MN1 Expression Is an Indepedent Prognostic Marker in FLT3-Mutated Acute Myeloid Leukemia and Is Involved in the Resistance to FLT3 Inhibitors
Authors: Pereira-Martins Diego A , Coelho-Silva Juan L , Weinhäuser Isabel , Thomé Carol Hassibe , Ortiz Rojas César Alexander , Simões Luíse A A , Traina Fabiola , Heuser Michael , Ganser Arnold , Lucena-Araujo Antonio R. , Rego Eduardo M 
Published: November 2019

Introduction: Meningioma 1 (MN1) gene was described as a prognostic marker for AML patients with normal karyotype (Carturan et al. Oncotarget 2016). In addition, MN1 high expression was linked to RA resistance and was shown to be necessary and sufficient to transform common myeloid progenitors in a MEIS1/AbdB-like HOX protein complex-dependent manner. However, the relevance of the copresence of MN1 overexpression in AML patients with mutations of the FLT3 and/or NPM1 genes is unknown. Moreover, it is also unknown the functional effects of MN1 in the biology of leukemic blasts harboring FLT3 mutations and if it may modulate the response to FLT3 inhibitors, such as quizartinib (AC220) and midostaurin (PKC). Aims: Herein, we investigated the prognostic impact of MN1 expression across multiple transcriptomic platforms and AML data sets. Additionally, we transduced different AML cell lines to evaluate the impact of MN1 on cell survival and differentiation. Methods: Three different AML series (1th: GSE6891, 240 patients, 2nd: TCGA, 113 patients and 3rd: BeatAML, 139 patients) were used. All patients presented similar gender distribution, were above 18y and treated by the 3+7 scheme. All genes from the RNAseq (TCGA) were pre-ranked according to their differential expression comparing tumors with high and low expression of MN1, using their median expression rate as the cutoff. GSEA was performed using the Reactome, KEGG and Hallmarks databases. Additionally, we transduced 07 AML cell lines with the MN1 gene and the control. For those cells, clonogenicity and proliferation rate was evaluated to identify which cell lines are sensitive and resistant to MN1. Drug induced apoptotic rate was assessed for FLT3-ITD- cell lines when treated with AraC (10 nM) and FLT3-ITD+ cell lines (MOLM13/MV411) upon treatment with PKC and AC220 over a time period of 24, 48 and 72 h. Apoptosis was further confirmed by cleaved Caspase-3/PARP detection. The myeloid differentiation in response to PMA treatment (100 ng/ml) was determined by the surface levels of CD11b, CD11c, CD14, CD15, CD61 and HLADR. Results: In all three AML series, high MN1 levels higher than the median (hereafter called High MN1) were associated with lower frequency of FLT3 and NPM1 mutations (P&lt;.01 for all comparisons). Baseline features were similar between patients with low and high MN1 levels in all the cohorts. The median 5y OS rates were 33%, 29% and 41% in the GSE6891, TCGA and BeatAML, respectively. Multivariate analysis showed that patients carrying the FLT3 mutation who expressed high MN1 levels exhibited increased OS rates in the GSE6891 (50%), TCGA (50%) and BeatAML (71%) cohorts in comparison with patients with lower MN1 levels taking ELN, sex and age as confounders (P&lt;.05). Regarding the patients with NPM1 and IDH1/2 mutations, high MN1 levels was associated with lower OS rates in all the cohorts. The analysis to predict genetic interactors in AML samples (BeatAML) revealed that MN1 is associated with cellular mediators of myeloid differentiation, such as FOXM1, CEBPA and MEIS1, and drug-resistance (CYP26A1). Additionally, GSEA indicated that the MN1 expression was associated with biological processes such as apoptosis, STAT5 signaling and Interferon response. In AML cell lines, induced MN1 overexpression strongly reduced growth, colony formation (basal and under treatment with PKC/AC220) and increased drug-induced apoptosis in both FLT3-ITD cells (P&lt;.01 for all). Forced MN1 expression in FLT3-ITD cells lead to decreased cytokine mediated resistance to FLT3 inhibitors (PKC/AC220; P&lt;.01 for all). Using a nonlinear regression analysis, IC50 for PKC and AC220 was significantly lower for MN1-expressing cells (PKC: 8.4 and 4.9 nM; AC220: 0.69 and 0.17 nM for MV411 and MOLM13, respectively) in comparison to control cells (PKC: 13.3 and 8.5 nM; AC220: 0.62 and 0.38 nM, respectively). 3 out of 7 cell lines showed increased cell proliferation when MN1 gene was overexpressed (P&lt;.05). In accordance, MN1 overexpression reduced the drug-induced apoptosis rate in those AML cells with increased proliferation in a time-dependent manner (P&lt;.05). Phorbol induced myeloid differentiation was decreased for all MN1 transduced cell lines (P&lt;.01 for all), except for the THP1 cells. Conclusion: In summary, our results demonstrated that MN1 overexpression decreases cell viability and proliferation in FLT3-ITD+ cells and decreased resistance to FLT3 inhibitors.

Disclosures
Heuser: Bayer Pharma AG, Berlin: Research Funding; Synimmune: Research Funding.