Structural variants involving MLLT10 fusion are associated with adverse outcomes in pediatric acute myeloid leukemia.

ABSTRACT: MLLT10 gene rearrangements with KMT2A occur in pediatric acute myeloid leukemia (AML) and confer poor prognosis, but the prognostic impact of MLLT10 in partnership with other genes is unknown. We conducted a retrospective study with 2080 children and young adults with AML registered on the Children's Oncology Group AAML0531 (NCT00372593) and AAML1031 trials (NCT01371981). Transcriptome profiling and/or karyotyping were performed to identify leukemia-associated fusions associated with prognosis. Collectively, 127 patients (6.1%) were identified with MLLT10 fusions: 104 (81.9%) with KMT2A::MLLT10, 13 (10.2%) with PICALM::MLLT10, and 10 (7.9%) X::MLLT10: (2 each of DDX3X and TEC), with 6 partners (DDX3Y, CEP164, SCN2B, TREH, NAP1L1, and XPO1) observed in single patients. Patients with MLLT10 (n = 127) demonstrated adverse outcomes, with 5-year event-free survival (EFS) of 18.6% vs 49% in patients without MLLT10 (n = 1953, P < .001), inferior 5-year overall survival (OS) of 38.2% vs 65.7% (P ≤ .001), and a higher relapse risk of 76% vs 38.6% (P < .001). Patients with KMT2A::MLLT10 had an EFS from study entry of 19.5% vs 12.7% (P = .628), and an OS from study entry of 40.4% vs 27.6% (P = .361) in those with other MLLT10 fusion partners. Patients with PICALM::MLLT10 had an EFS of 9.2% vs 20% in other MLLT10- without PICALM (X::MLLT10; P = .788). Patients with PICALM::MLLT10 and X::MLLT10 fusions exhibit a DNA hypermethylation signature resembling NUP98::NSD1 fusions, whereas patients with KMT2A::MLLT10 bear aberrations primarily affecting distal regulatory elements. Regardless of the fusion partner, patients with AML harboring MLLT10 fusions exhibit very high-risk features and should be prioritized for alternative therapeutic interventions.

Validation of Myc-Associated Protein X (MAX) regulation in growth hormone secreting and nonfunctional pituitary adenoma.

INTRODUCTION: Many patients with growth hormone-secreting pituitary adenoma (GHPA) fail to achieve biochemical remission, warranting investigation into epigenetic and molecular signatures associated with tumorigenesis and hormonal secretion. Prior work exploring the DNA methylome showed Myc-Associated Protein X (MAX), a transcription factor involved in cell cycle regulation, was differentially methylated between GHPA and nonfunctional pituitary adenoma (NFPA). We aimed to validate the differential DNA methylation and related MAX protein expression profiles between NFPA and GHPA. METHODS: DNA methylation levels were measured in 52 surgically resected tumors (37 NFPA, 15 GHPA) at ~100,000 known MAX binding sites derived using ChIP-seq analysis from ENCODE. Findings were correlated with MAX protein expression using a constructed tissue microarray (TMA). Gene ontology analysis was performed to explore downstream genetic and signaling pathways regulated by MAX. RESULTS: GHPA had more hypomethylation events across all known MAX binding sites. Of binding sites defined using ChIP-seq analysis, 1,551 sites had significantly different methylation patterns between the two cohorts; 432 occurred near promoter regions potentially regulated by MAX, including promoters of TNF and MMP9. Gene ontology analysis suggested enrichment in genes involved in oxygen response, immune system regulation, and cell proliferation. Thirteen MAX binding sites were within coding regions of genes. GHPA demonstrated significantly increased expression of MAX protein compared to NFPA. CONCLUSION: GHPA have significantly different DNA methylation and downstream protein expression levels of MAX compared to NFPA. These differences may influence mechanisms involved with cellular proliferation, tumor invasion and hormonal secretion.

Sensitive and reproducible cell-free methylome quantification with synthetic spike-in controls.

Cell-free methylated DNA immunoprecipitation sequencing (cfMeDIP-seq) identifies genomic regions with DNA methylation, using a protocol adapted to work with low-input DNA samples and with cell-free DNA (cfDNA). We developed a set of synthetic spike-in DNA controls for cfMeDIP-seq to provide a simple and inexpensive reference for quantitative normalization. We designed 54 DNA fragments with combinations of methylation status (methylated and unmethylated), fragment length (80 bp, 160 bp, 320 bp), G + C content (35%, 50%, 65%), and fraction of CpG dinucleotides within the fragment (1/80 bp, 1/40 bp, 1/20 bp). Using 0.01 ng of spike-in controls enables training a generalized linear model that absolutely quantifies methylated cfDNA in MeDIP-seq experiments. It mitigates batch effects and corrects for biases in enrichment due to known biophysical properties of DNA fragments and other technical biases.

Mesothelin is a novel cell surface disease marker and potential therapeutic target in acute myeloid leukemia.

In an effort to identify acute myeloid leukemia (AML)-restricted targets for therapeutic development in AML, we analyzed the transcriptomes of 2051 children and young adults with AML and compared the expression profile with normal marrow specimens. This analysis identified a large cohort of AML-restricted genes with high expression in AML, but low to no expression in normal hematopoiesis. Mesothelin (MSLN), a known therapeutic target in solid tumors, was shown to be highly overexpressed in 36% of the AML cohort (range, 5-1077.6 transcripts per million [TPM]) and virtually absent in normal marrow (range, 0.1-10.7 TPM). We verified MSLN transcript expression by quantitative reverse transcription polymerase chain reaction, confirmed cell surface protein expression on leukemic blasts by multidimensional flow cytometry, and demonstrated that MSLN expression was associated with promoter hypomethylation. MSLN was highly expressed in patients with KMT2A rearrangements (P < .001), core-binding factor fusions [inv(16)/t(16;16), P < .001; t(8;21), P < .001], and extramedullary disease (P = .001). We also demonstrated the presence of soluble MSLN in diagnostic serum specimens using an MSLN-directed enzyme-linked immunosorbent assay. In vitro and in vivo preclinical efficacy of the MSLN-directed antibody-drug conjugates (ADCs) anetumab ravtansine and anti-MSLN-DGN462 were evaluated in MSLN+ leukemia cell lines in vitro and in vivo, as well as in patient-derived xenografts. Treatment with ADCs resulted in potent target-dependent cytotoxicity in MSLN+ AML. In this study, we demonstrate that MSLN is expressed in a significant proportion of patients with AML and holds significant promise as a diagnostic and therapeutic target in AML, and that MSLN-directed therapeutic strategies, including ADCs, warrant further clinical investigation.

Distinctive epigenomic alterations in NF1-deficient cutaneous and plexiform neurofibromas drive differential MKK/p38 signaling.

Benign peripheral nerve sheath tumors are the clinical hallmark of Neurofibromatosis Type 1. They account for substantial morbidity and mortality in NF1. Cutaneous (CNF) and plexiform neurofibromas (PNF) share nearly identical histology, but maintain different growth rates and risk of malignant conversion. The reasons for this disparate clinical behavior are not well explained by recent genome or transcriptome profiling studies. We hypothesized that CNFs and PNFs are epigenetically distinct tumor types that exhibit differential signaling due to genome-wide and site-specific methylation events. We interrogated the methylation profiles of 45 CNFs and 17 PNFs from NF1 subjects with the Illumina EPIC 850K methylation array. Based on these profiles, we confirm that CNFs and PNFs are epigenetically distinct tumors with broad differences in higher-order chromatin states and specific methylation events altering genes involved in key biological and cellular processes, such as inflammation, RAS/MAPK signaling, actin cytoskeleton rearrangement, and oxytocin signaling. Based on our identification of two separate DMRs associated with alternative leading exons in MAP2K3, we demonstrate differential RAS/MKK3/p38 signaling between CNFs and PNFs. Epigenetic reinforcement of RAS/MKK/p38 was a defining characteristic of CNFs leading to pro-inflammatory signaling and chromatin conformational changes, whereas PNFs signaled predominantly through RAS/MEK. Tumor size also correlated with specific CpG methylation events. Taken together, these findings confirm that NF1 deficiency influences the epigenetic regulation of RAS signaling fates, accounting for observed differences in CNF and PNF clinical behavior. The extension of these findings is that CNFs may respond differently than PNFs to RAS-targeted therapeutics raising the possibility of targeting p38-mediated inflammation for CNF treatment.

Patient-derived xenografts of central nervous system metastasis reveal expansion of aggressive minor clones.

BACKGROUND: The dearth of relevant tumor models reflecting the heterogeneity of human central nervous system metastasis (CM) has hindered development of novel therapies. METHODS: We established 39 CM patient-derived xenograft (PDX) models representing the histological spectrum, and performed phenotypic and multi-omic characterization of PDXs and their original patient tumors. PDX clonal evolution was also reconstructed using allele-specific copy number and somatic variants. RESULTS: PDXs retained their metastatic potential, with flank-implanted PDXs forming spontaneous metastases in multiple organs, including brain, and CM subsequent to intracardiac injection. PDXs also retained the histological and molecular profiles of the original patient tumors, including retention of genomic aberrations and signaling pathways. Novel modes of clonal evolution involving rapid expansion by a minor clone were identified in 2 PDXs, including CM13, which was highly aggressive in vivo forming multiple spontaneous metastases, including to brain. These PDXs had little molecular resemblance to the patient donor tumor, including reversion to a copy number neutral genome, no shared nonsynonymous mutations, and no correlation by gene expression. CONCLUSIONS: We generated a diverse and novel repertoire of PDXs that provides a new set of tools to enhance our knowledge of CM biology and improve preclinical testing. Furthermore, our study suggests that minor clone succession may confer tumor aggressiveness and potentiate brain metastasis.

Lack of APC somatic mutation is associated with early-onset colorectal cancer in African Americans.

African Americans (AAs) have higher incidence and mortality rates of colorectal cancer (CRC) compared with other US populations. They present with more right-sided, microsatellite stable disease and are diagnosed at earlier ages compared with non-Hispanic Whites (NHWs). To gain insight into these trends, we conducted exome sequencing (n = 45), copy number (n = 33) and methylation analysis (n = 11) of microsatellite stable AA CRCs. Results were compared with data from The Cancer Genome Atlas (TCGA). Two of the 45 tumors contained POLE mutations. In the remaining 43 tumors, only 27 (63%) contained loss-of-function mutations in APC compared with 80% of TCGA NHW CRCs. APC-mutation-negative CRCs were associated with an earlier onset of CRC (P = 0.01). They were also associated with lower overall mutation burden, fewer copy number variants and a DNA methylation signature that was distinct from the CpG island methylator phenotype characterized in microsatellite unstable disease. Three of the APC-mutation-negative CRCs had loss-of-function mutations in BCL9L. Mutations in driver genes identified by TCGA exome analysis were less frequent in AA CRC cases than TCGA NHWs. Genes that regulate the WNT signaling pathway, including SOX9, GATA6, TET1, GLIS1 and FAT1, were differentially hypermethylated in APC-mutation-negative CRCs, suggesting a novel mechanism for cancer development in these tumors. In summary, we have identified a subtype of CRC that is associated with younger age of diagnosis, lack of APC mutation, microsatellite and chromosome stability, lower mutation burden and distinctive methylation changes.

Senescent human hematopoietic progenitors show elevated expression of transposable elements and inflammatory genes.

Genomic transposable elements (TEs) constitute the majority of the genome. Expression of TEs is known to activate the double-stranded RNA recognition pathway ("viral mimicry"), leading to the activation of interferon-stimulated genes, inflammation, and immune-mediated cell death. Recently, we showed that the expression of TEs is suppressed along with immune pathways in leukemic stem cells (LSCs) in acute myeloid leukemia, suggesting a potential mechanism for immune escape of LSCs. This indicated that, during oncogenesis, where there is escape from senescence, expression of TEs is suppressed. Senescence is known to activate the interferon response and inflammatory cytokines, known as the senescence-associated secretory phenotype (SASP). We characterized the transcriptome of senescent and active human hematopoietic stem and progenitor cells (HSPCs) in vivo and showed co-occurrence of overexpression of TEs, SASP genes, and gene pathways of inflammation in senescence. The percentage of circulating senescent HSPCs (s-HSPCs) did not increase with age, indicating active clearance. Induction of senescence in human HSPCs in vitro showed increased expression of TE and SASP genes. SASP is known to mediate clearance of senescent cells and active clearance of senescent cells has been shown to increase organismal fitness. We speculate that the expression of TEs in s-HSPCs could contribute to orderly clearance of the cells via activation of immune pathways, warranting further mechanistic studies. This is the first study to characterize the transcriptome of human s-HSPCs in vivo, revealing activated expression of TEs and inflammatory genes.

Methylome of human senescent hematopoietic progenitors.

Senescence, a state of permanent cell cycle arrest, can be induced by DNA damage. This process, which was initially described in fibroblasts, is now recognized to occur in stem cells. It has been well characterized in cell lines, but there is currently very limited data available on human senescence in vivo. We recently reported that the expression of transposable elements (TE), including endogenous retroviruses, was up-regulated along with inflammatory genes in human senescent hematopoietic stem and progenitor cells (HSPCs) in vivo. The mechanism of regulation of TE expression is not completely understood, but changes in DNA methylation and chromatin modifications are known to alter their expression. In order to elucidate the molecular mechanisms for TE up-regulation after senescence of HSPCs, we employed whole-genome bisulfite sequencing in paired senescent and active human HSPCs in vivo from healthy subjects. We found that the senescent HSPCs exhibited hypomethylated regions in the genome, which were enriched for TEs. This is the first report characterizing the methylome of senescent human HSPCs.

Characterizing the genetic basis of methylome diversity in histologically normal human lung tissue.

The genetic regulation of the human epigenome is not fully appreciated. Here we describe the effects of genetic variants on the DNA methylome in human lung based on methylation-quantitative trait loci (meQTL) analyses. We report 34,304 cis- and 585 trans-meQTLs, a genetic-epigenetic interaction of surprising magnitude, including a regulatory hotspot. These findings are replicated in both breast and kidney tissues and show distinct patterns: cis-meQTLs mostly localize to CpG sites outside of genes, promoters and CpG islands (CGIs), while trans-meQTLs are over-represented in promoter CGIs. meQTL SNPs are enriched in CTCF-binding sites, DNaseI hypersensitivity regions and histone marks. Importantly, four of the five established lung cancer risk loci in European ancestry are cis-meQTLs and, in aggregate, cis-meQTLs are enriched for lung cancer risk in a genome-wide analysis of 11,587 subjects. Thus, inherited genetic variation may affect lung carcinogenesis by regulating the human methylome.

Whole blood genomic biomarkers of acute cardiac allograft rejection.

BACKGROUND: Significant progress has been made in cardiac transplantation over the past 30 years; however, the means for detection of acute cardiac allograft rejection remains in need of improvement. At present, the endomyocardial biopsy, an invasive and inconvenient procedure for patients, is required for the surveillance and diagnosis of acute cardiac allograft rejection. In the Biomarkers in Transplantation initiative, we investigated gene expression profiles in peripheral blood of cardiac transplant subjects as potential biomarkers for diagnosis of allograft rejection. METHODS: Whole blood samples were obtained from 28 cardiac transplant subjects who consented to the study. Serial samples were collected from pre-transplant through 3 years post-transplant according to the standard protocol. Temporally correspondent biopsies were also collected, reviewed in a blinded manner, and graded according to current ISHLT guidelines. Blood samples were analyzed using Affymetrix microarrays. Genomic profiles were compared in subjects with acute rejection (AR; ISHLT Grade > or =2R) and no rejection (NR; Grade 0R). Biomarker panel genes were identified using linear discriminant analysis. RESULTS: We found 1,295 differentially expressed probe-sets between AR and NR samples and developed a 12-gene biomarker panel that classifies our internal validation samples with 83% sensitivity and 100% specificity. CONCLUSIONS: Based on our current results, we believe whole blood genomic biomarkers hold great potential in the diagnosis of acute cardiac allograft rejection. A prospective, Canada-wide trial will be conducted shortly to further evaluate the classifier panel in diverse patients and a range of clinical programs.