Mesomelic dysplasias associated with the HOXD locus are caused by regulatory reallocations.

Human families with chromosomal rearrangements at 2q31, where the human HOXD locus maps, display mesomelic dysplasia, a severe shortening and bending of the limb. In mice, the dominant Ulnaless inversion of the HoxD cluster produces a similar phenotype suggesting the same origin for these malformations in humans and mice. Here we engineer 1 Mb inversion including the HoxD gene cluster, which positioned Hoxd13 close to proximal limb enhancers. Using this model, we show that these enhancers contact and activate Hoxd13 in proximal cells, inducing the formation of mesomelic dysplasia. We show that a secondary Hoxd13 null mutation in-cis with the inversion completely rescues the alterations, demonstrating that ectopic HOXD13 is directly responsible for this bone anomaly. Single-cell expression analysis and evaluation of HOXD13 binding sites suggests that the phenotype arises primarily by acting through genes normally controlled by HOXD13 in distal limb cells. Altogether, these results provide a conceptual and mechanistic framework to understand and unify the molecular origins of human mesomelic dysplasia associated with 2q31.

Mammalian-specific ectodermal enhancers control the expression of Hoxc genes in developing nails and hair follicles.

Vertebrate Hox genes are critical for the establishment of structures during the development of the main body axis. Subsequently, they play important roles either in organizing secondary axial structures such as the appendages, or during homeostasis in postnatal stages and adulthood. Here, we set up to analyze their elusive function in the ectodermal compartment, using the mouse limb bud as a model. We report that the HoxC gene cluster was co-opted to be transcribed in the distal limb ectoderm, where it is activated following the rule of temporal colinearity. These ectodermal cells subsequently produce various keratinized organs such as nails or claws. Accordingly, deletion of the HoxC cluster led to mice lacking nails (anonychia), a condition stronger than the previously reported loss of function of Hoxc13, which is the causative gene of the ectodermal dysplasia 9 (ECTD9) in human patients. We further identified two mammalian-specific ectodermal enhancers located upstream of the HoxC gene cluster, which together regulate Hoxc gene expression in the hair and nail ectodermal organs. Deletion of these regulatory elements alone or in combination revealed a strong quantitative component in the regulation of Hoxc genes in the ectoderm, suggesting that these two enhancers may have evolved along with the mammalian taxon to provide the level of HOXC proteins necessary for the full development of hair and nail.

Activated ALK signals through the ERK-ETV5-RET pathway to drive neuroblastoma oncogenesis.

Activating mutations of the ALK receptor occur in a subset of neuroblastoma tumors. We previously demonstrated that Alk mutations cooperate with MYCN overexpression to induce neuroblastoma in mice and identified Ret as being strongly upregulated in MYCN/Alkmut tumors. By a genetic approach in vivo, we now document an oncogenic cooperation between activated Ret and MYCN overexpression in neuroblastoma formation. We show that MYCN/RetM919T tumors exhibit histological features and expression profiles close to MYCN/Alkmut tumors. We show that RET transcript levels decrease precedes RET protein levels decrease upon ALK inhibition in neuroblastoma cell lines. Etv5 was identified as a candidate transcription factor regulating Ret expression from murine MYCN/Alkmut tumor transcriptomic data. We demonstrate that ETV5 is regulated both at the protein and mRNA levels upon ALK activation or inhibition in neuroblastoma cell lines and that this regulation precedes RET modulation. We document that ALK activation induces ETV5 protein upregulation through stabilization in a MEK/ERK-dependent manner. We show that RNAi-mediated inhibition of ETV5 decreases RET expression. Reporter assays indicate that ETV5 is able to drive RET gene transcription. ChIP-seq analysis confirmed ETV5 binding on the RET promoter and identified an enhancer upstream of the promoter. Finally, we demonstrate that combining RET and ALK inhibitors reduces tumor growth more efficiently than each single agent in MYCN and AlkF1178L-driven murine neuroblastoma. Altogether, these results define the ERK-ETV5-RET pathway as a critical axis driving neuroblastoma oncogenesis downstream of activated ALK.

The ALK receptor in sympathetic neuron development and neuroblastoma.

The ALK gene encodes a tyrosine kinase receptor characterized by an expression pattern mainly restricted to the developing central and peripheral nervous systems. In 2008, the discovery of ALK activating mutations in neuroblastoma, a tumor of the sympathetic nervous system, represented a breakthrough in the understanding of the pathogenesis of this pediatric cancer and established mutated ALK as a tractable therapeutic target for precision medicine. Subsequent studies addressed the identity of ALK ligands, as well as its physiological function in the sympathoadrenal lineage, its role in neuroblastoma development and the signaling pathways triggered by mutated ALK. This review focuses on these different aspects of the ALK biology and summarizes the various therapeutic strategies relying on ALK inhibition in neuroblastoma, either as monotherapies or combinatory treatments.

Hyperactivation of Alk induces neonatal lethality in knock-in AlkF1178L mice.

The ALK (Anaplastic Lymphoma Kinase) gene encodes a tyrosine kinase receptor preferentially expressed in the central and peripheral nervous systems. A syndromic presentation associating congenital neuroblastoma with severe encephalopathy and an abnormal shape of the brainstem has been described in patients harbouring de novo germline F1174V and F1245V ALK mutations. Here, we investigated the phenotype of knock-in (KI) mice bearing the AlkF1178L mutation (F1174L in human). Although heterozygous KI mice did not reproduce the severe breathing and feeding difficulties observed in human patients, behavioral tests documented a reduced activity during dark phases and an increased anxiety of mutated mice. Matings of heterozygotes yielded the expected proportions of wild-type, heterozygotes and homozygotes at birth but a high neonatal lethality was noticed for homozygotes. We documented Alk expression in several motor nuclei of the brainstem involved in the control of sucking and swallowing. Evaluation of basic physiological functions 12 hours after birth revealed slightly more apneas but a dramatic reduced milk intake for homozygotes compared to control littermates. Overall, our data demonstrate that Alk activation above a critical threshold is not compatible with survival in mice, in agreement with the extremely severe phenotype of patients carrying aggressive de novo ALK germline mutations.

Activated Alk triggers prolonged neurogenesis and Ret upregulation providing a therapeutic target in ALK-mutated neuroblastoma.

Activating mutations of the ALK (Anaplastic lymphoma Kinase) gene have been identified in sporadic and familial cases of neuroblastoma, a cancer of early childhood arising from the sympathetic nervous system (SNS). To decipher ALK function in neuroblastoma predisposition and oncogenesis, we have characterized knock-in (KI) mice bearing the two most frequent mutations observed in neuroblastoma patients. A dramatic enlargement of sympathetic ganglia is observed in AlkF1178L mice from embryonic to adult stages associated with an increased proliferation of sympathetic neuroblasts from E14.5 to birth. In a MYCN transgenic context, the F1178L mutation displays a higher oncogenic potential than the R1279Q mutation as evident from a shorter latency of tumor onset. We show that tumors expressing the R1279Q mutation are sensitive to ALK inhibition upon crizotinib treatment. Furthermore, our data provide evidence that activated ALK triggers RET upregulation in mouse sympathetic ganglia at birth as well as in murine and human neuroblastoma. Using vandetanib, we show that RET inhibition strongly impairs tumor growth in vivo in both MYCN/KI AlkR1279Q and MYCN/KI AlkF1178L mice. Altogether, our findings demonstrate the critical role of activated ALK in SNS development and pathogenesis and identify RET as a therapeutic target in ALK mutated neuroblastoma.