Circulating, cell-free methylated DNA indicates cellular sources of allograft injury after liver transplant.

Post-transplant complications reduce allograft and recipient survival. Current approaches for detecting allograft injury non-invasively are limited and do not differentiate between cellular mechanisms. Here, we monitor cellular damages after liver transplants from cell-free DNA (cfDNA) fragments released from dying cells into the circulation. We analyzed 130 blood samples collected from 44 patients at different time points after transplant. Sequence-based methylation of cfDNA fragments were mapped to patterns established to identify cell types in different organs. For liver cell types DNA methylation patterns and multi-omic data integration show distinct enrichment in open chromatin and regulatory regions functionally important for the respective cell types. We find that multi-tissue cellular damages post-transplant recover in patients without allograft injury during the first post-operative week. However, sustained elevation of hepatocyte and biliary epithelial cfDNA beyond the first week indicates early-onset allograft injury. Further, cfDNA composition differentiates amongst causes of allograft injury indicating the potential for non-invasive monitoring and timely intervention.

AIB1/SRC-3/NCOA3 function in estrogen receptor alpha positive breast cancer.

The estrogen receptor alpha (ERα) is a steroid receptor that is pivotal in the initiation and progression of most breast cancers. ERα regulates gene transcription through recruitment of essential coregulators, including the steroid receptor coactivator AIB1 (Amplified in Breast Cancer 1). AIB1 itself is an oncogene that is overexpressed in a subset of breast cancers and is known to play a role in tumor progression and resistance to endocrine therapy through multiple mechanisms. Here we review the normal and pathological functions of AIB1 in regard to its ERα-dependent and ERα-independent actions, as well as its genomic conservation and protein evolution. We also outline the efforts to target AIB1 in the treatment of breast cancer.

Loss of ANCO1 Expression Regulates Chromatin Accessibility and Drives Progression of Early-Stage Triple-Negative Breast Cancer.

Mutations in the gene ankyrin repeat domain containing 11 (ANKRD11/ANCO1) play a role in neurodegenerative disorders, and its loss of heterozygosity and low expression are seen in some cancers. Here, we show that low ANCO1 mRNA and protein expression levels are prognostic markers for poor clinical outcomes in breast cancer and that loss of nuclear ANCO1 protein expression predicts lower overall survival of patients with triple-negative breast cancer (TNBC). Knockdown of ANCO1 in early-stage TNBC cells led to aneuploidy, cellular senescence, and enhanced invasion in a 3D matrix. The presence of a subpopulation of ANCO1-depleted cells enabled invasion of the overall cell population in vitro and they converted more rapidly to invasive lesions in a xenograft mouse model. In ANCO1-depleted cells, ChIP-seq analysis showed a global increase in H3K27Ac signals that were enriched for AP-1, TEAD, STAT3, and NFκB motifs. ANCO1-regulated H3K27Ac peaks had a significantly higher overlap with known breast cancer enhancers compared to ANCO1-independent ones. H3K27Ac engagement was associated with transcriptional activation of genes in the PI3K-AKT, epithelial-mesenchymal transition (EMT), and senescence pathways. In conclusion, ANCO1 has hallmarks of a tumor suppressor whose loss of expression activates breast-cancer-specific enhancers and oncogenic pathways that can accelerate the early-stage progression of breast cancer.

Circulating cell-free methylated DNA reveals tissue-specific, cellular damage from radiation treatment.

Radiation therapy is an effective cancer treatment, although damage to healthy tissues is common. Here we analyzed cell-free, methylated DNA released from dying cells into the circulation to evaluate radiation-induced cellular damage in different tissues. To map the circulating DNA fragments to human and mouse tissues, we established sequencing-based, cell-type-specific reference DNA methylation atlases. We found that cell-type-specific DNA blocks were mostly hypomethylated and located within signature genes of cellular identity. Cell-free DNA fragments were captured from serum samples by hybridization to CpG-rich DNA panels and mapped to the DNA methylation atlases. In a mouse model, thoracic radiation-induced tissue damage was reflected by dose-dependent increases in lung endothelial and cardiomyocyte methylated DNA in serum. The analysis of serum samples from patients with breast cancer undergoing radiation treatment revealed distinct dose-dependent and tissue-specific epithelial and endothelial responses to radiation across multiple organs. Strikingly, patients treated for right-sided breast cancers also showed increased hepatocyte and liver endothelial DNA in the circulation, indicating the impact on liver tissues. Thus, changes in cell-free methylated DNA can uncover cell-type-specific effects of radiation and provide a readout of the biologically effective radiation dose received by healthy tissues.

Impaired CXCL12 signaling contributes to resistance of pancreatic cancer subpopulations to T cell-mediated cytotoxicity.

Pancreatic cancer remains largely unresponsive to immune modulatory therapy attributable in part to an immunosuppressive, desmoplastic tumor microenvironment. Here, we analyze mechanisms of cancer cell-autonomous resistance to T cells. We used a 3D co-culture model of cancer cell spheroids from the KPC (LSL-KrasG12D/+ /LSL-Trp53R172H/+ /p48-Cre) pancreatic ductal adenocarcinoma (PDAC) model, to examine interactions with tumor-educated T cells isolated from draining lymph nodes of PDAC-bearing mice. Subpopulations of cancer cells resistant to these tumor-educated T cells were isolated from the in vitro co-culture and their properties compared with sensitive cancer cells. In co-culture with resistant cancer cell subpopulations, tumor-educated T cells showed reduced effector T cell functionality, reduced infiltration into tumor cell spheroids and decreased induction of apoptosis. A combination of comparative transcriptomic analyses, cytometric and immunohistochemistry techniques allowed us to dissect the role of differential gene expression and signaling pathways between sensitive and resistant cells. A decreased expression of the chemokine CXCL12 (SDF-1) was revealed as a common feature in the resistant cell subpopulations. Adding back CXCL12 reversed the resistant phenotype and was inhibited by the CXCR4 inhibitor AMD3100 (plerixafor). We conclude that reduced CXCL12 signaling contributes to PDAC subpopulation resistance to T cell-mediated attack.

Detection of Cell Types Contributing to Cancer From Circulating, Cell-Free Methylated DNA.

Detection of cellular changes in tissue biopsies has been the basis for cancer diagnostics. However, tissue biopsies are invasive and limited by inaccuracies due to sampling locations, restricted sampling frequency, and poor representation of tissue heterogeneity. Liquid biopsies are emerging as a complementary approach to traditional tissue biopsies to detect dynamic changes in specific cell populations. Cell-free DNA (cfDNA) fragments released into the circulation from dying cells can be traced back to the tissues and cell types they originated from using DNA methylation, an epigenetic regulatory mechanism that is highly cell-type specific. Decoding changes in the cellular origins of cfDNA over time can reveal altered host tissue homeostasis due to local cancer invasion and metastatic spread to distant organs as well as treatment responses. In addition to host-derived cfDNA, changes in cancer cells can be detected from cell-free, circulating tumor DNA (ctDNA) by monitoring DNA mutations carried by cancer cells. Here, we will discuss computational approaches to identify and validate robust biomarkers of changed tissue homeostasis using cell-free, methylated DNA in the circulation. We highlight studies performing genome-wide profiling of cfDNA methylation and those that combine genetic and epigenetic markers to further identify cell-type specific signatures. Finally, we discuss opportunities and current limitations of these approaches for implementation in clinical oncology.

AKT1 E17K Inhibits Cancer Cell Migration by Abrogating ß-Catenin Signaling.

Mutational activation of the PI3K/AKT pathway is among the most common pro-oncogenic events in human cancers. The clinical utility of PI3K and AKT inhibitors has, however, been modest to date. Here, we used CRISPR-mediated gene editing to study the biological consequences of AKT1 E17K mutation by developing an AKT1 E17K-mutant isogenic system in a TP53-null background. AKT1 E17K expression under the control of its endogenous promoter enhanced cell growth and colony formation, but had a paradoxical inhibitory effect on cell migration and invasion. The mechanistic basis by which activated AKT1 inhibited cell migration and invasion was increased E-cadherin expression mediated by suppression of ZEB1 transcription via altered ß-catenin subcellular localization. This phenotypic effect was AKT1-specific, as AKT2 activation had the opposite effect, a reduction in E-cadherin expression. Consistent with the opposing effects of AKT1 and AKT2 activation on E-cadherin expression, a pro-migratory effect of AKT1 activation was not observed in breast cancer cells with PTEN loss or expression of an activating PIK3CA mutation, alterations which induce the activation of both AKT isoforms. The results suggest that the use of AKT inhibitors in patients with breast cancer could paradoxically accelerate metastatic progression in some genetic contexts and may explain the frequent coselection for CDH1 mutations in AKT1-mutated breast tumors. IMPLICATIONS: AKT1 E17K mutation in breast cancer impairs migration/invasiveness via sequestration of ß-catenin to the cell membrane leading to decreased ZEB1 transcription, resulting in increased E-cadherin expression and a reversal of epithelial-mesenchymal transition.

Leveraging Systematic Functional Analysis to Benchmark an In Silico Framework Distinguishes Driver from Passenger MEK Mutants in Cancer.

Despite significant advances in cancer precision medicine, a significant hurdle to its broader adoption remains the multitude of variants of unknown significance identified by clinical tumor sequencing and the lack of biologically validated methods to distinguish between functional and benign variants. Here we used functional data on MAP2K1 and MAP2K2 mutations generated in real-time within a co-clinical trial framework to benchmark the predictive value of a three-part in silico methodology. Our computational approach to variant classification incorporated hotspot analysis, three-dimensional molecular dynamics simulation, and sequence paralogy. In silico prediction accurately distinguished functional from benign MAP2K1 and MAP2K2 mutants, yet drug sensitivity varied widely among activating mutant alleles. These results suggest that multifaceted in silico modeling can inform patient accrual to MEK/ERK inhibitor clinical trials, but computational methods need to be paired with laboratory- and clinic-based efforts designed to unravel variabilities in drug response. SIGNIFICANCE: Leveraging prospective functional characterization of MEK1/2 mutants, it was found that hotspot analysis, molecular dynamics simulation, and sequence paralogy are complementary tools that can robustly prioritize variants for biologic, therapeutic, and clinical validation.See related commentary by Whitehead and Sebolt-Leopold, p. 4042.

Distinct vascular genomic response of proton and gamma radiation-A pilot investigation.

The cardiovascular biology of proton radiotherapy is not well understood. We aimed to compare the genomic dose-response to proton and gamma radiation of the mouse aorta to assess whether their vascular effects may diverge. We performed comparative RNA sequencing of the aorta following (4 hrs) total-body proton and gamma irradiation (0.5-200 cGy whole body dose, 10 dose levels) of conscious mice. A trend analysis identified genes that showed a dose response. While fewer genes were dose-responsive to proton than gamma radiation (29 vs. 194 genes; q-value ≤ 0.1), the magnitude of the effect was greater. Highly responsive genes were enriched for radiation response pathways (DNA damage, apoptosis, cellular stress and inflammation; p-value ≤ 0.01). Gamma, but not proton radiation induced additionally genes in vasculature specific pathways. Genes responsive to both radiation types showed almost perfectly superimposable dose-response relationships. Despite the activation of canonical radiation response pathways by both radiation types, we detected marked differences in the genomic response of the murine aorta. Models of cardiovascular risk based on photon radiation may not accurately predict the risk associated with proton radiation.

Activating mutation of PDGFRB gene in a rare cardiac undifferentiated intimal sarcoma of the left atrium: a case report.

Cardiac sarcoma is a rare malignant tumor with undefined genetic mutations and no targeted therapy. Here in one rare case of undifferentiated cardiac intimal sarcoma (IS), a next-generation sequencing based assay, MSK-IMPACT (Memorial Sloan Kettering - Integrated Mutation Profiling of Actionable Cancer Targets), identified a somatic, activating mutation in PDGFRB, along with amplification of PDGFRA. This E472D mutation of PDGFRB was discovered for the first time in IS. These findings suggest that concurrent aberrant PDGFRA and PDGFRB signaling may be a diagnostic biomarker and molecular therapeutic target of IS of the heart.