MAPK8‐mediated stabilization of SP1 is essential for RUNX1‐RUNX1T1 – driven leukaemia

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 selfrenewal 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-jB/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 RUNX1RUNX1T1 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 lmol/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 KMT2AMLLT3 (MLL-AF9) fusion gene and showed very low SP1 levels (Fig S2B). After 24 h of treatment with 25 lmol/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. correspondence