Whole-exome sequencing of familial esophageal squamous cell carcinoma identified rare pathogenic variants in new predisposition genes.

PURPOSE: Esophageal squamous cell carcinoma (ESCC) is one of the most important causes of mortality in the developing world. Although hereditary forms arise from germ-line mutations in TP53, Rb, and the mismatch repair genes, many familial cases present with an unknown inherited cause. The new theory of rare, high-penetrance mutations in less known genes is a likely explanation for the underlying predisposition in some of these familial cases. METHODS: Exome sequencing was performed in 9 patients with esophageal squamous cancer from 9 families with strong disease aggregation without mutations in known hereditary esophageal cancer genes. Data analysis was limited to only really rare variants (0-0.01%), producing a putative loss of function and located in genes with a role compatible with carcinogenesis. RESULTS: Twenty-two final candidate variants were selected and validated by Sanger sequencing. Correct family segregation and somatic studies were used to categorize the most interesting variants in CDK11A, ARID1A, JMJD6, MAML3, CDKN2AIP, and PHLDA1. CONCLUSION: Together, we identified new potential esophageal squamous cancer predisposition variants in genes which may have a role in cancer and are involved in chromatin remodeling and cell-cycle pathway, which could increase the risk of ESCC.

Dnmt3a regulates T-cell development and suppresses T-ALL transformation.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic neoplasm resulting from the malignant transformation of T-cell progenitors, and comprises ~15% and 25% of pediatric and adult ALL cases, respectively. It is well-established that activating NOTCH1 mutations are the major genetic lesions driving T-ALL in most patients, but efforts to develop targeted therapies against this pathway have produced limited success in decreasing leukemic burden and come with significant clinical side effects. A finer detailed understanding of the genetic and molecular mechanisms underlying T-ALL is required identify patients at increased risk for treatment failure and the development of precision medicine strategies. Generation of genetic models that more accurately reflect the normal developmental history of T-ALL are necessary to identify new avenues for treatment. The DNA methyltransferase enzyme DNMT3A is also recurrently mutated in T-ALL patients, and we show here that inactivation of Dnmt3a combined with Notch1 gain-of-function leads to an aggressive T-ALL in mouse models. Moreover, conditional inactivation of Dnmt3a in mouse hematopoietic cells leads to an accumulation of immature progenitors in the thymus, which are less apoptotic. These data demonstrate that Dnmt3a is required for normal T-cell development, and acts as a T-ALL tumor suppressor.

Genetic variation in MKL2 and decreased downstream PCTAIRE1 expression in extreme, fatal primary human microcephaly.

The genetic mechanisms driving normal brain development remain largely unknown. We performed genomic and immunohistochemical characterization of a novel, fatal human phenotype including extreme microcephaly with cerebral growth arrest at 14-18 weeks gestation in three full sisters born to healthy, non-consanguineous parents. Analysis of index cases and parents included familial exome sequencing, karyotyping, and genome-wide single nucleotide polymorphism (SNP) array. From proband, control and unrelated microcephalic fetal cortical tissue, we compared gene expression of RNA and targeted immunohistochemistry. Each daughter was homozygous for a rare, non-synonymous, deleterious variant in the MKL2 gene and heterozygous for a private 185 kb deletion on the paternal allele, upstream and in cis with his MKL2 variant allele, eliminating 24 CArG transcription factor binding sites and MIR4718. MKL1 was underexpressed in probands. Dysfunction of MKL2 and its transcriptional coactivation partner, serum response factor (SRF), was supported by a decrease in gene and protein expression of PCTAIRE1, a downstream target of MKL2:SRF heterodimer transcriptional activation, previously shown to result in severe microcephaly in murine models. While disruption of the MKL2:SRF axis has been associated with severe microcephaly and disordered brain development in multiple model systems, the role of this transcription factor complex has not been previously demonstrated in human brain development.

Excess congenital non-synonymous variation in leukemia-associated genes in MLL- infant leukemia: a Children's Oncology Group report.

Infant leukemia (IL) is a rare sporadic cancer with a grim prognosis. Although most cases are accompanied by MLL rearrangements and harbor very few somatic mutations, less is known about the genetics of the cases without MLL translocations. We performed the largest exome-sequencing study to date on matched non-cancer DNA from pairs of mothers and IL patients to characterize congenital variation that may contribute to early leukemogenesis. Using the COSMIC database to define acute leukemia-associated candidate genes, we find a significant enrichment of rare, potentially functional congenital variation in IL patients compared with randomly selected genes within the same patients and unaffected pediatric controls. IL acute myeloid leukemia (AML) patients had more overall variation than IL acute lymphocytic leukemia (ALL) patients, but less of that variation was inherited from mothers. Of our candidate genes, we found that MLL3 was a compound heterozygote in every infant who developed AML and 50% of infants who developed ALL. These data suggest a model by which known genetic mechanisms for leukemogenesis could be disrupted without an abundance of somatic mutation or chromosomal rearrangements. This model would be consistent with existing models for the establishment of leukemia clones in utero and the high rate of IL concordance in monozygotic twins.

Early outcomes after allogeneic hematopoietic SCT in pediatric patients with hematologic malignancies following single fraction TBI.

Fractionated TBI (FTBI) followed by allogeneic hematopoietic SCT results in donor engraftment and improves survival in children with high-risk hematologic malignancies. However, acute toxicities (skin, lung and mucosa) are common after FTBI. Late complications include cataracts, endocrine dysfunction, sterility and impaired neurodevelopment. Instead of FTBI, we used low-dose single fraction TBI (550 cGy) with CY as transplant conditioning for pediatric hematologic malignancies. GVHD prophylaxis included CYA and short-course MTX; methylprednisolone was added for unrelated donor transplants. A total of 55 children in first (40%) or second remission and beyond (60%) underwent transplantation from BM (65%) or peripheral blood; 62% from unrelated donors; 22% were mismatched. Median follow-up was 18.5 months (1-68). Overall survival and disease-free survival at 1 year were 60 and 47%, respectively. Acute toxicities included grade 3-4 mucositis (18%), invasive infections (11%), multiorgan failure/shock (11%), hemolytic anemia (7%), veno-occlusive disease (4%) and renal failure (4%). TRM was 11% at 100 days. Non-relapse mortality was 6% thereafter. Graft rejection occurred in 2%. Three patients (5%) died of GVHD. The regimen was well tolerated even in heavily pretreated children and supported donor cell engraftment; long-term follow up is in progress.

From MDR to MXR: new understanding of multidrug resistance systems, their properties and clinical significance.

The ATP binding cassette (ABC) superfamily of membrane transporters is one of the largest protein classes known, and counts numerous proteins involved in the trafficking of biological molecules across cell membranes. The first known human ABC transporter was P-glycoprotein (P-gp), which confers multidrug resistance (MDR) to anticancer drugs. In recent years, we have obtained an increased understanding of the mechanism of action of P-gp as its ATPase activity, substrate specificity and pharmacokinetic interactions have been investigated. This review focuses on the functional characterization of P-gp, as well as other ABC transporters involved in MDR: the family of multidrug-resistance-associated proteins (MRP1-7), and the recently discovered ABC half-transporter MXR (also known as BCRP, ABCP and ABCG2). We describe recent progress in the analysis of protein structure-function relationships, and consider the conceptual problem of defining and identifying substrates and inhibitors of MDR. An in-depth discussion follows of how coupling of nucleotide hydrolysis to substrate transport takes place, and we propose a scheme for the mechanism of P-gp function. Finally, the clinical correlations, both for reversal of MDR in cancer and for drug delivery, are discussed.

P-glycoprotein-mediated colchicine resistance in different cell lines correlates with the effects of colchicine on P-glycoprotein conformation.

The multidrug transporter P-glycoprotein (Pgp) is an ATPase efflux pump for multiple cytotoxic agents, including vinblastine and colchicine. We have found that resistance to vinblastine but not to colchicine in cell lines derived from different types of tissues and expressing the wild-type human Pgp correlates with the Pgp density. Vinblastine induces a conformational change in Pgp, evidenced by increased reactivity with a conformation-sensitive monoclonal antibody UIC2, in all the tested cell lines. In contrast, colchicine increases the UIC2 reactivity in only some of the cell lines. In those lines where colchicine alone did not affect UIC2 reactivity, this drug was, however, able to reverse the vinblastine-induced increase in UIC2 reactivity. The magnitude of the increase in UIC2 reactivity in the presence of saturating concentrations of colchicine correlates with the relative ability of Pgp to confer colchicine resistance in different cell lines, suggesting the existence of some cell-specific factors that have a coordinate effect on the ability of colchicine to induce conformational transitions and to be transported by Pgp. Colchicine, like vinblastine, reverses the decrease in UIC2 reactivity produced by nonhydrolyzable nucleotides, but unlike vinblastine, it does not reverse the effect of ATP at a high concentration. Colchicine, however, decreases the Hill number for the effect of ATP on the UIC2 reactivity from 2 to 1. Colchicine increases the UIC2 reactivity and reverses the effect of ATP in ATPase-deficient Pgp mutants, but not in the wild-type Pgp expressed in the same cellular background, suggesting that ATP hydrolysis counteracts the effects of colchicine on the Pgp conformation.

Analysis of MDR1 P-glycoprotein conformational changes in permeabilized cells using differential immunoreactivity.

The reactivity of the ATP-dependent multidrug transporter P-glycoprotein (Pgp) with the conformation-sensitive monoclonal antibody UIC2 is increased in the presence of Pgp transport substrates, ATP-depleting agents, or mutations that reduce the level of nucleotide binding by Pgp. We have investigated the effects of nucleotides and vinblastine, a Pgp transport substrate, on the UIC2 reactivity of Pgp in cells permeabilized by Staphylococcus aureus alpha-toxin. ATP, ADP, and nonhydrolyzable ATP analogues decreased the UIC2 reactivity; this effect was potentiated by vanadate, a nucleotide-trapping agent. The Hill number for the nucleotide-induced conformational transition was 2 for ATP and ADP but 1 for nonhydrolyzable ATP analogues. The Hill numbers for ATP and ADP were decreased to 1 by mutations in one of the two nucleotide binding sites of Pgp, whereas mutation of both sites greatly diminished the overall effect of nucleotides. Vinblastine reversed the decrease in the UIC2 reactivity brought about by all the nucleotides, including nonhydrolyzable analogues; this effect of vinblastine was blocked by vanadate. These data indicate that UIC2-detectable conformational changes of Pgp are driven by binding and debinding of nucleotides, that nucleotide hydrolysis affects the Hill number for its Pgp interactions, and that Pgp transport substrates promote nucleotide dissociation from Pgp. These findings are consistent with a conventional E1/E2 model that explains conformational transitions of a transporter protein through a series of linked equilibria.