Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation.

Depleting the microenvironment of important nutrients such as arginine is a key strategy for immune evasion by cancer cells. Many tumors overexpress arginase, but it is unclear how these cancers, but not T cells, tolerate arginine depletion. In this study, we show that tumor cells synthesize arginine from citrulline by upregulating argininosuccinate synthetase 1 (ASS1). Under arginine starvation, ASS1 transcription is induced by ATF4 and CEBPß binding to an enhancer within ASS1. T cells cannot induce ASS1, despite the presence of active ATF4 and CEBPß, as the gene is repressed. Arginine starvation drives global chromatin compaction and repressive histone methylation, which disrupts ATF4/CEBPß binding and target gene transcription. We find that T cell activation is impaired in arginine-depleted conditions, with significant metabolic perturbation linked to incomplete chromatin remodeling and misregulation of key genes. Our results highlight a T cell behavior mediated by nutritional stress, exploited by cancer cells to enable pathological immune evasion.

H3K79me2/3 controls enhancer-promoter interactions and activation of the pan-cancer stem cell marker PROM1/CD133 in MLL-AF4 leukemia cells.

MLL gene rearrangements (MLLr) are a common cause of aggressive, incurable acute lymphoblastic leukemias (ALL) in infants and children, most of which originate in utero. The most common MLLr produces an MLL-AF4 fusion protein. MLL-AF4 promotes leukemogenesis by activating key target genes, mainly through recruitment of DOT1L and increased histone H3 lysine-79 methylation (H3K79me2/3). One key MLL-AF4 target gene is PROM1, which encodes CD133 (Prominin-1). CD133 is a pentaspan transmembrane glycoprotein that represents a potential pan-cancer target as it is found on multiple cancer stem cells. Here we demonstrate that aberrant PROM1/CD133 expression is essential for leukemic cell growth, mediated by direct binding of MLL-AF4. Activation is controlled by an intragenic H3K79me2/3 enhancer element (KEE) leading to increased enhancer-promoter interactions between PROM1 and the nearby gene TAPT1. This dual locus regulation is reflected in a strong correlation of expression in leukemia. We find that in PROM1/CD133 non-expressing cells, the PROM1 locus is repressed by polycomb repressive complex 2 (PRC2) binding, associated with reduced expression of TAPT1, partially due to loss of interactions with the PROM1 locus. Together, these results provide the first detailed analysis of PROM1/CD133 regulation that explains CD133 expression in MLLr ALL.

DOT1L inhibition reveals a distinct subset of enhancers dependent on H3K79 methylation.

Enhancer elements are a key regulatory feature of many important genes. Several general features including the presence of specific histone modifications are used to demarcate potentially active enhancers. Here we reveal that putative enhancers marked with H3 lysine 79 (H3K79) di or trimethylation (me2/3) (which we name H3K79me2/3 enhancer elements or KEEs) can be found in multiple cell types. Mixed lineage leukemia gene (MLL) rearrangements (MLL-r) such as MLL-AF4 are a major cause of incurable acute lymphoblastic leukemias (ALL). Using the DOT1L inhibitor EPZ-5676 in MLL-AF4 leukemia cells, we show that H3K79me2/3 is required for maintaining chromatin accessibility, histone acetylation and transcription factor binding specifically at KEEs but not non-KEE enhancers. We go on to show that H3K79me2/3 is essential for maintaining enhancer-promoter interactions at a subset of KEEs. Together, these data implicate H3K79me2/3 as having a functional role at a subset of active enhancers in MLL-AF4 leukemia cells.

Genetically distinct leukemic stem cells in human CD34- acute myeloid leukemia are arrested at a hemopoietic precursor-like stage.

Our understanding of the perturbation of normal cellular differentiation hierarchies to create tumor-propagating stem cell populations is incomplete. In human acute myeloid leukemia (AML), current models suggest transformation creates leukemic stem cell (LSC) populations arrested at a progenitor-like stage expressing cell surface CD34. We show that in ∼25% of AML, with a distinct genetic mutation pattern where >98% of cells are CD34(-), there are multiple, nonhierarchically arranged CD34(+) and CD34(-) LSC populations. Within CD34(-) and CD34(+) LSC-containing populations, LSC frequencies are similar; there are shared clonal structures and near-identical transcriptional signatures. CD34(-) LSCs have disordered global transcription profiles, but these profiles are enriched for transcriptional signatures of normal CD34(-) mature granulocyte-macrophage precursors, downstream of progenitors. But unlike mature precursors, LSCs express multiple normal stem cell transcriptional regulators previously implicated in LSC function. This suggests a new refined model of the relationship between LSCs and normal hemopoiesis in which the nature of genetic/epigenetic changes determines the disordered transcriptional program, resulting in LSC differentiation arrest at stages that are most like either progenitor or precursor stages of hemopoiesis.

MLL-AF9 Expression in Hematopoietic Stem Cells Drives a Highly Invasive AML Expressing EMT-Related Genes Linked to Poor Outcome.

To address the impact of cellular origin on acute myeloid leukemia (AML), we generated an inducible transgenic mouse model for MLL-AF9-driven leukemia. MLL-AF9 expression in long-term hematopoietic stem cells (LT-HSC) in vitro resulted in dispersed clonogenic growth and expression of genes involved in migration and invasion. In vivo, 20% LT-HSC-derived AML were particularly aggressive with extensive tissue infiltration, chemoresistance, and expressed genes related to epithelial-mesenchymal transition (EMT) in solid cancers. Knockdown of the EMT regulator ZEB1 significantly reduced leukemic blast invasion. By classifying mouse and human leukemias according to Evi1/EVI1 and Erg/ERG expression, reflecting aggressiveness and cell of origin, and performing comparative transcriptomics, we identified several EMT-related genes that were significantly associated with poor overall survival of AML patients.

Double Relapsed and/or Refractory Multiple Myeloma: Clinical Outcomes and Real World Healthcare Costs.

Double relapsed and/or refractory multiple myeloma (DRMM), MM that is relapsed and/or refractory to bortezomib and lenalidomide, carries a poor prognosis. The healthcare costs of DRMM have not previously been reported. We analyzed detailed medical resource utilization (MRU) costs, drug costs and outcomes for 39 UK patients receiving standard DRMM therapy. Median OS in this cohort was 5.6 months. The mean cost of DRMM treatment plus MRU until death was £23,472 [range: £1,411-£90,262], split between drug costs £11,191 and other resource use costs £12,281. The cost per assumed quality-adjusted life year (QALY) during DRMM was £66,983. These data provide a standard of care comparison when evaluating the cost-effectiveness of new drugs in DRMM.

Management and controversies of classical Hodgkin lymphoma in pregnancy.

The goal of managing classical Hodgkin lymphoma (cHL) in pregnancy is to obtain good long-term outcomes for both the mother and fetus. Given the excellent outcomes outside of pregnancy, the goal of treatment should remain curative. There remains a tension and debate regarding the timing of chemotherapy, the curative nature of such treatment and the timing of delivery. Moreover, the aim during pregnancy should be to minimize fetal toxicity and optimize perinatal outcomes. The management of cHL within pregnancy was covered within the excellent recent British Committee for Standards in Haematology guidelines, but with necessary brevity. By reviewing the literature over the last 30 years, herein we discuss the options for management during each trimester. Critical organogenesis occurs between 2 and 8 weeks post-conception; during which time the immature fetus is vulnerable to cytotoxic exposure. We discuss the evidence for using ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine) and single agent vinblastine in the first trimester. cHL presenting in pregnancy raises complex and difficult ethical dilemmas that can cause anxiety for patients, families and physicians. Decision-making must be multi-disciplinary and holistic, taking into account the patient's wishes, psycho-social and religious beliefs and personal circumstances. Clear communication between the haemato-oncologist, medical obstetrician, nurse specialists, midwives and neonatologists is paramount to a successful outcome.

Bendamustine in combination with thalidomide and dexamethasone is a viable salvage option in myeloma relapsed and/or refractory to bortezomib and lenalidomide.

Treatment options are limited in myeloma relapsed or refractory to both bortezomib and lenalidomide (double-relapsed/refractory multiple myeloma; DRMM). Bendamustine is an antitumour agent that has efficacy in relapsed myeloma. We retrospectively analysed data from 30 DRMM patients who received a combination of bendamustine, thalidomide and dexamethasone (BTD) in 28-day treatment cycles. Bendamustine was administered with a cumulative dose of up to 200 mg/m(2). Thalidomide (50-150 mg) was given daily as tolerated, and dexamethasone was given at an equivalent dose of up to 160 mg per cycle. A median of 5 (2-9) treatment cycles were administered per patient. Twenty-six patients (87 %) achieved stable disease or better. At a median follow-up time of 12.1 (2.3-21.5) months, median (95 % CI) progression-free survival and overall survival were 4.0 (2.6-5.3) months and 7.2 (5.2-9.2) months, respectively. The most common grade 3-4 adverse events were haematological: anaemia (n = 8, 34.8 %), neutropenia (n = 16, 69.6 %) and thrombocytopenia (n = 10, 43.5 %). Non-haematological toxicities included pain (n = 3, 13.0 %), infection (n = 7, 30.4 %) and sensory neuropathy (n = 1, 4.3 %). We propose that BTD is a viable salvage treatment option for DRMM patients.

Local cell interactions and self-amplifying individual cell ingression drive amniote gastrulation.

Gastrulation generates three layers of cells (ectoderm, mesoderm, endoderm) from a single sheet, while large scale cell movements occur across the entire embryo. In amniote (reptiles, birds, mammals) embryos, the deep layers arise by epithelial-to-mesenchymal transition (EMT) at a morphologically stable midline structure, the primitive streak (PS). We know very little about how these events are controlled or how the PS is maintained despite its continuously changing cellular composition. Using the chick, we show that isolated EMT events and ingression of individual cells start well before gastrulation. A Nodal-dependent 'community effect' then concentrates and amplifies EMT by positive feedback to form the PS as a zone of massive cell ingression. Computer simulations show that a combination of local cell interactions (EMT and cell intercalation) is sufficient to explain PS formation and the associated complex movements globally across a large epithelial sheet, without the need to invoke long-range signalling.DOI: http://dx.doi.org/10.7554/eLife.01817.001.