Immunotherapies based on immune checkpoint inhibitors are emerging as an innovative treatment for different types of advanced cancers. While the utility of immune checkpoint inhibitors has been clearly demonstrated, the response rate is highly variable across individuals. Due to the cost and toxicity of these immunotherapies, a critical challenge in this field is the identification of predictive biomarkers to discriminate which patients may respond to immunotherapy. Recently, a high tumor mutational burden (TMB) has been identified as a genetic signature that is associated with a favorable outcome for immune checkpoint inhibitor therapy. The TMB is defined as the total number of nonsynonymous mutations per coding area of a tumor genome. Initially, it was determined using whole exome sequencing, but due to the high costs and long turnaround time of this method, targeted panel sequencing is currently being explored to measure TMB. In the near future, TMB evaluation may play an important role in immuno-oncology, but its implementation in a routine setting involves robust analytical and clinical validation. Standardization is also needed in order to make informed decisions about patients. This review presents the methodologies employed for determining TMB and discusses the factors that may have an impact on its measurement.

Highlights d In somatosensory ganglia, Prdm12 is specific to the nociceptive lineage d Prdm12 is necessary for the survival of developing nociceptors d Prdm12 initiates and maintains the expression of TrkA in developing nociceptors d Prdm12 acts in conjunction with bHLH proteins Ngn1/2 to promote a nociceptor fate

V1 interneurons are inhibitory neurons that play an essential role in vertebrate locomotion. The molecular mechanisms underlying their genesis remain, however, largely undefined. Here, we show that the transcription factor Prdm12 is selectively expressed in p1 progenitors of the hindbrain and spinal cord in the frog embryo, and that a similar restricted expression profile is observed in the nerve cord of other vertebrates as well as of the cephalochordate amphioxus. Using frog, chick and mice, we analyzed the regulation of Prdm12 and found that its expression in the caudal neural tube is dependent on retinoic acid and Pax6, and that it is restricted to p1 progenitors, due to the repressive action of Dbx1 and Nkx6-1/2 expressed in the adjacent p0 and p2 domains. Functional studies in the frog, including genomewide identification of its targets by RNA-seq and ChIP-Seq, reveal that vertebrate Prdm12 proteins act as a general determinant of V1 cell fate, at least in part, by directly repressing Dbx1 and Nkx6 genes. This probably occurs by recruiting the methyltransferase G9a, an activity that is not displayed by the amphioxus Prdm12 protein. Together, these findings indicate that Prdm12 promotes V1 interneurons through cross-repressive interactions with Dbx1 and Nkx6 genes, and suggest that this function might have only been acquired after the split of the vertebrate and cephalochordate lineages.

Mutational screens are an effective means used in the functional annotation of a genome. We present a method for a mutational screen of the mouse X chromosome using gene trap technologies. This method has the potential to screen all of the genes on the X chromosome without establishing mutant animals, as all gene-trapped embryonic stem (ES) cell lines are hemizygous null for mutations on the X chromosome. Based on this method, embryonic morphological phenotypes and expression patterns for 58 genes were assessed, ∼10% of all human and mouse syntenic genes on the X chromosome. Of these, 17 are novel embryonic lethal mutations and nine are mutant mouse models of genes associated with genetic disease in humans, including BCOR and PORCN. The rate of lethal mutations is similar to previous mutagenic screens of the autosomes. Interestingly, some genes associated with X-linked mental retardation (XLMR) in humans show lethal phenotypes in mice, suggesting that null mutations cannot be responsible for all cases of XLMR. The entire data set is available via the publicly accessible website (http://xlinkedgenes.ibme.utoronto.ca/).

These authors contributed equally to this work.Keywords: Hamlet, nociception, PRDM12, sensory neurons, TRHDE Abbreviation: PRDM12, PR homology domain-containing member 12; HSAN, hereditary and sensory autonomic neuropathy; TRHDE, tyrotropin-releasing hormone degrading enzyme; RA, retinoic acid; Brn3d, brain 3d; Tlx3, T-cell leukemia homeobox 3; Hmx3, H6 family homeobox 3; Drgx, dorsal root ganglia homeobox; Sncg, Synuclein Gamma (Breast Cancer-Specific Protein 1); En1, engrailed-1; RT-qPCR, real-time quantitative polymerase chain reaction; MO, morpholino oligonucleotide; ChIP, chromatin immunoprecipitation; S1PR1, Sphi8ngosine-1-phosphate receptor 1; CGNL1, cyclin L1; IL1R1, interleukin 1 receptor type 1; TRH(DE), tryrotropin-releasing hormone degrading enzyme; pCMV6, plasmid cytomegalovirus; DDK, DYKDDDDK epitope; GAPDH, glyceraldehyde 3-phospate dehydrogenase; NBT/BCIP, nitro blue tetrazolium / 5-bromo-4-chloro-3-indolyl-phosphate; SET, Su(var)3-9 and 'Enhancer of zeste'; PDB, protein data base; HEK293, human embryonic kidney cell 293; PBS, phosphate buffered saline; DAPI, 4 0 ,6-diamidino-2-phenylindole; GFP, green fluorescent protein; BSA, bovine serum albumin; HRP, horseraddish peroxidase; ECL, enhanced chemiluminescence; FPKM, fragments per kilobase exon; FDR, false discovery rate; GEO, gene expression omnibus; PMID, pubmed identification.PR homology domain-containing member 12 (PRDM12) belongs to a family of conserved transcription factors implicated in cell fate decisions. Here we show that PRDM12 is a key regulator of sensory neuronal specification in Xenopus. Modeling of human PRDM12 mutations that cause hereditary sensory and autonomic neuropathy (HSAN) revealed remarkable conservation of the mutated residues in evolution. Expression of wild-type human PRDM12 in Xenopus induced the expression of sensory neuronal markers, which was reduced using various human PRDM12 mutants. In Drosophila, we identified Hamlet as the functional PRDM12 homolog that controls nociceptive behavior in sensory neurons. Furthermore, expression analysis of human patient fibroblasts with PRDM12 mutations uncovered possible downstream target genes. Knockdown of several of these target genes including thyrotropin-releasing hormone degrading enzyme (TRHDE) in Drosophila sensory neurons resulted in altered cellular morphology and impaired nociception. These data show that PRDM12 and its functional fly homolog Hamlet are evolutionary conserved master regulators of sensory neuronal specification and play a critical role in pain perception. Our data also uncover novel pathways in multiple species that regulate evolutionary conserved nociception.

Notch signaling has been shown to play a role in cell fate decisions in the Xenopus pronephros anlagen. Here, we show that the Xenopus Hairy-related transcription factor (HRT) gene XHRT1, and the Hairy/Enhancer of split (HES) genes Xhairy1, Xhairy2b, esr9 and esr10, have distinct restricted dynamic expression patterns during pronephros development, and that their expression is regulated by Notch. XHRT1, which is the earliest and strongest gene expressed in the pronephric region, is initially transcribed predominantly in the forming glomus, where it is downregulated by antisense morpholino oligonucleotide inhibition of xWT1. Later, it is activated in the most dorsoanterior part of the pronephros anlagen that gives rise to the proximal tubules. In agreement with this dynamic expression profile, we found that early activation of Notch favors glomus, whereas only later activation promotes proximal tubule formation. We show that, among the bHLH-O factors tested, only XHRT1 efficiently inhibits distal tubule and duct formation, and that only its translational inhibition causes a reduction of the expression of proximal tubule and glomus markers. Using domain swap experiments, we found that the XHRT1 C-terminal region is crucial for its activity. Together, our results provide evidence that XHRT1 plays an important role in glomerular development and early proximodistal patterning that is distinct from those of the other pronephric bHLH repressors.

The ecotropic viral integration site 1 (Evi1) and related MEL1 (MDS1/Evi1-like gene 1) genes are zinc finger oncogenic transcription factors involved in myeloid leukaemia. Here, we show that in Xenopus, Evi1 and MEL1 have partially overlapping restricted embryonic expression profiles. Within the pronephros, Evi1 and MEL1 are sequentially expressed within the distal tubule and duct compartments, Evi1 transcription being detected prior to any sign of pronephric morphogenesis. In the pronephros of zebrafish embryos, Evi1 expression is restricted to the posterior portion of the duct, the anterior portion having characteristics of proximal tubules. In the Xenopus pronephros, Evi1 expression is upregulated by retinoid signaling and repressed by overexpression of xWT1 and by Notch signaling. Overexpression of Evi1 from late neurula stage specifically inhibits the expression of proximal tubule and glomus pronephric markers. We show that the first zinc finger and CtBP interaction domains are required for this activity. Overexpression of a hormone-inducible Evi1-VP16 antimorphic fusion with activation at neurula stage disrupts distal tubule and duct formation and expands the expression of glomus markers. Although overexpression of this construct also causes in many embryos a reduction of proximal tubule markers, embryos with expanded and ectopic staining have been also observed. Together, these data indicate that Evi1 plays a role in the proximo-distal patterning of the pronephros and suggest that it may do so by functioning as a CtBP dependent repressor.

Wnt signaling pathways are essential for embryonic patterning, and they are disturbed in a wide spectrum of diseases, including cancer. An unresolved question is how the different Wnt pathways are supported and regulated. We previously established that the postsynaptic density 95/disc-large/zona occludens (PDZ) protein syntenin binds to syndecans, Wnt coreceptors, and known stimulators of protein kinase C (PKC)␣ and CDC42 activity. Here, we show that syntenin also interacts with the C-terminal PDZ binding motif of several Frizzled Wnt receptors, without compromising the recruitment of Dishevelled, a key downstream Wnt-signaling component. Syntenin is coexpressed with cognate Frizzled during early development in Xenopus. Overexpression and down-regulation of syntenin disrupt convergent extension movements, supporting a role for syntenin in noncanonical Wnt signaling. Syntenin stimulates c-jun phosphorylation and modulates Frizzled 7 signaling, in particular the PKC␣/CDC42 noncanonical Wnt signaling cascade. The syntenin-Frizzled 7 binding mode indicates syntenin can accommodate Frizzled 7-syndecan complexes. We propose that syntenin is a novel component of the Wnt signal transduction cascade and that it might function as a direct intracellular link between Frizzled and syndecans. INTRODUCTIONWnt proteins are involved in cell proliferation, differentiation, polarity, migration, and apoptosis, controlling a variety of processes during embryonic development and adult homeostasis (Logan and Nusse, 2004). Various inborn and acquired diseases are based on aberrant Wnt signaling (Johnson and Rajamannan, 2006). Wnt signaling requires the interplay of multiple proteins (http://www.stanford.edu/ ϳrnusse/wntwindow.html), but the reception and transduction of Wnt signals are predominantly based on the binding of Wnt proteins to Frizzled (Fz) cell surface receptors (Yang-Snyder et al., 1996). The various Fz receptors differ in their spatial and temporal expression patterns and in their relative affinities for ligand. Receptor activation by Wnt somehow activates Dishevelled (Dsh), the most upstream component of the cytosolic signal transduction cascade. The role of Dsh is intricate because downstream of Dsh, the signaling branches into either the canonical Wnt/ ␤-catenin or the noncanonical (␤-catenin-independent) pathway (Boutros and Mlodzik, 1999). Activation of the canonical pathway drives ␤-catenin-dependent transcription of target genes, controls tissue-specific cell fate decisions during embryogenesis, and regulates cell proliferation in adult tissues (Logan and Nusse, 2004). The noncanonical pathway controls reorganization of the actin cytoskeleton, tissue polarity, and cell movement (Strutt, 2003). Several molecular cascades, which may overlap, seem to function in noncanonical Wnt signaling (Veeman et al., 2003;Kohn and Moon, 2005). One is similar to the planar cell polarity or PCP pathway in Drosophila, and it activates small GTPases of the Rho family and the c-Jun-NH 2 -terminal kinase (JNK). Another, triggered by Wn...