Frat2 mediates the oncogenic activation of Rac by MLL fusions

Abstract

IntroductionTranslocations affecting the mixed lineage leukemia (MLL) gene on chromosome band 11q23 are the most common chromosomal aberrations in pediatric acute myeloid leukemia (AML). 1 This cytogenetic subgroup is associated with an intermediate risk, 2 intensive chemotherapy having improved overall survival, although MLL-rearranged AML is still unfavorable in older patients. 3 Taken together, these data suggest that novel therapeutic interventions based on targeting MLL fusion function would benefit a significant number of patients.The MLL fusion genes resulting from these translocations are potent oncogenes. [4][5][6] They are sufficient to induce aberrant selfrenewal programs in mouse hematopoietic progenitor cells (HPCs) leading to transformation and leukemic progression. 7,8 Recent studies have demonstrated that this is also true for human HPCs. 9,10 We and others have previously shown that inhibition of MLL fusion expression reverses HPC immortalization [11][12][13] and abrogates established leukemia in vivo. 14,15 This suggests that the transcriptional and signaling pathways controlled by MLL fusions are necessary for maintenance as well as initiation of leukemia. Recent studies have demonstrated that progression of preleukemic to leukemic MLL fusion cells involves increased canonical Wnt signaling 16 and Rac GTPase activation 17 ; both of these signaling pathways play critical roles in leukemia progression. 10,16,[18][19][20] The sensitivity of MLL-rearranged AML cells to Rac inhibition suggests that targeting Rac activity or signaling pathways leading to Rac activation may represent novel therapeutic opportunities. In support of this, pharmacologic Rac inhibition was found to interfere with leukemic engraftment of MLL-rearranged human AML cell lines. 19 The available evidence indicates that MLL fusions can induce Rac activity. Thus, transformation of human cord blood-derived HPCs by MLL-AF9 was accompanied by increased Rac activation. 10,19 However, little is known about the molecular details of how this is achieved. In the present study, we have used conditionally immortalized preleukemic and leukemic cells to address the dependency of Rac GTPase activity on continued MLL fusion expression and to establish components of the signaling pathway linking the 2. These experiments identify multiple points potentially suitable for therapeutic targeting of MLL-rearranged AML. Methods MiceAll mice were maintained in the animal facilities of the UCL Institute of Child Health and experiments were performed according to United Kingdom Home Office regulations. The generation and characterization of Frat1/2/3 triple-knockout (TKO) mice have been reported previously. 21 These mice have been backcrossed onto the C57BL/6 background 20 times. Age-and sex-matched C57BL/6 J mice were used as controls. Retrovirus and lentivirus cloning and productionThe pMSCV-MLL-ENL-pgk-neo retroviral construct has been previously described. 14 Oligonucleotides for shRNAs targeting Frat1, Frat2, and Dvl1 were designed using RNAi centra...

TMEM98 is a negative regulator of FRAT mediated Wnt/ß-catenin signalling

“…Both FRAT and TMEM98 have been shown to have oncogenic activity [16,23,24,62,63]. While the physiological role of FRAT proteins is still elusive [22], TMEM98 has been genetically linked to autosomal dominant nanophthalmos [25,26], a developmental disorder resulting in small eyes, hyperopia and an increased risk of angle closure glaucoma.…”

“…However, FRAT function is dispensable for Wnt/ß-catenin signalling in mice [22], indicating that FRAT is a modulator, rather than a core component of the Wnt/ß-catenin pathway in mammals. Moreover, the oncogenic activities of FRAT in lymphomagenesis may at least partially be GSK3 independent [23,24]. To date, the precise role and regulation of FRAT1, and its close homologue FRAT2, remain to be resolved.…”

Insulin-like growth factor 1 is a direct HOXA9 target important for hematopoietic transformation

“…The shortlist also included Frat2, a guanine nucleotide exchange factor activating RAC in MLL-induced leukemia. 24 Whereas Myb and Frat2 expression levels were not proportional to the in vivo leukemogenic potential of the respective HOX genes, Igf1 clearly complied with this prerequisite (Figure 1a). HOXA9 cells contained an order of magnitude more Igf1 RNA than HOXA1 and HOXA6 transformed populations.…”

FRAT1 expression regulates proliferation in colon cancer cells

“…High FRAT1 expression is associated with the increased malignancy and a higher clinical stage, thus FRAT1 affects tumor biological characteristics (19). However, studies investigating FRAT1 expression in digestive tract tissue are limited.…”

“…The specific sequence of the shRNA targeting FRAT1 was designed as previously described (19)(20)(21)(22) using the following primer sequences: Forward, 5'-GCA GTT ACG TGC AAA GCT TTT CAA GAG AAA GCT TTG CAC GTA CTGC-3' and reverse, 3'-CGT CAA TGC ACG TTT CGA AAA GTT CTC TTT CGA AAC GTG CAT TGA CG-5'. The shRNA template was then added to two endonucleases, HindIII and BamH1, for digestion.…”