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PML is a stress-responsive protein that coordinates assembly of phase-separated nuclear aggregates, known as PML nuclear bodies (PML-NBs), where a large number of protein interactors and chromatin processes are finely regulated. Tampering with the PML gene produces a variety of phenotypic consequences that include promoting or interfering with tumor progression but the molecular underpinnings of PML pleiotropy are still elusive. In this review, we explore the contribution of PML splicing isoforms to PML-NB assorted activities. We describe recent literature indicating that distinct PML isoforms drive formation of specialized PML-NBs and perform unique functions and we suggest that future research efforts should delve into the contribution of isoform specificity to help elucidate the complex functionality of the PML gene.

Supplementary Figure Legends

Supplementary Figure S1. Candidate genes that couple with p53 loss to transform MSCs. Supplementary Figure S2. Lrf plays as oncosuppressor triggering tumorigenesis in MSCs. Supplementary Figure S3. Lrf expression does not affect the CFU-F capacities of MSCs nor it triggers MSCs to senescence or apoptosis. Supplementary Figure S4. Lrf regulates the differentiation process of MSCs through the repression of Dlk1. Supplementary Figure S5. Lrf plays a role as oncosuppressor in triggering the tumorigenesis of MSCs through Sox9 and Dlk1.

The regulatory factors governing adult mesenchymal stem cell (MSC) physiology and their tumorigenic potential are still largely unknown, which substantially delays the identification of effective therapeutic approaches for the treatment of aggressive and lethal forms of MSC-derived mesenchymal tumors, such as undifferentiated sarcomas. Here, we have developed a novel platform to screen and quickly identify genes and pathways responsible for adult MSC transformation, modeled undifferentiated sarcoma in vivo, and, ultimately, tested the efficacy of targeting the identified oncopathways. Importantly, by taking advantage of this new platform, we demonstrate the key role of an aberrant LRF–DLK1–SOX9 pathway in the pathogenesis of undifferentiated sarcoma, with important therapeutic implications.Significance: The paucity of therapeutic options for the treatment of sarcoma calls for a rapid and effective preclinical assessment of new therapeutic modalities. We have here developed a new platform to deconstruct the molecular genetics underlying the pathogenesis of sarcoma and to evaluatein vivo the efficacy of novel targeted therapies. Cancer Discov; 5(4); 396–409. ©2015 AACR.This article is highlighted in the In This Issue feature, p. 333

Supplementary Figure 3: EZN-2208 affects leukemic stromal cell numbers.

Supplementary Figure 4: Combined effect of EZN-2208 and ATRA on NB4 cell viability and differentiation.

Supplementary Figure 1: EZN-2208 does not impair cell migration and colony formation in U937-MT control cells

Supplementary Figure 2: High doses EZN-2208 induce apoptosis in leukemic bone marrow cells.

Purpose: Retinoic acid-arsenic trioxide (ATRA-ATO) combination therapy is the current standard of care for patients with acute promyelocytic leukemia (APL) carrying the oncogenic fusion protein PML-RARα. Despite the high cure rates obtained with this drug combination, resistance to arsenic is recently emerging. Moreover, patients with APL carrying the PLZF-RARα fusion protein are partially resistant to ATRA treatment. Hypoxia-inducible factor-1α (HIF-1α) activation has been recently reported in APL, and EZN-2208 (PEG-SN38) is a compound with HIF-1α inhibitory function currently tested in clinical trials. This study investigates the effect of EZN-2208 in different preclinical APL models, either alone or in combination with ATRA.Experimental Design: Efficacy of EZN-2208 in APL was measured in vitro by assessing expression of HIF-1α target genes, cell migration, clonogenicity, and differentiation, vis a vis the cytotoxic and cytostatic effects of this compound. In vivo, EZN-2208 was used in mouse models of APL driven by PML-RARα or PLZF-RARα, either alone or in combination with ATRA.Results: Treatment of APL cell lines with noncytotoxic doses of EZN-2208 causes dose-dependent downregulation of HIF-1α bona fide target genes and affects cell migration and clonogenicity in methylcellulose. In vivo, EZN-2208 impairs leukemia progression and prolongs mice survival in APL mouse models. More importantly, when used in combination with ATRA, EZN-2208 synergizes in debulking leukemia and eradicating leukemia-initiating cells.Conclusions: Our preclinical data suggest that the combination ATRA-EZN-2208 may be tested to treat patients with APL who develop resistance to ATO or patients carrying the PLZF-RARα fusion protein. Clin Cancer Res; 21(16); 3685–94. ©2015 AACR.

Differentiation arrest along the myeloid lineage is a defining feature of acute myeloid leukemia (AML), but its molecular determinants remain poorly defined. Pharmacological removal of the differentiation block leads to leukemia eradication in acute promyelocytic leukemia (APL) patients but has not yet been successfully translated to non-APL AMLs. Here, by investigating the function of hypoxia-inducible transcription factors HIF1α and HIF2α, we identify HIF2α as a new regulator of the AML differentiation block. Mechanistically, HIF2α cooperates with EZH2, the catalytic subunit of polycomb repressive complex 2, to promote deposition of the repressive H3K27me3 histone mark at the regulatory regions of myeloid differentiation genes. Thus, inhibiting HIF2α releases an epigenetic break to leukemic blasts maturation and cooperates with the pro-differentiation agent all-trans retinoic acid to trigger AML differentiation. We propose that HIF2α inhibition may open new therapeutic avenues for AML treatment by licensing blasts maturation and forcing leukemia debulking.

The zinc finger E-box binding homeobox 1 (ZEB1) transcription factor is a master regulator of the epithelial to mesenchymal transition (EMT). In solid tumors, its overexpression induces EMT, which activates cellular motility and is also associated with the maintenance of stem cell properties, driving tumor progression and metastasis. A double-negative feed-forward loop circuit has been described where, to promote EMT, ZEB1 suppresses the expression of miR-200 family members, which in turn inhibits its activity. Albeit recent evidence highlighted the existence of EMT processes also in hematological malignancies, the role of ZEB1 is not completely defined yet. Hence, this study aims to characterize ZEB1 behavior in acute myeloid leukemia (AML). To this purpose, we retrieved and analyzed RNA-Seq data from public repositories to evaluate the expression levels of the main EMT master regulator genes in normal and malignant hematopoietic cells. We observed a heterogeneous expression of these genes among AML morphological subtypes (from M0 to M5) defined by the French-American-British (FAB) classification system. Specifically, TWIST1 has the highest expression value in M3 and ZEB2 in M5, as already observed in published studies, while ZEB1 has the highest expression in M0, which significantly decreases until M5. To evaluate the ZEB1 transcriptional program, we performed a gene set enrichment analysis of hallmark signatures based on gene-wise correlation with ZEB1 among FABs that revealed the up-regulation of EMT in the M0 subtype. Moreover, the highest value of correlation was found between ZEB1 and its antisense lncRNA (ZEB1-AS1). This trait emerged as peculiar to AML both with respect to other cancer types and normal hematopoietic cells. Since ZEB1 orchestrates its transcriptional program by inhibiting the activity of specific miRNAs, we selected putative candidates that may take part in this circuit in AML by calculating their bimodality distribution, which reflects the toggle switch behavior typical of the double-negative feed-forward loop. We identified miR-425-5p, which was coherently down-regulated in M0 with respect to other FABs, and miR-151-3p which was surprisingly up-regulated. Specifically, the simultaneous up-regulation in the M0 subtype of miR-151-3p and its target ZEB1 is inconsistent with the toggle switch behavior of the circuit. Thus, we hypothesized that miR-151-3p is not able to inhibit ZEB1 expression in M0 due to the presence of ZEB1-AS1, which is supposed to disrupt the circuit mechanism protecting ZEB1 from miRNA-mediated inhibition. This hypothesis is also supported by the fact that all target genes of miR-151-3p were significantly up-regulated. A better comprehension of this regulatory circuit could allow identifying aberrant epigenetic mechanisms during leukemic development and discriminating of the different AML subtypes. Citation Format: Stefano Percio, Daniela Magliulo, Silvia Martini, Sandro Pasquali, Rosa Bernardi. ZEB1-AS1 lncRNA prevents ZEB1 suppression and leads to tumor progression in undifferentiated AML subtype. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6552.

Acute myeloid leukemia (AML) is characterized by epigenetic silencing of differentiation genes and accumulation of aberrant myeloid cells arrested at different stages of myeloid development. The combination of epigenetic and differentiation therapies represents an attractive opportunity for AML patients by promoting chromatin remodeling at differentiation genes and induction of terminal maturation and leukemia debulking. The aim of our work is to define the function of the hypoxia inducible transcription factor HIF2alpha in the pathogenesis of AML. By its genetic inactivation and pharmacological inhibition in AML cell lines and patient-derived xenograft models, we found that HIF2alpha acts as a novel regulator of the AML differentiation block and promotes leukemia progression. Gene expression profiling of HIF2alpha-dependent transcriptome revealed that HIF2alpha mainly promotes the expression of genes involved in transcriptional modulation and epigenetic modifications, whilst inhibiting genes of myeloid maturation and activation. Mechanistically, inhibition of HIF2alpha results in decreased global levels of the heterochromatin marker H3K27me3, which is specifically deposited by the catalytic subunit EZH2 of the Polycomb repressive complex 2. Intriguingly, we found that HIF2alpha and EZH2 cooperate at favoring EZH2-mediated deposition of H3K27me3 at reverse hypoxia responsive elements in the regulatory regions of myeloid maturation genes. Additionally, we demonstrate that HIF2alpha is positively regulated by the pro-differentiation agent all-trans retinoic acid (ATRA), and its inhibition cooperates with ATRA in triggering AML cell differentiation. In conclusion, we provide new mechanistic insights into the role of HIF2alpha in the pathogenesis of AML, by promoting an undifferentiated state via EZH2-mediated epigenetic silencing of myeloid differentiation genes. Importantly, small molecule inhibitors of HIF2alpha have been recently generated and are tested for solid cancers. Therefore, HIF2alpha inhibition may open new therapeutic avenues for AML patients and add therapeutic value to ATRA-based therapies by interrupting HIF2alpha upregulation and promoting an epigenetic state permissive to maturation of leukemic blasts and leukemia exhaustion. Citation Format: Daniela Magliulo, Carolina Caserta, Serena Belluschi, Cristina Fracassi, Kety Giannetti, Raffaella Pini, Ettore Zapparoli, Stefano Beretta, Eleonora Draghi, Marco J. Morelli, Raffaella Di Micco, Bernhard Gentner, Luca Vago, Rosa Bernardi. The transcription factor HIF2alpha partakes in the differentiation block of acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B027.

Background Pre-eclampsia has a major impact on renal function as shown by the development of proteinuria and podocyturia. How the systemic, soluble Fms-like tyrosine kinase-1 (sFlt-1)-driven inhibition of vascular endothelial growth factor (VEGF) activity detected in pre-eclampsia directly affects renal function remains unknown. The aim of the study was to clarify whether a non-canonical, renal-centred escape from VEGF inhibition in the case of pre-eclamptic pregnancy might have a direct impact on renal function. Methods We evaluated plasma and urinary VEGF and placental growth factor (PlGF), plasma sFlt-1 and carbonic anhydrase IX (CAIX), albuminuria and podocyturia in 18 women with uncomplicated pregnancy, 21 with pre-eclampsia and 18 non-pregnant. The three groups were matched for age and the pregnant groups also for gestational age at enrolment. Results Plasma VEGF was reduced in uncomplicated (P = 0.001) and pre-eclamptic (P = 0.0003) pregnancies when compared with controls. In uncomplicated pregnancy, the dysfunction was balanced by an increase (P = 0.009) of plasma PlGF. Increased (P = 0.0001) plasma CAIX in pre-eclampsia was in line with hypoxia. Pre-eclampsia resulted in a paradoxical increase (P = 0.0004) of urinary excretion of VEGF. Urinary concentrations of VEGF and podocytes were correlated to each other (r2 = 0.48, P Conclusions In the case of pre-eclampsia, the systemic VEGF inhibition leads the kidney, possibly the podocyte, to increase the VEGF synthesis. The mechanisms leading to local VEGF overproduction or the overproduced VEGF itself are reasonably involved in the pathogenesis of podocyturia and, as a consequence, renal dysfunction in pre-eclampsia.

Background: The accurate detection of nodal invasion is an unmet need in the clinical staging of renal cancer. Positron emission tomography (PET) with 18F-fluoroazomycin arabinoside (18F-FAZA), a hypoxia specific tracer, is a non-invasive imaging method that detects tumour hypoxia. The aim of this work was to evaluate the role of 18F-FAZA PET/CT in the identification of lymph node metastases in renal cancer. Methods: A proof-of-concept phase 2 study including 20 kidney cancer patients (ClinicalTrials.gov Identifier: NCT03955393) was conducted. Inclusion criteria were one or more of the following three criteria: (1) clinical tumour size > 10cm, (2) evidence of clinical lymphadenopathies at preoperative CT scan and (3) clinical T4 cancer. Before surgery, 18F-FAZA PET/CT was performed, 2h after the intravenous injection of the radiotracer. An experienced nuclear medicine physician, aware of patient’s history and of all available diagnostic imaging, performed a qualitative and semi-quantitative analysis on 18F-FAZA images. Histopathological analysis was obtained in all patients on surgical specimen. Results: Fourteen/19 (74%) patients had a non-organ confined renal cell carcinoma (RCC) at final pathology (either pT3 or pT4). Median number of nodes removed was 12 (IQR 7–15). The rate of lymph node invasion was 16%. No patient with pN1 disease showed positive 18F-FAZA PET, thus suggesting the non-hypoxic behaviour of the lesions. In addition, neither primary tumour nor distant metastases presented a pathological 18F-FAZA uptake. No adverse events were recorded during the study. Conclusions: 18F-FAZA PET/CT scan did not detect RCC lymph neither nodal nor distant metastases and did not show any uptake in the primary renal tumour.

Há diferentes tendências político-pedagógicas de Educação Ambiental no Brasil, que foram desenvolvidas historicamente junto ao movimento ambientalista. O presente artigo, por meio de análise documental, quais dessas tendências são usadas para embasar as ações educativas nos Parques Estaduais paulistas, ambientes propícios para processos educativos. Os resultados contribuem para a reflexão da Educação Ambiental a ser exercida nesses Parques, indicando uma predominância da abordagem conservacionista e uma presença mais modesta das abordagens crítica e pragmática. Portanto, a Educação Ambiental se mostra ainda conservadora, com indícios de tendência crítica, mas que ainda não tem atingido as ações práticas.

In chronic lymphocytic leukemia (CLL), the hypoxia-inducible factor 1 (HIF-1) regulates the response of tumor cells to hypoxia and their protective interactions with the leukemic microenvironment. In this study, we demonstrate that CLL cells from TP53-disrupted (TP53 dis) patients have constitutively higher expression levels of the α-subunit of HIF-1 (HIF-1α) and increased HIF-1 transcriptional activity compared to the wild-type counterpart. In the TP53 dis subset, HIF-1α upregulation is due to reduced expression of the HIF-1α ubiquitin ligase von Hippel-Lindau protein (pVHL). Hypoxia and stromal cells further enhance HIF-1α accumulation, independently of TP53 status. Hypoxia acts through the downmodulation of pVHL and the activation of the PI3K/AKT and RAS/ERK1-2 pathways, whereas stromal cells induce an increased activity of the RAS/ERK1-2, RHOA/RHOA kinase and PI3K/AKT pathways, without affecting pVHL expression. Interestingly, we observed that higher levels of HIF-1A mRNA correlate with a lower susceptibility of leukemic cells to spontaneous apoptosis, and associate with the fludarabine resistance that mainly characterizes TP53 dis tumor cells. The HIF-1α inhibitor BAY87-2243 exerts cytotoxic effects toward leukemic cells, regardless of the TP53 status, and has anti-tumor activity in Em-TCL1 mice. BAY87-2243 also overcomes the constitutive fludarabine resistance of TP53 dis leukemic cells and elicits a strongly synergistic cytotoxic effect in combination with ibrutinib, thus providing preclinical evidence to stimulate further investigation into use as a potential new drug in CLL.

A Política Nacional de Meio Ambiente (PNMA) é a principal lei ambiental do Brasil, a qual estabelece treze instrumentos para alcançar seus objetivos, sendo um dos mais aplicados o licenciamento ambiental. Associado a ele está outro instrumento, a Avaliação de Impacto Ambiental. As esferas federal, estadual e municipal possuem competência para licenciar variadas tipologias de empreendimentos, conforme o estabelecido pela Lei Complementar nº 140/2011. Embora estejam ocorrendo mudanças nos órgãos licenciadores municipais que começaram a exercer sua competência, é reportado pela literatura que faltam legislações, carecem de órgãos específicos, bem como mão de obra qualificada para exercer tal atividade. Os desafios existentes indicam a necessidade de pesquisar a aptidão dos órgãos municipais de meio ambiente, sua capacidade institucional e a existência de legislação específica, a fim de aprimorar o licenciamento ambiental local. Diante disto, esta pesquisa teve como objetivo principal descrever e caracterizar o licenciamento ambiental descentralizado e a gestão ambiental local, tendo como estudo de caso a cidade de Uberaba/MG. Para isso foi verificada a efetividade do licenciamento ambiental com AIA descentralizado e qual a influência que este instrumento trouxe para a gestão ambiental do município como um todo. Isto foi realizado por meio de levantamento e análise de legislações e documentos orientadores de 22 processos de licenciamento ambiental classes 3 e 4, realização de entrevistas, revisão de literatura e de legislações ambientais e urbanísticas do município, juntamente com a escolha de critérios apropriados para avaliar a forma como tem funcionado o sistema de licenciamento com AIA municipal em Uberaba, além da verificação da sua efetividade. Assim, constatou-se que Uberaba apresenta indícios de efetividade em seu sistema de licenciamento ambiental com AIA, sob o olhar da efetividade procedimental, visto que atendeu a maioria dos critérios aplicados, como, por exemplo, o fato de possuir os elementos do sistema estabelecidos em lei, assim como o objetivo da AIA, e uma secretaria estruturada e com agentes capacitados. Ademais, percebeu-se que a realização do licenciamento ambiental não trouxe novos elementos para a gestão ambiental do município, pois esta já contava com uma robusta estrutura antes mesmo que o convênio para a realização do licenciamento fosse assinado. Isto remete à importância de estudos desta natureza, a fim de avaliar a efetividade de sistemas de licenciamento e a gestão ambiental, para que sejam identificadas as suas carências para a proposição de melhorias. The National Environmental Policy (PNMA) is Brazil\'s main environmental law which establishes thirteen instruments to achieve its objectives, one of the most applied being environmental licensing. Associated with its there are another instrument, the Environmental Impact Assessment (EIA). The federal, state and municipal spheres have the authority to license various typologies of enterprises as established by Complementary Law nº140/2011. Although changes are occurring in the municipal licensing agencies that have begun to exercise their competence, it is reported in the literature that they lack legislation, lack specific bodies, as well as skilled labor to carry out such activity. Existing challenges indicate the need to investigate the suitability of municipal environmental agencies, their institutional capacity and the existence of specific legislation in order to improve local environmental licensing. In view of this, the main objective of this research was to describe and characterize decentralized environmental licensing and local environmental management having as a case study the city of Uberaba/MG. For this, the effectiveness of environmental licensing with decentralized EIA was verified and what influence this instrument brought to the environmental management of the municipality as a whole. This was done by surveying and analyzing legislation and guiding documents of 22 environmental licensing processes classes 3 and 4, conducting interviews, reviewing the literature and environmental and town planning legislation of the municipality, along with choosing appropriate criteria to evaluate the way in which the licensing system with municipal EIA in Uberaba has functioned, in addition to verifying its effectiveness. Thus, it was verified that Uberaba presents indications of effectiveness in its environmental licensing system with EIA, under the view of procedural effectiveness, since it met most of the applied criteria, such as the fact that it has the established system elements in law, as well as the purpose of the EIA, and a structured secretariat with trained agents. In addition, it was realized that the environmental licensing did not bring new elements for the environmental management of the municipality, since it already had a robust structure before the agreement for the accomplishment of the licensing was signed. This refers to the importance of studies of this nature in order to evaluate the effectiveness of licensing systems and environmental management, so that their shortcomings can be identified for the proposition of improvements.

Our previous findings indicate that A2A and D2 receptors are co-expressed on adult rat striatal astrocytes and on the astrocyte processes, and that A2A-D2 receptor−receptor interaction can control the release of glutamate from the processes. Functional evidence suggests that the receptor−receptor interaction was based on heteromerization of native A2A and D2 receptors at the plasma membrane of striatal astrocyte processes. We here provide biochemical and biophysical evidence confirming that receptor−receptor interaction between A2A and D2 receptors at the astrocyte plasma membrane is based on A2A-D2 heteromerization. To our knowledge, this is the first direct demonstration of the ability of native A2A and D2 receptors to heteromerize on glial cells. As striatal astrocytes are recognized to be involved in Parkinson’s pathophysiology, the findings that adenosine A2A and dopamine D2 receptors can form A2A-D2 heteromers on the astrocytes in the striatum (and that these heteromers can play roles in the control of the striatal glutamatergic transmission) may shed light on the molecular mechanisms involved in the pathogenesis of the disease.

Hypoxia inducible transcription factors (HIFs) are the main regulators of adaptive responses to hypoxia and are often activated in solid tumors, but their role in leukemia is less clear. In acute myeloid leukemia (AML), in particular, controversial new findings indicate that HIF-1α can act either as an oncogene or a tumor suppressor gene, and this may depend on the stage of leukemia development and/or the AML sub-type. In this study, we find that HIF-1α promotes leukemia progression in the acute monocytic leukemia sub-type of AML through activation of an invasive phenotype. By applying a list of validated HIF-1α-target genes to different AML sub-types, we identified a HIF-1α signature that typifies acute monocytic leukemia when compared with all other AML sub-types. We validated expression of this signature in cell lines and primary cells from AML patients. Interestingly, this signature is enriched for genes that control cell motility at different levels. As a consequence, inhibiting HIF-1α impaired leukemia cell migration, chemotaxis, invasion and transendothelial migration in vitro, and this resulted in impaired bone marrow homing and leukemia progression in vivo. Our data suggest that in acute monocytic leukemia an active HIF-1α-dependent pro-invasive pathway mediates the ability of leukemic cells to migrate and invade extramedullary sites and may be targeted to reduce leukemia dissemination.

The transcription factor HIF-1α is overexpressed in chronic lymphocytic leukaemia (CLL), where it promotes leukaemia progression by favouring the interaction of leukaemic cells with protective tissue microenvironments. Here, we tested the hypothesis that a pharmacological compound previously shown to inhibit HIF-1α may act as a chemosensitizer by interrupting protective microenvironmental interactions and exposing CLL cells to fludarabine-induced cytotoxicity. We found that the camptothecin-11 analogue EZN-2208 sensitizes CLL cells to fludarabine-induced apoptosis in cytoprotective in vitro cultures; in vivo EZN-2208 improves fludarabine responses, especially in early phases of leukaemia expansion, and exerts significant anti-leukaemia activity, thus suggesting that this or similar compounds may be considered as effective CLL therapeutic approaches.

INTRODUCTION Hypoxia-inducible transcription factors have been identified as regulators of adaptive responses to hypoxia. Over the past 20 years, more than 8000 papers have described their increasingly complex role and regulation in cancer. Presently, it is recognized that hypoxia-inducible factors (HIFs) are regulated by oxygen-dependent and oxygen-independent mechanisms in cancer development; the list of their targets has increased to include more than 500 genes involved in most hallmarks of cancer. Areas covered: Most literature describes the function of HIF factors in solid tumors; however, in the past 10 years, evidence has steadily accumulated to indicate that HIFs are implicated in hematological malignancies. This review summarizes our current understanding of the function and regulation of HIF factors in hematopoiesis and leukemia. Moreover, we provide an update on pharmacological inhibitors of this pathway that have shown promising therapeutic effects in clinical trials or leukemia pre-clinical models. Expert opinion: The inhibition of the function of HIF factors may provide an interesting approach for treating leukemia. We posit that before moving into the clinic, we should (i) fully characterize the outcome of HIF inhibition in specific leukemia contexts (ii) test the possibility of combining HIF-targeting strategies with cytotoxic compounds and (iii) consider patient selection to increase therapeutic efficacy.

In chronic lymphocytic leukemia (CLL), the non-hematopoietic stromal microenvironment plays a critical role in promoting tumor cell recruitment, activation, survival, and expansion. However, the nature of the stromal cells and molecular pathways involved remain largely unknown. Here, we demonstrate that leukemic B lymphocytes induce the activation of retinoid acid synthesis and signaling in the microenvironment. Inhibition of RA-signaling in stromal cells causes deregulation of genes associated with adhesion, tissue organization and chemokine secretion including the B-cell chemokine CXCL13. Notably, reducing retinoic acid precursors from the diet or inhibiting RA-signaling through retinoid-antagonist therapy prolong survival by preventing dissemination of leukemia cells into lymphoid tissues. Furthermore, mouse and human leukemia cells could be distinguished from normal B-cells by their increased expression of Rarγ2 and RXRα, respectively. These findings establish a role for retinoids in murine CLL pathogenesis, and provide new therapeutic strategies to target the microenvironment and to control disease progression., The stromal microenvironment plays a key role in the expansion of chronic lymphocytic leukemia. Here, the authors use the Eµ-TCL1 mouse model to show that leukemic B-cells induce the activation of retinoic acid synthesis in stromal cells of the lymphoid microenvironment, and that impacting on retinoic acid signalling via diet or chemical inhibition prolonged survival by preventing leukemia dissemination and accumulation in lymphoid tissues.

The regulatory factors governing adult mesenchymal stem cell (MSC) physiology and their tumorigenic potential are still largely unknown, which substantially delays the identification of effective therapeutic approaches for the treatment of aggressive and lethal forms of MSC-derived mesenchymal tumors, such as undifferentiated sarcomas. Here, we have developed a novel platform to screen and quickly identify genes and pathways responsible for adult MSC transformation, modeled undifferentiated sarcoma in vivo, and, ultimately, tested the efficacy of targeting the identified oncopathways. Importantly, by taking advantage of this new platform, we demonstrate the key role of an aberrant LRF–DLK1–SOX9 pathway in the pathogenesis of undifferentiated sarcoma, with important therapeutic implications. Significance: The paucity of therapeutic options for the treatment of sarcoma calls for a rapid and effective preclinical assessment of new therapeutic modalities. We have here developed a new platform to deconstruct the molecular genetics underlying the pathogenesis of sarcoma and to evaluatein vivo the efficacy of novel targeted therapies. Cancer Discov; 5(4); 396–409. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 333

Elucidating the molecular basis of tumor metastasis is pivotal for eradicating cancer-related mortality. Triple-negative breast cancer (TNBC) encompasses a class of aggressive tumors characterized by high rates of recurrence and metastasis, as well as poor overall survival. Here, we find that the promyelocytic leukemia protein PML exerts a prometastatic function in TNBC that can be targeted by arsenic trioxide. We found that, in TNBC patients, constitutive HIF1A activity induces high expression of PML, along with a number of HIF1A target genes that promote metastasis at multiple levels. Intriguingly, PML controls the expression of these genes by binding to their regulatory regions along with HIF1A. This mechanism is specific to TNBC cells and does not occur in other subtypes of breast cancer where PML and prometastatic HIF1A target genes are underexpressed. As a consequence, PML promotes cell migration, invasion, and metastasis in TNBC cell and mouse models. Notably, pharmacological inhibition of PML with arsenic trioxide, a PML-degrading agent used to treat promyelocytic leukemia patients, delays tumor growth, impairs TNBC metastasis, and cooperates with chemotherapy by preventing metastatic dissemination. In conclusion, we report identification of a prometastatic pathway in TNBC and suggest clinical development toward the use of arsenic trioxide for TNBC patients.

Cells in the tumor microenvironment may be reprogrammed by tumor-derived metabolites. Cholesterol-oxidized products, namely oxysterols, have been shown to favor tumor growth directly by promoting tumor cell growth and indirectly by dampening antitumor immune responses. However, the cellular and molecular mechanisms governing oxysterol generation within tumor microenvironments remain elusive. We recently showed that tumorderived oxysterols recruit neutrophils endowed with protumoral activities, such as neoangiogenesis. Here, we show that hypoxia inducible factor-1a (HIF-1α) controls the overexpression of the enzyme Cyp46a1, which generates the oxysterol 24-hydroxycholesterol (24S-HC) in a pancreatic neuroendocrine tumor (pNET) model commonly used to study neoangiogenesis. The activation of the HIF-1α-24S-HC axis ultimately leads to the induction of the angiogenic switch through the positioning of proangiogenic neutrophils in proximity to Cyp46a1+islets. Pharmacologic blockade or genetic inactivation of oxysterols controls pNET tumorigenesis by dampening the 24S-HC-neutrophil axis. Finally, we show that in some human pNET samples Cyp46a1 transcripts are overexpressed, which correlate with the HIF-1α target VEGF and with tumor diameter. This study reveals a layer in the angiogenic switch of pNETs and identifies a therapeutic target for pNET patients.

Hypoxia-inducible transcription factors (HIFs) regulate a wide array of adaptive responses to hypoxia and are often activated in solid tumors and hematologic malignancies due to intratumoral hypoxia and emerging new layers of regulation. We found that in chronic lymphocytic leukemia (CLL), HIF-1α is a novel regulator of the interaction of CLL cells with protective leukemia microenvironments and, in turn, is regulated by this interaction in a positive feedback loop that promotes leukemia survival and propagation. Through unbiased microarray analysis, we found that in CLL cells, HIF-1α regulates the expression of important chemokine receptors and cell adhesion molecules that control the interaction of leukemic cells with bone marrow and spleen microenvironments. Inactivation of HIF-1α impairs chemotaxis and cell adhesion to stroma, reduces bone marrow and spleen colonization in xenograft and allograft CLL mouse models, and prolongs survival in mice. Of interest, we found that in CLL cells, HIF-1α is transcriptionally regulated after coculture with stromal cells. Furthermore, HIF-1α messenger RNA levels vary significantly within CLL patients and correlate with the expression of HIF-1α target genes, including CXCR4, thus further emphasizing the relevance of HIF-1α expression to CLL pathogenesis.

The promyelocytic leukemia (PML) tumor suppressor gene was initially identified as part of the t(15:17) chromosomal translocation associated with acute promyelocytic leukemia (APL). The PML protein is responsible for the assembly and function of characteristic nuclear domains known as PML-nuclear bodies (PML-NBs), which have been implicated in a variety of cellular functions, including growth suppression, apoptosis, and cellular senescence. PML’s many roles have been linked, at least in part, to its functional interaction with the tumor suppressor p53. It has been shown that PML favors both p53 accumulation and transcriptional activity; in turn, PML expression is directly regulated by p53, and this reciprocal regulation contributes to p53-mediated apoptosis and senescence. Nevertheless, genetic proof and in vivo assessment of the relevance of this functional crosstalk are still missing. Here we show that complete Pml inactivation, in a context of p53 heterozygosity, redistributes and expands the tumor spectrum leading to the formation of angiosarcomas and increased lymphomagenesis. Importantly, we find that Pml inactivation decreases the rate of loss of heterozygosity (LOH) in the remaining p53 allele, revealing the relevancy of p53 haploinsufficiency to tumorigenesis. Our results thus lend in vivo genetic support to the importance of the crosstalk between these two critical tumor suppressor genes.

Impaired ribosome biogenesis is attributed to nucleolar disruption and diffusion of a subset of 60S ribosomal proteins, particularly ribosomal protein (rp)L11, into the nucleoplasm, where they inhibit MDM2, leading to p53 induction and cell-cycle arrest1–4. Previously, we demonstrated that deletion of the 40S rpS6 gene in mouse liver prevents hepatocytes from re-entering the cell cycle after partial hepatectomy5. Here, we show that this response leads to an increase in p53, which is recapitulated in culture by rpS6-siRNA treatment and rescued by the simultaneous depletion of p53. However, disruption of biogenesis of 40S ribosomes had no effect on nucleolar integrity, although p53 induction was mediated by rpL11, leading to the finding that the cell selectively upregulates the translation of mRNAs with a polypyrimidine tract at their 5´-transcriptional start site (5´-TOP mRNAs), including that encoding rpL11, on impairment of 40S ribosome biogenesis. Increased 5´-TOP mRNA translation takes place despite continued 60S ribosome biogenesis and a decrease in global translation. Thus, in proliferative human disorders involving hypomorphic mutations in 40S ribosomal proteins6,7, specific targeting of rpL11 upregulation would spare other stress pathways that mediate the potential benefits of p53 induction8.

Mutations leading to aberrant cytoplasmic localization of Nucleophosmin 1 (NPM1) have been recently identified as the most frequent genetic alteration in acute myelogenous leukemia. However, the oncogenic potential of this nucleophosmin mutant (NPMc+) has never been established, which casts doubt on its role in leukemogenesis. By performing classical transformation assays, we find that NPMc+, but not wild-type NPM, cooperates specifically with adenovirus E1A to transform primary mouse embryonic fibroblasts in soft agar. We demonstrate that NPMc+ blocks the p19(Arf) (Arf) induction elicited by E1A. Surprisingly, however, we find that NPMc+ induces cellular senescence and that E1A is able to overcome this response. We propose a model whereby the NPMc+ pro-senescence activity needs to be evaded for oncogenic transformation, even though NPMc+ can concomitantly blunt the Arf/p53 pathway. These findings identify for the first time NPMc+ as an oncogene and shed new unexpected light on its mechanism of action.

Loss of the promyelocytic leukaemia (PML) tumour suppressor has been observed in several human cancers. The tumour-suppressive function of PML has been attributed to its ability to induce growth arrest, cellular senescence and apoptosis. Here we identify PML as a critical inhibitor of neoangiogenesis (the formation of new blood vessels) in vivo, in both ischaemic and neoplastic conditions, through the control of protein translation. We demonstrate that in hypoxic conditions PML acts as a negative regulator of the synthesis rate of hypoxia-inducible factor 1α (HIF-1α) by repressing mammalian target of rapamycin (mTOR). PML physically interacts with mTOR and negatively regulates its association with the small GTPase Rheb by favouring mTOR nuclear accumulation. Notably, Pml-/- cells and tumours display higher sensitivity both in vitro and in vivo to growth inhibition by rapamycin, and lack of PML inversely correlates with phosphorylation of ribosomal protein S6 and tumour angiogenesis in mouse and human tumours. Thus, our findings identify PML as a novel suppressor of mTOR and neoangiogenesis. The tumour suppressor PML ('promyelocytic leukaemia') is shown to be an inhibitor of blood vessel formation in tumours and in tissue recovering from ischaemia. Loss of PML is associated with a range of human cancers. Its role in angiogenesis control marks it as a drug target in cancers, where upregulation is the aim, and in encouraging blood vessel formation following ischaemic heart attack or stroke, by downregulation. The tumour suppressor gene PML is shown to normally restrain angiogenesis, both in tumours and under ischaemic conditions. This involves a new pathway in which during hypoxia, PML regulates mTOR and the transcription factor HIF-1α

Purpose: Retinoic acid-arsenic trioxide (ATRA-ATO) combination therapy is the current standard of care for patients with acute promyelocytic leukemia (APL) carrying the oncogenic fusion protein PML-RARα. Despite the high cure rates obtained with this drug combination, resistance to arsenic is recently emerging. Moreover, patients with APL carrying the PLZF-RARα fusion protein are partially resistant to ATRA treatment. Hypoxia-inducible factor-1α (HIF-1α) activation has been recently reported in APL, and EZN-2208 (PEG-SN38) is a compound with HIF-1α inhibitory function currently tested in clinical trials. This study investigates the effect of EZN-2208 in different preclinical APL models, either alone or in combination with ATRA. Experimental Design: Efficacy of EZN-2208 in APL was measured in vitro by assessing expression of HIF-1α target genes, cell migration, clonogenicity, and differentiation, vis a vis the cytotoxic and cytostatic effects of this compound. In vivo, EZN-2208 was used in mouse models of APL driven by PML-RARα or PLZF-RARα, either alone or in combination with ATRA. Results: Treatment of APL cell lines with noncytotoxic doses of EZN-2208 causes dose-dependent downregulation of HIF-1α bona fide target genes and affects cell migration and clonogenicity in methylcellulose. In vivo, EZN-2208 impairs leukemia progression and prolongs mice survival in APL mouse models. More importantly, when used in combination with ATRA, EZN-2208 synergizes in debulking leukemia and eradicating leukemia-initiating cells. Conclusions: Our preclinical data suggest that the combination ATRA-EZN-2208 may be tested to treat patients with APL who develop resistance to ATO or patients carrying the PLZF-RARα fusion protein. Clin Cancer Res; 21(16); 3685–94. ©2015 AACR.

PML is a tumor suppressor implicated in leukemia and cancer pathogenesis. PML epitomizes a multiprotein nuclear structure, the PML-nuclear body (PML-NB), whose proper formation and function depends on PML. Studies in knockout (KO) mice and cells unraveled an essential pleiotropic role for PML in multiple p53-dependent and -independent apoptotic pathways. As a result, Pml(-/-) mice and cells are protected from apoptosis triggered by a number of stimuli such as ionizing radiation, interferon, ceramide, Fas and TNF. It is becoming apparent that PML and the PML-NB act as molecular hubs for the induction and/or reinforcement of programmed cell death through a selective and dynamic regulation of proapoptotic transcriptional events. In addition, recent observations propose a role for PML in checkpoint responses upon DNA damage. Moreover, PML and the PML-NB have also been implicated in the control of genomic stability and DNA repair. Here, we will discuss the molecular mechanisms by which PML regulates these processes and the implication of these findings for cancer pathogenesis and therapy.

This introductory article has been written in the form of a conversation between Pier Paolo Pandolfi, Director of the Cancer Center of Beth Israel Deaconess Medical Center in Boston, and Rosa Bernardi, a former post-doctoral fellow in the laboratory of Dr. Pandolfi, now principal investigator at San Raffaele Scientific Institute in Milan, Italy. We have chosen this atypical review format because we want to offer to our readers a more direct and personal perspective on the first 20 years of research over the promyelocytic leukemia gene. This article begins as an interview, but soon transforms into a dialog where we exchange our thoughts on a number of issues around the past, present, and future research over the biology of PML. We were particularly keen on emphasizing the aspects that we find most interesting or challenging, therefore, we warn our readers that this will not be a comprehensive essay but rather a very personal view of what has been, is, and will be exciting and interesting in the PML world, in our opinion.

Acute promyelocytic leukemia (APL) is epitomized by the chromosomal translocation t(15;17) and the resulting oncogenic fusion protein PML-RARa. Although acting primarily as a transcriptional repressor, PML-RARa can also exert functions of transcriptional co-activation. Here, we find that PML-RARa stimulates transcription driven by HIF factors, which are critical regulators of adaptive responses to hypoxia and stem cell maintenance. Consistently, HIF-related gene signatures are upregulated in leukemic promyelocytes from APL patients compared to normal promyelocytes. Through in vitro and in vivo studies, we find that PML-RARa exploits a number of HIF-1a-regulated pro-leukemogenic functions that include cell migration, bone marrow (BM) neo-angiogenesis and self-renewal of APL blasts. Furthermore, HIF-1a levels increase upon treatment of APL cells with all-trans retinoic acid (ATRA). As a consequence, inhibiting HIF-1a in APL mouse models delays leukemia progression and exquisitely synergizes with ATRA to eliminate leukemia-initiating cells (LICs).

To analyze relevant features of HeLa and HL60 cells driven into apoptosis by etoposide, we have developed a new “tricolor” assay, based on the simultaneous analysis in the single cell of chromatin condensation, DNA degradation, and cellular poly(ADP-ribose) synthesis. The latter reaction is catalyzed by poly(ADP-ribose)polymerase (E.C. 2.4.2.30), an enzyme which is activated by the presence of DNA free ends. The protocol consists in the visualization of apoptotic cells by Hoechst staining, TUNEL assay, and immunoreaction with anti-poly(ADP-ribose) antibody. We thus provide the first evidence that endogenous poly(ADP-ribose) production is indeed stimulated in cells undergoing apoptosis after treatment with antitumoral drugs, and that the monitoring of this endogenous enzymatic reaction, combined with morphological and other biochemical parameters, should facilitate the detection of apoptotic cells.