Arid1a-dependent canonical BAF complex suppresses inflammatory programs to drive efficient germinal center B cell responses.

The mammalian Brg1/Brm-associated factor (BAF) complexes are major regulators of nucleosomal remodeling that are commonly mutated in several cancers, including germinal center (GC)-derived B cell lymphomas. However, the specific roles of different BAF complexes in GC B cell biology are not well understood. Here we show that the AT-rich interaction domain 1a (Arid1a) containing canonical BAF (cBAF) complex is required for maintenance of GCs and high-affinity antibody responses. While Arid1a-deficient B cells undergo initial activation, they fail to sustain the GC program. Arid1a establishes permissive chromatin landscapes for B cell activation and is concomitantly required to suppress inflammatory gene programs. The inflammatory signatures instigated by Arid1a deficiency promoted the recruitment of neutrophils and inflammatory monocytes. Dampening of inflammatory cues through interleukin-1ß blockade or glucocorticoid receptor agonist partially rescued Arid1a-deficient GCs, highlighting a critical role for inflammation in impeding GCs. Our work reveals essential functions of Arid1a-dependent cBAF in promoting efficient GC responses.

Alternative DNA structures in hematopoiesis and adaptive immunity.

Besides the canonical B-form, DNA also adopts alternative non-B form conformations which are highly conserved in all domains of life. While extensive research over decades has centered on the genomic functions of B-form DNA, understanding how non-B-form conformations influence functional genomic states remains a fundamental and open question. Recent studies have ascribed alternative DNA conformations such as G-quadruplexes and R-loops as important functional features in eukaryotic genomes. This review delves into the biological importance of alternative DNA structures, with a specific focus on hematopoiesis and adaptive immunity. We discuss the emerging roles of G-quadruplex and R-loop structures, the two most well-studied alternative DNA conformations, in the hematopoietic compartment and present evidence for their functional roles in normal cellular physiology and associated pathologies.

Arid1a-dependent canonical BAF complex suppresses inflammatory programs to drive efficient Germinal Center B cell responses.

Differentiating B cells in germinal centers (GC) require tightly coordinated transcriptional and epigenetic transitions to generate efficient humoral immune responses. The mammalian Brg1/Brm-associated factor (BAF) complexes are major regulators of nucleosomal remodeling, crucial for cellular differentiation and development, and are commonly mutated in several cancers, including GC-derived B cell lymphomas. However, the specific roles of distinct BAF complexes in GC B cell biology and generation of functional humoral immune responses are not well understood. Here, we show that the A-T Rich Interaction Domain 1a (Arid1a) containing canonical BAF (cBAF) complex is required for maintenance of GCs and therefore high affinity antibody responses. While Arid1a-deficient B cells undergo activation to initiate GC responses, they fail to sustain the GC program resulting in premature GC collapse. We discovered that Arid1a-dependent cBAF activity establishes permissive chromatin landscapes during B cell activation and is concomitantly required to suppress inflammatory gene programs to maintain transcriptional fidelity in early GC B cells. Interestingly, the inflammatory signatures instigated by Arid1a deficiency in early GC B cells recruited neutrophils and inflammatory monocytes and eventually disrupted GC homeostasis. Dampening of inflammatory cues with anti-inflammatory glucocorticoid receptor signaling rescued GC B cell differentiation of Arid1a-deficient B cells, thus highlighting a critical role of inflammation in impeding GC responses. In sum, our work identifies essential functions of Arid1a-dependent BAF activity in promoting efficient GC responses. These findings further support an emerging paradigm in which unrestrained inflammation limits GC-derived humoral responses, as reported in the context of severe bacterial and viral infections.

An Unusual Antenatal Presentation of a Mature Pericardial Teratoma Masquerading as Congenital Pulmonary Airway Malformation.

We report an antenatal presentation of a huge pericardial mature teratoma that was referred as congenital pulmonary airway malformation (CPAM) in the late third trimester of pregnancy. Initial ultrasound evaluation revealed a huge predominantly cystic lesion with mixed echogenicity in the left hemithorax. A provisional diagnosis of pleural tumor was considered in view of previous scans at 20‒28 weeks being normal and associated pleural effusion. Magnetic resonance imaging of the fetus reported the lesion to be CPAM which was supported by postnatal computed tomographic imaging done on day 2 of life. However, intraoperatively, the lesion was found to be of pericardial origin which on subsequent histopathological examination was confirmed to be mature teratoma. We recommend considering potential differential diagnosis other than CPAM, especially when the lesion is found for the first time in the late third trimester.

Toward a systems-level probing of tumor clonality.

Cancer has been described as a genetic disease that clonally evolves in the face of selective pressures imposed by cell-intrinsic and extrinsic factors. Although classical models based on genetic data predominantly propose Darwinian mechanisms of cancer evolution, recent single-cell profiling of cancers has described unprecedented heterogeneity in tumors providing support for alternative models of branched and neutral evolution through both genetic and non-genetic mechanisms. Emerging evidence points to a complex interplay between genetic, non-genetic, and extrinsic environmental factors in shaping the evolution of tumors. In this perspective, we briefly discuss the role of cell-intrinsic and extrinsic factors that shape clonal behaviors during tumor progression, metastasis, and drug resistance. Taking examples of pre-malignant states associated with hematological malignancies and esophageal cancer, we discuss recent paradigms of tumor evolution and prospective approaches to further enhance our understanding of this spatiotemporally regulated process.

Low c-Kit expression identifies primitive, therapy-resistant CML stem cells.

Despite the efficacy of tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML), malignant long-term hematopoietic stem cells (LT-HSCs) persist as a source of relapse. However, LT-HSCs are heterogenous and the most primitive, drug-resistant LT-HSC subpopulations are not well characterized. In normal hematopoiesis, self-renewal and long-term reconstitution capacity are enriched within LT-HSCs with low c-Kit expression (c-KITlo). Here, using a transgenic CML mouse model, we found that long-term engraftment and leukemogenic capacity were restricted to c-KITlo CML LT-HSCs. CML LT-HSCs demonstrated enhanced differentiation with expansion of mature progeny following exposure to the c-KIT ligand, stem cell factor (SCF). Conversely, SCF deletion led to depletion of normal LT-HSCs but increase in c-KITlo and total CML LT-HSCs with reduced generation of mature myeloid cells. CML c-KITlo LT-HSCs showed reduced cell cycling and expressed enhanced quiescence and inflammatory gene signatures. SCF administration led to enhanced depletion of CML primitive progenitors but not LT-HSCs after TKI treatment. Human CML LT-HSCs with low or absent c-KIT expression were markedly enriched after TKI treatment. We conclude that CML LT-HSCs expressing low c-KIT levels are enriched for primitive, quiescent, drug-resistant leukemia-initiating cells and represent a critical target for eliminating disease persistence.

Hedgehog Signaling in Myeloid Malignancies.

Myeloid malignancies arise from normal hematopoiesis and include several individual disorders with a wide range of clinical manifestations, treatment options, and clinical outcomes. The Hedgehog (HH) signaling pathway is aberrantly activated in many of these diseases, and glasdegib, a Smoothened (SMO) antagonist and HH pathway inhibitor, has recently been approved for the treatment of acute myeloid leukemia (AML). The efficacy of SMO inhibitors in AML suggests that they may be broadly active, but clinical studies in other myeloid malignancies have been largely inconclusive. We will discuss the biological role of the HH pathway in normal hematopoiesis and myeloid malignancies and review clinical studies targeting HH signaling in these diseases. In addition, we will examine SMO-independent pathway activation and highlight potential strategies that may expand the clinical utility of HH pathway antagonists.

Dnmt3b catalytic activity is critical for its tumour suppressor function in lymphomagenesis and is associated with c-Met oncogenic signalling.

BACKGROUND: DNA methylation regulates gene transcription in many physiological processes in mammals including development and haematopoiesis. It is catalysed by several DNA methyltransferases, including Dnmt3b that mediates both methylation-dependant and independent gene repression. Dnmt3b is critical for mouse embryogenesis and functions as a tumour suppressor in haematologic malignancies in mice. However, the extent to which Dnmt3b's catalytic activity (CA) is involved in development and cancer is unclear. METHODS: We used a mouse model expressing catalytically inactive Dnmt3b (Dnmt3bCI) to study a role of Dnmt3b's CA in development and cancer. We utilized global approaches including Whole-genome Bisulfite sequencing and RNA-seq to analyse DNA methylation and gene expression to identify putative targets of Dnmt3b's CA. To analyse postnatal development and haematopoiesis, we used tissue staining, histological and FACS analysis. To determine potential involvement of selected genes in lymphomagenesis, we used overexpression and knock down approaches followed by in vitro growth assays. FINDINGS: We show that mice expressing Dnmt3bCI only, survive postnatal development and develop ICF (the immunodeficiency-centromeric instability-facial anomalies) -like syndrome. The lack of Dnmt3b's CA promoted fibroblasts transformation in vitro, accelerated MLL-AF9 driven Acute Myeloid Leukaemia and MYC-induced T-cell lymphomagenesis in vivo. The elimination of Dnmt3b's CA resulted in decreased methylation of c-Met promoter and its upregulation, activated oncogenic Met signalling, Stat3 phosphorylation and up-regulation of Lin28b promoting lymphomagenesis. INTERPRETATION: Our data demonstrates that Dnmt3b's CA is largely dispensable for mouse development but critical to prevent tumourigenesis by controlling events involved in cellular transformation. FUNDING: This study was supported by Department of Anatomy and Cell Biology and Cancer Centre at the University of Florida start-up funds, NIH/NCI grant 1R01CA188561-01A1 (R.O.).

Decreases in different Dnmt3b activities drive distinct development of hematologic malignancies in mice.

DNA methylation regulates gene transcription and is involved in various physiological processes in mammals, including development and hematopoiesis. It is catalyzed by DNA methyltransferases including Dnmt1, Dnmt3a, and Dnmt3b. For Dnmt3b, its effects on transcription can result from its own DNA methylase activity, the recruitment of other Dnmts to mediate methylation, or transcription repression in a methylation-independent manner. Low-frequency mutations in human DNMT3B are found in hematologic malignancies including cutaneous T-cell lymphomas, hairy cell leukemia, and diffuse large B-cell lymphomas. Moreover, Dnmt3b is a tumor suppressor in oncogene-driven lymphoid and myeloid malignancies in mice. However, it is poorly understood how the different Dnmt3b activities contribute to these outcomes. We modulated Dnmt3b activity in vivo by generating Dnmt3b+/- mice expressing one wild-type allele as well as Dnmt3b+/CI and Dnmt3bCI/CI mice where one or both alleles express catalytically inactive Dnmt3bCI. We show that 43% of Dnmt3b+/- mice developed T-cell lymphomas, chronic lymphocytic leukemia, and myeloproliferation over 18 months, thus resembling phenotypes previously observed in Dnmt3a+/- mice, possibly through regulation of shared target genes. Interestingly, Dnmt3b+/CI and Dnmt3bCI/CI mice survived postnatal development and were affected by B-cell rather than T-cell malignancies with decreased penetrance. Genome-wide hypomethylation, increased expression of oncogenes such as Jdp2, STAT1, and Trip13, and p53 downregulation were major events contributing to Dnmt3b+/- lymphoma development. We conclude that Dnmt3b catalytic activity is critical to prevent B-cell transformation in vivo, whereas accessory and methylation-independent repressive functions are important to prevent T-cell transformation.

Catalytically inactive Dnmt3b rescues mouse embryonic development by accessory and repressive functions.

DNA methylation regulates gene expression in a variety of processes, including mouse embryonic development. Four catalytically active enzymes function in mice as DNA methyltransferases (Dnmts) and as transcriptional regulators. Inactivation of Dnmt3b results in mouse embryonic lethality, but which activities are involved is unclear. Here we show that catalytically inactive Dnmt3b restores a majority of methylation and expression changes deregulated in the absence of Dnmt3b, and as a result, mice survive embryonic development. Thus, Dnmt3b functions as an accessory cofactor supporting catalytic activities performed by other Dnmts. We further demonstrate that Dnmt3b is linked to a control of major developmental pathways, including Wnt and hedgehog signaling. Dnmt3b directly represses Wnt9b whose aberrant up-regulation contributes to embryonic lethality of Dnmt3b knockout embryos. Our results highlight that Dnmt3b is a multifaceted protein that serves as an enzyme, an accessory factor for other methyltransferases, and as a transcriptional repressor in mouse embryogenesis.

SIRT1 regulates metabolism and leukemogenic potential in CML stem cells.

Chronic myeloid leukemia (CML) results from hematopoietic stem cell transformation by the BCR-ABL kinase. Despite the success of BCR-ABL tyrosine kinase inhibitors (TKIs) in treating CML patients, leukemia stem cells (LSCs) resist elimination and persist as a major barrier to cure. Previous studies suggest that overexpression of the sirtuin 1 (SIRT1) deacetylase may contribute to LSC maintenance in CML. Here, by genetically deleting SIRT1 in transgenic CML mice, we definitively demonstrated an important role for SIRT1 in leukemia development. We identified a previously unrecognized role for SIRT1 in mediating increased mitochondrial oxidative phosphorylation in CML LSCs. We showed that mitochondrial alterations were kinase independent and that TKI treatment enhanced inhibition of CML hematopoiesis in SIRT1-deleted mice. We further showed that the SIRT1 substrate PGC-1α contributed to increased oxidative phosphorylation and TKI resistance in CML LSCs. These results reveal an important role for SIRT1 and downstream signaling mechanisms in altered mitochondrial respiration in CML LSCs.

Role of NF-E2 related factor 2 (Nrf2) on chemotherapy resistance in acute myeloid leukemia (AML) and the effect of pharmacological inhibition of Nrf2.

Cytarabine (Ara-C) and Daunorubicin (Dnr) forms the backbone of acute myeloid leukemia (AML) therapy. Drug resistance and toxic side effects pose a major threat to treatment success and hence alternate less toxic therapies are warranted. NF-E2 related factor-2 (Nrf2), a master regulator of antioxidant response is implicated in chemoresistance in solid tumors. However, little is known about the role of Nrf2 in AML chemoresistance and the effect of pharmacological inhibitor brusatol in modulating this resistance. Primary AML samples with high ex-vivo IC50 to Ara-C, ATO, Dnr had significantly high NRF2 RNA expression. Gene-specific knockdown of NRF2 improved sensitivity to these drugs in resistant AML cell lines by decreasing the expression of downstream antioxidant targets of Nrf2 by compromising the cell's ability to scavenge the ROS. Treatment with brusatol, a pharmacological inhibitor of Nrf2, improved sensitivity to Ara-C, ATO, and Dnr and reduced colony formation capacity. AML cell lines stably overexpressing NRF2 showed increased resistance to ATO, Dnr and Ara-C and increased expression of downstream targets. This study demonstrates that Nrf2 could be an ideal druggable target in AML, more so to the drugs that function through ROS, suggesting the possibility of using Nrf2 inhibitors in combination with chemotherapeutic agents to modulate drug resistance in AML.

ATP-binding casette transporter expression in acute myeloid leukemia: association with in vitro cytotoxicity and prognostic markers.

INTRODUCTION: Drug resistance and relapse are considered to be the major reasons for treatment failure in acute myeloid leukemia (AML). There is limited data on the role of ABC transporter expression on in vitro sensitivity to cytarabine (Ara-C) and daunorubicin (Dnr) in primary AML cells. PATIENTS & METHODS: RNA expression levels of 12 ABC transporters were analyzed by real-time quantitative PCR in 233 de novo adult acute myeloid leukemia patients. Based on cytarabine or Dnr IC50, the samples were categorized as sensitive, intermediate and resistant. Role of candidate ABC transporter RNA expression on in vitro cytotoxicity, treatment outcome post therapy as well as the influence of various prognostic markers on ABC transporter expression were analyzed. RESULTS: Expression of ABCC3 and ABCB6 were significantly higher in Dnr-resistant samples when compared with Dnr-sensitive samples. Increased ABCC1 expression was associated with poor disease-free survival in this cohort of patients. CONCLUSION: This comprehensive analysis suggests ABCC1, ABCC3, ABCB6 and ABCA5 as probable targets which can be modulated for improving chemotherapeutic responses.

Population pharmacokinetics of Daunorubicin in adult patients with acute myeloid leukemia.

PURPOSE: Chemotherapy drug resistance and relapse of the disease have been the major factors limiting the success of acute myeloid leukemia (AML) therapy. Several factors, including the pharmacokinetics (PK) of Cytarabine (Ara-C) and Daunorubicin (Dnr), could contribute to difference in treatment outcome in AML. METHODS: In the present study, we evaluated the plasma PK of Dnr, the influence of genetic polymorphisms of genes involved in transport and metabolism of Dnr on the PK, and also the influence of these factors on clinical outcome. Plasma levels of Dnr and its major metabolite, Daunorubicinol (DOL), were available in 70 adult de novo AML patients. PK parameters (Area under curve (AUC) and clearance (CL)) of Dnr and DOL were calculated using nonlinear mixed-effects modeling analysis performed with Monolix. Genetic variants in ABCB1, ABCG2, CBR1, and CBR3 genes as well as RNA expression of CBR1, ABCB1, and ABCG2 were compared with Dnr PK parameters. RESULTS: The AUC and CL of Dnr and DOL showed wide inter-individual variation. Patients with an exon1 variant of rs25678 in CBR1 had significantly higher plasma Dnr AUC [p = 0.05] compared to patients with wild type. Patients who achieved complete remission (CR) had significantly lower plasma Dnr AUC, Cmax, and higher CL compared to patients who did not achieve CR. CONCLUSION: Further validation of these findings in a larger cohort of AML patients is warranted before establishing a therapeutic window for plasma Dnr levels and targeted dose adjustment.

RNA expression of genes involved in cytarabine metabolism and transport predicts cytarabine response in acute myeloid leukemia.

BACKGROUND: Variation in terms of outcome and toxic side effects of treatment exists among acute myeloid leukemia (AML) patients on chemotherapy with cytarabine (Ara-C) and daunorubicin (Dnr). Candidate Ara-C metabolizing gene expression in primary AML cells is proposed to account for this variation. METHODS: Ex vivo Ara-C sensitivity was determined in primary AML samples using MTT assay. mRNA expression of candidate Ara-C metabolizing genes were evaluated by RQPCR analysis. Global gene expression profiling was carried out for identifying differentially expressed genes between exvivo Ara-C sensitive and resistant samples. RESULTS: Wide interindividual variations in ex vivo Ara-C cytotoxicity were observed among samples from patients with AML and were stratified into sensitive, intermediately sensitive and resistant, based on IC50 values obtained by MTT assay. RNA expression of deoxycytidine kinase (DCK), human equilibrative nucleoside transporter-1 (ENT1) and ribonucleotide reductase M1 (RRM1) were significantly higher and cytidine deaminase (CDA) was significantly lower in ex vivo Ara-C sensitive samples. Higher DCK and RRM1 expression in AML patient's blast correlated with better DFS. Ara-C resistance index (RI), a mathematically derived quotient was proposed based on candidate gene expression pattern. Ara-C ex vivo sensitive samples were found to have significantly lower RI compared with resistant as well as samples from patients presenting with relapse. Patients with low RI supposedly highly sensitive to Ara-C were found to have higher incidence of induction death (p = 0.002; RR: 4.35 [95% CI: 1.69-11.22]). Global gene expression profiling undertaken to find out additional contributors of Ara-C resistance identified many apoptosis as well as metabolic pathway genes to be differentially expressed between Ara-C resistant and sensitive samples. CONCLUSION: This study highlights the importance of evaluating expression of candidate Ara-C metabolizing genes in predicting ex vivo drug response as well as treatment outcome. RI could be a predictor of ex vivo Ara-C response irrespective of cytogenetic and molecular risk groups and a potential biomarker for AML treatment outcome and toxicity. Original submitted 22 December 2014; Revision submitted 9 April 2015.

Population prevalence of ultrasound features of osteoarthritis in the hand, knee and hip at age 63 years: the Newcastle thousand families birth cohort.

BACKGROUND: Musculoskeletal ultrasound has been found to be more sensitive than radiographs in detecting osteophytes. Our objective was to measure the prevalence of features of osteoarthritis (OA), in the dominant hand, knees and hips using ultrasound, within the Newcastle Thousand Families birth cohort. METHODS: Participants were aged 61-63 (mean 63) years. Knee images were scored for presence of osteophytes and effusion. Hip images were scored for the presence of osteophytes and femoral head abnormality. The first carpometacarpal joint, metacarpophalangeal, proximal interphalangeal and distal interphalangeal joints of the index finger (dominant hand) were imaged for osteophytes. RESULTS: Among 311 participants, prevalence of osteophytes at the distal interphalangeal joint was 70% while it was 23%, 10% and 41% for index proximal interphalangeal and metacarpophalangeal and thumb base carpometacarpal joints respectively. Prevalence of knee osteophytes was 30%, hip OA was 41%. Prevalence of knee effusions was 24% (right) and 20% (left). Ultrasound evidence of generalised OA (48%) and isolated hand OA (31%) was common, compared to isolated hip or knee OA (5%) and both hip and knee OA (3%). CONCLUSION: This is the first study to assess prevalence of ultrasound features of OA in a population-based sample. The higher prevalence of hand/hip OA, when compared to previous radiographic studies, supports the hypothesis that ultrasound is more sensitive than radiography in detecting OA, particularly for osteophytes.

Carbonyl reductase 1 expression influences daunorubicin metabolism in acute myeloid leukemia.

PURPOSE: The present study aimed to investigate the role of expression of daunorubicin-metabolizing enzymes carbonyl reductase 1 and 3 (CBR1 and CBR3) on the in vitro cytotoxicity of daunorubicin in primary acute myeloid leukemia (AML) cells and the effect of genetic variants in CBR1 and CBR3 on the plasma pharmacokinetics of daunorubicin and daunorubicinol (DOL) in AML patients. METHODS: RNA expression of CBR1 and CBR3, intracellular daunorubicin and DOL levels, and in vitro cytotoxicity of daunorubicin were measured in bone marrow mononuclear cells of 104 adult AML patients. Plasma pharmacokinetics of daunorubicin and DOL was measured in 24 patients receiving daunorubicin-based induction chemotherapy for AML. RESULTS: Increased expression of CBR1 significantly reduced the in vitro cytotoxicity of daunorubicin and also positively correlated with intracellular DOL levels. Polymorphisms in CBR1 and CBR3 did not show any association with intracellular daunorubicin or DOL levels, but there was a trend towards significant increase in plasma daunorubicin systemic exposure in patients with a variant genotype for CBR1 polymorphism rs25678. CONCLUSIONS: This pilot study suggests that CBR1 RNA expression may be helpful in identifying AML patients at risk of developing resistance or toxicity to daunorubicin due to increased formation of DOL. Further confirmation of these findings in a larger sample pool would be required to determine the applicability of these results. Inhibition of CBR1 can be an option to improve the efficacy and prevent toxicity related to the treatment. Influence of daunorubicin and DOL plasma levels on clinical outcome, if any, remains to be evaluated.