The pattern of somatic mutations observed at diagnosis of acute myeloid leukemia (AML) has been well-characterized. However, the premalignant mutational landscape of AML and its impact on risk and time to diagnosis is unknown. Here we identified 212 women from the Women's Health Initiative who were healthy at study baseline, but eventually developed AML during follow-up (median time: 9.6 years). Deep sequencing was performed on peripheral blood DNA of these cases and compared to age-matched controls that did not develop AML. We discovered that mutations in IDH1, IDH2, TP53, DNMT3A, TET2 and spliceosome genes significantly increased the odds of developing AML. All subjects with TP53 mutations (n = 21 out of 21 patients) and IDH1 and IDH2 (n = 15 out of 15 patients) mutations eventually developed AML in our study. The presence of detectable mutations years before diagnosis suggests that there is a period of latency that precedes AML during which early detection, monitoring and interventional studies should be considered.

Genetic heterogeneity contributes to clinical outcome and progression of most tumors. Yet, little is known regarding allelic diversity for epigenetic compartments and almost no data exists for acute myeloid leukemia (AML). Here we examined epigenetic heterogeneity as assessed by cytosine methylation within defined genomic loci with four CpGs (epigenetic alleles), somatic mutations and transcriptomes of AML patient samples at serial time points. We observe that epigenetic allele burden is linked to inferior outcome and varies considerably during disease progression. Epigenetic and genetic allelic burden and patterning follow different patterns and kinetics during disease progression. We observed a subset of AMLs with high epiallele and low somatic mutation burden at diagnosis, a subset with high somatic mutation and lower epiallele burdens at diagnosis, and a subset with a mixed profile, suggesting distinct modes of tumor heterogeneity. Genes linked to promoter-associated epiallele shifts during tumor progression display increased single-cell transcriptional variance and differential expression, suggesting functional impact on gene regulation. Thus, genetic and epigenetic heterogeneity can occur with distinct kinetics, each likely able to impact biological and clinical features of tumors.

Tumor microvasculature tends to be malformed, more permeable, and more tortuous than vessels in healthy tissue, effects that have been largely attributed to up-regulated VEGF expression. However, tumor tissue tends to stiffen during solid tumor progression, and tissue stiffness is known to alter cell behaviors including proliferation, migration, and cell-cell adhesion, which are all requisite for angiogenesis. Using in vitro, in vivo, and ex ovo models, we investigated the effects of matrix stiffness on vessel growth and integrity during angiogenesis. Our data indicate that angiogenic outgrowth, invasion, and neovessel branching increase with matrix cross-linking. These effects are caused by increased matrix stiffness independent of matrix density, because increased matrix density results in decreased angiogenesis. Notably, matrix stiffness up-regulates matrix metalloproteinase (MMP) activity, and inhibiting MMPs significantly reduces angiogenic outgrowth in stiffer crosslinked gels. To investigate the functional significance of altered endothelial cell behavior in response to matrix stiffness, we measured endothelial cell barrier function on substrates mimicking the stiffness of healthy and tumor tissue. Our data indicate that barrier function is impaired and the localization of vascular endothelial cadherin is altered as function of matrix stiffness. These results demonstrate that matrix stiffness, separately from matrix density, can alter vascular growth and integrity, mimicking the changes that exist in tumor vasculature. These data suggest that therapeutically targeting tumor stiffness or the endothelial cell response to tumor stiffening may help restore vessel structure, minimize metastasis, and aid in drug delivery.tumor stiffness | endothelial cells | vascular permeability | glycation | extracellular matrix T he ingrowth of newly sprouted blood vessels is necessary for solid tumor growth, and tumor vasculature is typically malformed, leakier, and more tortuous than the vasculature of normal tissues (1-3). Generally, aberrant tumor vasculature is considered to be caused by up-regulated VEGF expression resulting in chaotic vascular growth and failure to establish mature, well-regulated networks (4, 5). Here, we propose a different hypothesis, namely that extracellular matrix (ECM) mechanical properties also contribute to the aberrant vascular phenotype seen in tumors.Solid tumor tissue is typically stiffer than native, healthy tissue (1, 6). Increased ECM stiffness within tumors is caused primarily by both increased collagen deposition and increased cross-linking within the tumor stroma (7). Increased ECM density and crosslinking are associated with poor prognosis in a number of cancers (8, 9). Many studies have investigated the role of matrix density on angiogenesis and, in both collagen and fibrin matrices, have shown that angiogenesis decreases with increasing matrix concentration (10-13). Increased matrix density appears to act as a physical barrier that restricts cell migration, and cells rely on matr...

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Leukemia stem cells (LSCs) are thought to play a central role in the pathogenesis of acute leukemia and likely contribute to both disease initiation and relapse. Therefore, identification of agents that target LSCs is an important consideration for the development of new therapies. To this end, we have previously demonstrated that the naturally occurring compound parthenolide (PTL) can induce death of human LSCs in vitro while sparing normal hematopoietic cells. However, PTL has relatively poor pharmacologic properties that limit its potential clinical use.Consequently, we generated a family of PTL analogs designed to improve solubility and bioavailability. These studies identified an analog, dimethylaminoparthenolide (DMAPT), which induces rapid death of primary human LSCs from both myeloid and lymphoid leukemias, and is also highly cytotoxic to bulk leukemic cell populations. Molecular studies indicate the prevalent activities of DMAPT include induction of oxidative stress responses, inhibition of NF-B, and activation of p53. The compound has approximately 70% oral bioavailability, and pharmacologic studies using both mouse xenograft models and spontaneous acute canine leukemias demonstrate in vivo bioactivity as determined by functional assays and multiple biomarkers. Therefore, based on the collective preclinical data, we propose that the novel compound DMAPT has the potential to target human LSCs in vivo. IntroductionRecent studies have demonstrated that myeloid leukemia and certain forms of lymphoid leukemia arise from malignant stem cells (called leukemia stem cells [LSCs]). [1][2][3] LSCs are typically found in a quiescent state and are thus unlikely to respond to standard chemotherapeutic agents that preferentially eradicate actively cycling cells. [4][5][6][7] Indeed, the persistence of LSCs following chemotherapy may be a major factor contributing to clinical relapse. 8,9 In addition, conventional leukemia therapy is also substantially toxic to normal hematopoietic cells and frequently results in severe myelosuppression. Therefore, given the drugrefractory nature of LSCs, and the importance of normal hematopoiesis, identification of less toxic and more specific forms of therapy are important priorities for the development of better therapeutic regimens.As a foundation for developing more selective leukemia treatments, our previous experiments have investigated basic properties of primitive acute myelogenous leukemia (AML) cells. These studies showed that LSCs from different AML subtypes share characteristics 10 that are unique to AML and thus represent potential therapeutic targets for the selective ablation of LSCs relative to their normal counterparts. 11,12 Specifically, we reported that NF-B, a known regulator of growth and survival, is constitutively active in LSCs but not in normal hematopoietic stem cells (HSCs). 13 Notably, many traditional cancer therapies induce activation of NF-B, a potentially undesirable characteristic likely to facilitate survival of malignant cells. 14,15 Given the abi...