Interplay between transcription regulators RUNX1 and FUBP1 activates an enhancer of the oncogene c-KIT and amplifies cell proliferation.

Runt-related transcription factor 1 (RUNX1) is a well-known master regulator of hematopoietic lineages but its mechanisms of action are still not fully understood. Here, we found that RUNX1 localizes on active chromatin together with Far Upstream Binding Protein 1 (FUBP1) in human B-cell precursor lymphoblasts, and that both factors interact in the same transcriptional regulatory complex. RUNX1 and FUBP1 chromatin localization identified c-KIT as a common target gene. We characterized two regulatory regions, at +700 bp and +30 kb within the first intron of c-KIT, bound by both RUNX1 and FUBP1, and that present active histone marks. Based on these regions, we proposed a novel FUBP1 FUSE-like DNA-binding sequence on the +30 kb enhancer. We demonstrated that FUBP1 and RUNX1 cooperate for the regulation of the expression of the oncogene c-KIT. Notably, upregulation of c-KIT expression by FUBP1 and RUNX1 promotes cell proliferation and renders cells more resistant to the c-KIT inhibitor imatinib mesylate, a common therapeutic drug. These results reveal a new mechanism of action of RUNX1 that implicates FUBP1, as a facilitator, to trigger transcriptional regulation of c-KIT and to regulate cell proliferation. Deregulation of this regulatory mechanism may explain some oncogenic function of RUNX1 and FUBP1

Reduction of RUNX1 transcription factor activity by a CBFA2T3-mimicking peptide: application to B cell precursor acute lymphoblastic leukemia.

Funder: FP7 People: Marie-Curie Actions; doi: http://dx.doi.org/10.13039/100011264; Grant(s): 291851BACKGROUND: B Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) is the most common pediatric cancer. Identifying key players involved in proliferation of BCP-ALL cells is crucial to propose new therapeutic targets. Runt Related Transcription Factor 1 (RUNX1) and Core-Binding Factor Runt Domain Alpha Subunit 2 Translocated To 3 (CBFA2T3, ETO2, MTG16) are master regulators of hematopoiesis and are implicated in leukemia. METHODS: We worked with BCP-ALL mononuclear bone marrow patients' cells and BCP-ALL cell lines, and performed Chromatin Immunoprecipitations followed by Sequencing (ChIP-Seq), co-immunoprecipitations (co-IP), proximity ligation assays (PLA), luciferase reporter assays and mouse xenograft models. RESULTS: We demonstrated that CBFA2T3 transcript levels correlate with RUNX1 expression in the pediatric t(12;21) ETV6-RUNX1 BCP-ALL. By ChIP-Seq in BCP-ALL patients' cells and cell lines, we found that RUNX1 is recruited on its promoter and on an enhancer of CBFA2T3 located - 2 kb upstream CBFA2T3 promoter and that, subsequently, the transcription factor RUNX1 drives both RUNX1 and CBFA2T3 expression. We demonstrated that, mechanistically, RUNX1 and CBFA2T3 can be part of the same complex allowing CBFA2T3 to strongly potentiate the activity of the transcription factor RUNX1. Finally, we characterized a CBFA2T3-mimicking peptide that inhibits the interaction between RUNX1 and CBFA2T3, abrogating the activity of this transcription complex and reducing BCP-ALL lymphoblast proliferation. CONCLUSIONS: Altogether, our findings reveal a novel and important activation loop between the transcription regulator CBFA2T3 and the transcription factor RUNX1 that promotes BCP-ALL proliferation, supporting the development of an innovative therapeutic approach based on the NHR2 subdomain of CBFA2T3 protein

ETV6-RUNX1 and RUNX1 directly regulate RAG1 expression: one more step in the understanding of childhood B-cell acute lymphoblastic leukemia leukemogenesis.

Funder: Société Française de Biochimie et Biologie Moléculaire ; French Research MinistryFunder: Cancéropole Grand Ouest ; Région Bretagne ; Société Française d’HématologieFunder: Ligue Régionale contre le cancer ;ETV6-RUNX1 and RUNX1 directly promote RAG1 expression. ETV6-RUNX1 and RUNX1 preferentially bind to the −1200 bp enhancer of RAG1 and the −80 bp promoter of RAG1 gene respectively, and compete for these bindings. ETV6-RUNX1 and RUNX1 induce an excessive RAG recombinase activity. ETV6-RUNX1 participates directly in two events of the multi-hit ALL leukemogenesis: as an initiating event and as an activator of RAG1 expression

Effects of Hypoxia on the Regulation of the Expression and the Function of the CD9 Tetraspanin in Childhood Acute Lymphoblastic Leukemias

Les leucémies aiguës lymphoblastiques (LAL) sont le cancer le plus fréquent chez l’enfant et dérivent le plus souvent de précurseurs lymphoïdes B. D’importants progrès thérapeutiques ont permis d’améliorer considérablement le pronostic. Néanmoins, 15 à 20 % des enfants rechutent encore. Ces rechutes peuvent survenir de façon isolée ou combinée dans la moelle osseuse, le site primitif des lymphoblastes, et/ou dans des organes extramédullaires tels que le testicule ou le système nerveux central. Notre équipe a montré que la protéine transmembranaire CD9 jouait un rôle majeur dans la migration des blastes dans ces sites et notamment le testicule, par l’activation de la voie RAC1 en réponse à la stimulation des cellules par le CXCL12. Ici, nous avons mis en évidence qu’un faible niveau d’oxygène, caractéristique commune aux niches médullaire et extramédullaires, régulait positivement l’expression de CD9 aux niveaux transcriptionnel et protéique, via la voie majeure de réponse à l’hypoxie, dépendante du facteur de transcription Hypoxia Inducible Factor 1a (HIF1a). Nous montrons que HIF1a se fixe directement sur le promoteur de CD9 pour induire sa transcription. Nous montrons aussi que la protéine CD9 est essentielle aux propriétés d’adhérence et de migration des blastes dans des conditions de basse oxygénation, et que son action pourrait s’exercer à travers RAC1 comme en normoxie. Nos résultats dans des expériences de xénogreffe à des souris indiquent que la voie HIF1a favorise la dissémination des blastes, possiblement à travers la régulation qu’elle exerce sur CD9. Ainsi, ce travail contribue à mieux comprendre le rôle de CD9 dans la pathogenèse des LAL de l’enfant.Acute lymphoblastic leukemia (ALL) are the most frequent cancer in children and derive most often from B-cell precursors. Huge therapeutic improvements have allowed to reach high survival rates near 90% at 10 years from diagnosis. However, 15-20% of children still relapse with a significant risk of death. Relapses can occur in bone marrow and/or extramedullary sites such as testis or central nervous system, usually referred as “sanctuary sites”. Our previous work showed that the transmembrane protein CD9 plays a major role in lymphoblasts migration into these sites, especially in testis, through the activation of RAC1 signaling upon blasts stimulation with C-X-C chemokine ligand 12 (CXCl12). Here, we addressed the question of putative common factors shared by bone marrow and extramedullary niches which could upregulate CD9 expression and function. Consequently, we found that low oxygen levels could actually enhance CD9 expression both at mRNA and protein levels. We further determined that Hypoxia Inducible Factor 1a (HIF1a), the master transcription factor involved in hypoxia response, binds directly CD9 promoter to induce its transcription. We also showed that CD9 protein is crucial for leukemic cell adhesion and migration at low oxygen levels, possibly through its action on RAC1 signaling. Mouse xenograft experiments indicate that HIF1a signaling pathway favors ALL cells dissemination, which may involve CD9 as well. The present work increments our understanding of CD9 implication in ALL pathogenesis

Complete and Repeated Response of a Metastatic ALK-rearranged Inflammatory Myofibroblastic Tumor to Crizotinib in a Teenage Girl

International audienceInflammatory myofibroblastic tumors (IMT) are rare tumors in children and young adults, considered by the World Health Organization to be intermediate malignancies and rarely metastasizing, with the presence of an anaplastic lymphoma kinase rearrangement in about 50% of the cases. We report the case of a teenager who presented with a metastatic aggressive IMT that was life-threatening despite multiple treatments, and which responded repeatedly to anaplastic lymphoma kinase–targeted crizotinib therapy. Crizotinib induced drastic primary tumor regression, which was sufficient to allow surgical resection and to control distant disease. This case shows that crizotinib is a promising therapy in IMT, even in adolescents and young adults

Care management for foreign children, adolescents, young adults with cancer, and their families

International audienceBACKGROUND: Little is known about care management for foreign patients in pediatric oncology in European centers. We aimed to describe care given to children, adolescents, and young adults who came to France for cancer treatment, and to determine whether their geographical origin had an influence on decision making. PROCEDURE: We conducted a monocentric retrospective study on all foreign patients aged 0-25 years and hospitalized for at least one night in Institut Curie (Paris, France) from 2009 to 2013. We analyzed the potential advantages of receiving treatment in France as well as their social and familial consequences. RESULTS: A total of 93 foreign patients' files were retrieved. Most of these patients came from Africa (70%). In accord with the specific expertise of the institution, retinoblastoma was the most frequent tumor type (39%). An antitumor treatment had already been administrated in the native country in 44% of patients. We considered that 66% of patients received a significant medical advantage from care in our institution. The treatment provided in France was considered impossible in the native country in 44% of cases. The social and familial impact on the patients' families was high (59%). Almost all patients (96%) received the treatment that would have been proposed to their French counterparts. CONCLUSIONS: There were notable medical advantages for foreign patients who come to France for their oncologic treatment despite important familial consequences. Patients' geographical origin did not have an influence on medical decisions

DZNep co-operates <i>in vivo</i> with bortezomib.

<p>(<b>a</b>) RPMI 8226-GFP-Luc cells were injected into the caudal vein of NSG mice (n = 20) at day 1 (5×10⁶ cells were injected per animal). Ten days later, mice were separated into four groups (n = 5 in each group), one received vehicle for control, one was treated with 12.5 µg bortezomib i.p. twice a week, one was treated with 50 µg DZNep i.p. every two days, one was treated with bortezomib plus 50 µg DZNep i.p. every two days. Mice were imaged at days 15, 20, 32 and 39. At day 40, all mice (except three, see below) were euthanized. Two mice in the bortezomib-treated group and one in the bortezomib plus DZNep group died at day 32. BLI of the dorsal and the ventral sides of mice was taken at these time points and added. The luciferase activity (in arbitrary unit) representative of tumor growth in each series (mean ± SD) is represented in the graph; in blue the control group, in red the DZNep-treated group, in green the bortezomib-treated group and in purple the bortezomib/DZNep group. *<i>p</i><0.05 with the Student’s <i>t</i>-test. (<b>b</b>) Examples of histological analyses (HES, CD138 and cl. caspase 3 staining) from rachis (mouse #27955 from control group and mouse #745 from DZNep/borezomib group), left femur (mouse #827) from DZNep-treated group, and right femur (mouse #780) from bortezomib group. CD138-positive MM cells invaded bone tissues causing destruction and disorganization. In DZNep- or bortezomib-treated animals a high caspase 3 activity underlined therapeutic efficacy. Impressively, in mice #745 treated by both compounds, little CD138-positive cells were detected suggesting a possible cure.</p

DZNep induces myeloma cells death.

<p>Exponentially growing MM cells were either treated with vehicle (DMSO 0.1%) or DZNep 1 µM for 72 h (<b>a left part</b>) or 48–144 h (<b>a right part</b>). (<b>a</b>) Cell viability was assessed by Trypan blue exclusion. The percentage of viable cells referred to control experiments assigned to 100%. The experiment has been repeated three times, histograms show means ± SD, *<i>p</i><0.05. (<b>b</b>) Responsive 8226 and resistant LP1 cells were either treated with vehicle or DZNep (1 µM for 24 h) then examined by transmission electronic microscopy. (<b>c</b>) RPMI 8226 cells were treated with vehicle, the PI3K inhibitor LY94002 (1 µM for 24 h, <b>left part of the figure</b>), the mTOR inhibitor everolimus (10 nM for 24 h, <b>right part of the figure</b>), DZNep (1 µM for 24 h) or both and cell proliferation assayed by a MTS assay. For each culture condition, cells were seeded in three to five wells. The experiments have been repeated two or three times. A representative experiment is shown with the mean and SD values. ns, not significant.</p

DZNep-induced cell death is apoptosis.

<p>(<b>a</b>) Exponentially growing MM cells were either treated with vehicle (DMSO 0.1%) or DZNep 1 µM for 72 h. Apoptosis was analyzed by FACS after APO2.7 staining. At least, 2×10⁴ events were gated. The percentage of apoptotic cells (stained for APO2.7) is indicated on the graph. (<b>b</b>) Exponentially growing JJN3 and 8226 cells were either pretreated with vehicle or Q-VD-OPh 10 µM for 1 h then with vehicle or DZNep 1 µM for 48 h. Cell proliferation was estimated by a MTS assay. Control samples referred to 100%. Here is shown a representative example from three independent experiments; each culture condition being in triplicate. Histograms show mean ± SD, *<i>p</i><0.05. (<b>c</b>) 8226, JJN3 and LP1 cells were treated with vehicle or Q-VD-OPh (10 µM for 1 h) and/or treated with DZNep (1 µM for 72 h). Cells were then stained with anti-APO2.7 Ab and analyzed by FACS. At least, 2×10⁴ events were gated. The percentage of apoptotic cells (stained for APO2.7) is indicated on the graph. (<b>d</b>) Western blots were performed with the indicated antibodies to study the caspase cascade; β-actin Ab was used as control of charge and transfer. White arrows show proforms of PARP and caspase 3/9 and black arrows the cleaved (cl.) and activated forms of proteins.</p

Localization of tumoral foci in NSG mice.

<p>The distribution of tumoral cells was assessed by BLI in ten mice (five per group), 45 days after the inoculation of 5×10⁶ RPMI 8226-GFP-Luc cells in the caudal vein of immunodeficient NSG mice.</p><p>Localization of tumoral foci in NSG mice.</p