional pathway for regulation of HLA‐A allotypes and provide a link between ubiquitination and mRNA degradation.

The LKB1/AMPK polarity pathway.

The LKB1 tumor suppressor kinase is an activator of the AMP‐activated protein kinase (AMPK), a metabolic gauge that responds to variations of cellular energetic levels by favoring catabolic versus anabolic processes. Recent studies have provided substantial evidence that LKB1 and AMPK control cell polarity from invertebrates to mammals. This review examines how the LKB1–AMPK pathway, in conjunction with other positional signals, converts energy‐sensing information into the activation of Myosin II to maintain epithelial‐cell architecture but also to complete cell division. This molecular link between polarity and metabolism may constitute an ancient stress‐response protective mechanism that was co‐opted for tumor suppression during evolution.

Azaindole derivatives are inhibitors of microtubule dynamics, with anti-cancer and anti-angiogenic activities.

Drugs targeting microtubules are commonly used for cancer treatment. However, the potency of microtubule inhibitors used clinically is limited by the emergence of resistance. We thus designed a strategy to find new cell‐permeable microtubule‐targeting agents.

RNA-Binding Proteins in Cancer: Old Players and New Actors

RNA-binding proteins (RBPs) are key players in post-transcriptional events. The combination of versatility of their RNA-binding domains with structural flexibility enables RBPs to control the metabolism of a large array of transcripts. Perturbations in RBP-RNA networks activity have been causally associated with cancer development, but the rational framework describing these contributions remains fragmented. We review here the evidence that RBPs modulate multiple cancer traits, emphasize their functional diversity, and assess future trends in the study of RBPs in cancer.

Identification of Novel Small Molecule Inhibitors of Oncogenic RET Kinase

Oncogenic mutation of the RET receptor tyrosine kinase is observed in several human malignancies. Here, we describe three novel type II RET tyrosine kinase inhibitors (TKI), ALW-II-41-27, XMD15-44 and HG-6-63-01, that inhibit the cellular activity of oncogenic RET mutants at two digit nanomolar concentration. These three compounds shared a 3-trifluoromethyl-4-methylpiperazinephenyl pharmacophore that stabilizes the ‘DFG-out’ inactive conformation of RET activation loop. They blocked RET-mediated signaling and proliferation with an IC50 in the nM range in fibroblasts transformed by the RET/C634R and RET/M918T oncogenes. They also inhibited autophosphorylation of several additional oncogenic RET-derived point mutants and chimeric oncogenes. At a concentration of 10 nM, ALW-II-41-27, XMD15-44 and HG-6-63-01 inhibited RET kinase and signaling in human thyroid cancer cell lines carrying oncogenic RET alleles; they also inhibited proliferation of cancer, but not non-tumoral Nthy-ori-3-1, thyroid cells, with an IC50 in the nM range. The three compounds were capable of inhibiting the ‘gatekeeper’ V804M mutant which confers substantial resistance to established RET inhibitors. In conclusion, we have identified a type II TKI scaffold, shared by ALW-II-41-27, XMD15-44 and HG-6-63-01, that may be used as novel lead for the development of novel agents for the treatment of cancers harboring oncogenic activation of RET.

LIM Kinase Inhibitor Pyr1 Reduces the Growth and Metastatic Load of Breast Cancers

LIM kinases (LIMK) are emerging targets for cancer therapy, and they function as network hubs to coordinate actin and microtubule dynamics. When LIMKs are inhibited, actin microfilaments are disorganized and microtubules are stabilized. Owing to their stabilizing effect on microtubules, LIMK inhibitors may provide a therapeutic strategy to treat taxane-resistant cancers. In this study, we investigated the effect of LIMK inhibition on breast tumor development and on paclitaxel-resistant tumors, using a novel selective LIMK inhibitor termed Pyr1. Treatment of breast cancer cells, including paclitaxel-resistant cells, blocked their invasion and proliferation in vitro and their growth in vivo in tumor xenograft assays. The tumor-invasive properties of Pyr1 were investigated in vivo by intravital microscopy of tumor xenografts. A striking change of cell morphology was observed with a rounded phenotype arising in a subpopulation of cells, while other cells remained elongated. Notably, although Pyr1 decreased the motility of elongated cells, it increased the motility of rounded cells in the tumor. Pyr1 administration prevented the growth of metastasis but not their spread. Overall, our results provided a preclinical proof of concept concerning how a small-molecule inhibitor of LIMK may offer a strategy to treat taxane-resistant breast tumors and metastases. Cancer Res; 76(12); 3541-52. ©2016 AACR.

MEX-3 proteins: recent insights on novel post-transcriptional regulators

RNA-binding proteins of the evolutionarily-conserved MEX-3 family are mediators of post-transcriptional regulation in different organisms. Recent studies highlight their involvement in diverse physiological settings, including the maintenance of a balance between stem cell self-renewal and differentiation. Here, we draw attention to their putative role in tissue homeostasis and disease, particularly cancer.

Analysis of NUAK1 and NUAK2 expression during early chick development reveals specific patterns in the developing head

Several human diseases are associated with the NUAK1 and NUAK2 genes. These genes encode kinases, members of the AMPK-related kinases (ARK) gene family. Both NUAK1 and NUAK2 are known targets of the serine threonine kinase LKB1, a tumor suppressor involved in regulating cell polarity. While much is known about their functions in disease, their expression pattern in normal development has not been extensively studied. Here, we present the expression patterns for NUAK1 and NUAK2 in the chick during early-stage embryogenesis, until day 3 (Hamburger and Hamilton stage HH20). Several embryonic structures, in particular the nascent head, showed distinct expression levels. NUAK1 expression was first detected at stage HH6 in the rostral neural folds. It was then expressed (HH7-11) throughout the encephalalon, predominantly in the telencephalon and mesencephalon. NUAK1 expression was also detected in the splanchnic endoderm area at HH8-10, and in the vitellin vein derived from this area, but not in the heart. NUAK2 expression was first detected at stage HH6 in the neural folds. It was then found throughout the encephalon at stage HH20. Particular attention was paid in this study to the dorsal ectoderm at stages HH7 and HH8, where a local deficit or accumulation of NUAK2 mRNA were found to correlate with the direction of curvature of the neural plate. This is the first description of NUAK1 and NUAK2 expression patterns in the chick during early development; it reveals non-identical expression profiles for both genes in neural development.

LKB1 signaling in cephalic neural crest cells is essential for vertebrate head development.

Head development in vertebrates proceeds through a series of elaborate patterning mechanisms and cell-cell interactions involving cephalic neural crest cells (CNCC). These cells undergo extensive migration along stereotypical paths after their separation from the dorsal margins of the neural tube and they give rise to most of the craniofacial skeleton. Here, we report that the silencing of the LKB1 tumor suppressor affects the delamination of pre-migratory CNCC from the neural primordium as well as their polarization and survival, thus resulting in severe facial and brain defects. We further show that LKB1-mediated effects on the development of CNCC involve the sequential activation of the AMP-activated protein kinase (AMPK), the Rho-dependent kinase (ROCK) and the actin-based motor protein myosin II. Collectively, these results establish that the complex morphogenetic processes governing head formation critically depends on the activation of the LKB1 signaling network in CNCC.