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• W1944693766 abstract "The presence of the RUNX1-RUNX1T1 (AML1-ETO) fusion protein indicates acute myeloid leukaemia (AML) with t(8;21)(q22;q22), which accounts for approximately 4% of all cases of AML and 40% of all cases of French-American-British M2 type AML. This product is the consequence of a reciprocal translocation that fuses the DNA-binding domain of the RUNX1 transcription factor – essential for transcriptional activation of genes involved in normal haematopoiesis – in frame with nearly the entire RUNX1T1 transcriptional repressor protein. In human haematopoietic stem and progenitor cells (HSPC), RUNX1-RUNX1T1 promotes self-renewal and interferes with efficient myeloid and erythroid differentiation, although it is not sufficient to cause leukaemic transformation (Mulloy et al, 2002). Several studies have reinforced the importance of the interaction between RUNX1-RUNX1T1 and other transcription factors on its DNA-binding profile, and we previously identified the Sp1 transcription factor (SP1) binding site in more than 50% of DNA-bound RUNX1-RUNX1T1 protein (Maiques-Diaz et al, 2012). SP1 induces the expression of essential haematopoietic genes (i.e. KIT, DNMT1) and forms part of the SP1/NF-κB/HDAC1/MIR29B network (Liu et al, 2008, 2010). It also controls CDKN1A expression depending on and independently of TP53 binding (Huang et al, 2000; Koutsodontis et al, 2001). In the present study, we further explored the importance of the SP1 protein in the RUNX1-RUNX1T1 leukaemogenic process and found that not only is SP1 directly regulated by the fusion protein but that it is also a key factor for RUNX1-RUNX1T1 AML cell maintenance. Details of the materials and methods are provided in Data S1. We previously reported relatively low SP1 mRNA levels in human haematopoetic stem progenitor cells expressing RUNX1-RUNX1T1 (referred to hereafter as HSPC-RR) and the SKNO1 cell line compared with HSPC (selected CD34+ cells with an empty vector transduction) (Maiques-Diaz et al, 2012). However, abundant SP1 protein levels were observed in RUNX1-RUNX1T1 – expressing cells compared with normal HSPC (Fig 1A). To determine whether SP1 protein accumulation was a direct effect of RUNX1-RUNX1T1 expression, we selected normal CD34+ cells and transduced them with RUNX1-RUNX1T1 – expressing retroviruses (for construct information, see Mulloy et al, 2002). A significant increase in SP1 protein levels was observed 1 week after transduction (Fig 1B), confirming that SP1 protein accumulates as a direct effect of RUNX1-RUNX1T1 expression in HSPCs. SP1 protein regulation is mediated by a variety of posttranslational modifications that have been reported to control SP1 protein levels, transactivation and DNA binding affinity. MAPK8 (also termed JNK1) is known to impair SP1 proteasomal degradation by inducing phosphorylation of Thr278 and Thr739 (Chuang et al, 2008). Interestingly, high levels of MAPK8 mRNA and MAPK8 protein relative to HSPC cells were observed in HSPC-RR and SKNO1 (Fig 1A and C). We analysed several AML cell lines harbouring different chromosomal translocations and, with the exception of the BCR-ABL1–positive K562 cell line, none showed high SP1 protein levels (Fig S1A), suggesting that this event is associated with specific AML contexts. Increased MAPK8 activation (measured as phospho-MAPK8) was also observed in both SKNO1 and K562 cells, correlating with the higher SP1 levels observed (Fig S1A). To further investigate the role of MAPK8 in SP1 stabilization in this cellular context, we treated SKNO1 cells with JNK/MAPK8 inhibitor II (SP600125; Calbiochem, San Diego, CA, USA), which competes reversibly with ATP, abrogating MAPK8 and MAPK9 (also termed JNK2) activity. Two days’ exposure of SKNO1 cells with 12 or 25 μmol/l of MAPK inhibitor (MAPKi) led to a significant reduction in levels of SP1, especially of the non-phosphorylated form, not impaired for proteasomal degradation (Fig 1D). Washing out the compound returned SP1 levels to normal (Fig 1D). Interestingly, RUNX1-RUNX1T1 – expressing cells were significantly more sensitive to JNK inhibition than the THP1 AML cell line, which is driven by the expression of the KMT2A-MLLT3 (MLL-AF9) fusion gene and showed very low SP1 levels (Fig S2B). After 24 h of treatment with 25 μmol/l of MAPKi, less than 40% of RUNX1-RUNX1T1 cells were viable, whereas 80% of THP1 cells remained alive (Fig 1E). The biological importance of SP1 in t(8;21) leukaemia was further studied using two lentiviral short hairpin constructs on three independent HSPC-RR clones. After antibiotic selection, the cells showed a complete reduction in viable SP1 knockdown cells, which was not observed in the non-targeting transduced HSPC-RR clones (Fig S2A). Moreover, the stable knockdown of SP1 in the SKNO1 cell line (Fig 2A) resulted in significantly reduced growth and increased apoptosis (Fig 2B–C and S2B). When the same SP1 shRNA constructs were used in THP1 cells, SP1 knockdown had no effect in this cellular context (Fig S2C–D), indicating that the effect is directly related to RUNX1-RUNX1T1 expression. As t(8;21) patients consistently exhibit more intense expression of CDKN1A than other AML patients (Berg et al, 2008), and HSPC-RR cells show higher amounts of CDKN1A and TP53 proteins, along with more double-strand breaks (Krejci et al, 2008), we hypothesize that the observed dependence of RUNX1-RUNX1T1 cells on SP1 could be related to their role in regulating CDKN1A gene expression. Indeed, CDKN1A mRNA levels were augmented in HSPC-RR cells (Fig 2D) and decreased when SP1 was knocked-down (Fig 2E). Furthermore, SP1 knockdown consistently alters the SKNO1 cell cycle with a significantly increased number of cells entering the G2 phase (Fig 2F). These observations further explain that we were unable to establish HSPC-RR clones with SP1 knockdown (Fig S2A): our HSPC-RR cells represent a preleukaemic model and are thus highly sensitive to CDNK1A downregulation induced by SP1 knockdown, previously found as an essential cyclin for self-renewal and initiation of RUNX1T/RUNX1T1 leukaemia (Viale et al, 2009). Finally, HSPC-RR and SKNO1 cells were treated with increased concentrations (0–75 ng/ml) of mithramycin A, a known SP1 binding inhibitor (Liu et al, 2010). Compared with the THP1 cell line, RR cells showed greater than threefold higher sensitivity to the inhibitor [50% inhibitory concentration (IC50) = 25 ng/ml vs. IC50 > 75 ng/ml] (Fig 2G). These data indicate SP1 as a good therapeutic target in this AML context. In summary, we deciphered a new function for the activation of MAPK8 observed in RUNX1-RUNX1T1 cells, namely, that it is responsible for stabilization of SP1. Our data show for the first time the essential role of SP1 in RUNX1-RUNX1T1 cell maintenance through the regulation of key genes, such as CDKN1A. These results provide new evidence for the inclusion of pharmacological approaches leading to degradation of SP1 in the treatment of patients harbouring a RUNX1-RUNX1T1 fusion protein. We thank Thomas O'Boyle for reviewing the manuscript. This work was supported by the Spanish Government through grants AP2008-00339 (A.M.D.), PI11/00944 (S.A.), and PI12-0425 (J.C.C.). Funding was also received through a Centre of Excellence in Molecular Haematology P30 award (DK090971) (JCM), a Hyundai Hope on Wheels award (JCM) and a grant from the Asociación Española Contra el Cáncer (AECC). J.C.M. is a Leukemia and Lymphoma Society Scholar. The authors declare that they have no conflicts of interest. A.M.D. designed the experiments, performed the research and prepared the manuscript. M.H, A.L.S., A.R-M. and M.S. participated in the performance of some experiments. J.C.M. provided key reagents. J.C.C. participated in the design of the study. S.A. was responsible of the overall design of the study and experiments and prepared the manuscript. Fig S1. Related to Fig 1. Levels of p-SP1 are proportional of MAPK8 activation in AML cells. Fig S2. Related to Fig 2. Effect of SP1-knockdown on AML cells. Fig S3. Related to Fig 2. SP1 protein degradation is induced by proteasomal inhibitor independently of MAPK8 activation. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article." @default.
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• W1944693766 title "MAPK8-mediated stabilization of SP1 is essential for RUNX1-RUNX1T1 - driven leukaemia" @default.
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