Identification of miRNA-mRNA network in NPM1 mutated acute myeloid leukemia Syed Khizer Hasan 1 , Nikhil Gadewal 1 , Swapnil Aher 1 , Anagha Gardane 1 , Rohit Kumar 1 , Ashok Varma 1 and Navin Khattry 2 1 ACTREC, Tata Memorial Centre, Navi-Mumbai, India. 2 Adult Hematolymphoid Disease Management Group, Department of Medical Oncology, Tata Memorial Centre, Mumbai, India BACKGROUND AIM OF THE STUDY  AML with NPM1 mutation is a disease driving genetic alteration with good prognosis.  Mutant NPM1 AML has unique microRNA and their target gene (mRNA) signature compared to wildtype NPM1.  Dynamic regulation of miRNA-mRNA has been reported to influence prognostic outcome.  NPM1 mutation induces chemo-sensitivity in leukemic cells but the underlying cause for the better survival of NPM1 mutated patients is still not clear.  To evaluate the pairwise correlations of differential expression between miRNA and mRNA and if the strength of differential (negative or positive) regulation of a miRNA on its target gene can be modulated by NPM1 mutation in AML. MATERIALS AND METHODS In silico data AML patients data from GDC portal (n =183) In vitro data Clinico-biological data Expression datasets NPM1 +ve (n=21) NPM1 -ve (n=162) miRNA (1881) mRNA (55640) miRNA & mRNA datasets derived from RNA-Seq (NPM1 mut scramble & NPM1 mut wildtype-mimic) miRNA-mRNA network analyses be- tween in silico and in vitro datasets High throughput transcriptional profiling (RNA sequencing)- In-vitro analyses  Knockdown of NPM1 mutation in OCI/AML3 cells by NPM1 mutation specific siRNA using lipid based reverse transfection method  Evaluation of NPM1 mutation knockdown efficiency by real time quantitative PCR (RQ-PCR) and Western blotting  High throughput RNA sequencing using NPM1 mut scramble and NPM1 mut wildtype-mimic and data analyses. Patient samples, cDNA synthesis and RQ-PCR  To validate the impact of SMC1A expression on survival between NPM1 mut and NPM1 wt patients in an independent cohort of AML patients, bone marrow samples from 67 AML patients at the time of diagnosis were collected. All patients provided written informed consent. The SYBR green based RQ-PCR assays for SMC1A was carried out using in house designed primers and standard protocols. STATISTICAL ANALYSES  Overall Survival (OS): From the date of start of induction therapy to date of last follow up or death. Results were analyzed by Kaplan-Meier method and com- pared using log-rank test. Statistical analysis was done using IBM SPSS v21. RESULTS Transcriptomics analysis from Genomic Data Commons (GDC)  In silico analyses (Schematic representation shown below)  Quantitative expression of NPM1 mutation A by RQ-PCR (Left panel) and Western blot (Right panel) OCI-AML3 siNPM1 mut OCI-AML3 siRNA Scramble Anti-NPM1 Mutation A β Actin miRNA In-silico dataset In-vitro dataset Log2 FC p-value FDR Log2 FC hsa-mir-363 1.9336 0.0002 0.0105 -2.4921 hsa-mir-1291 -1.3753 0.0004 0.0176 3.9508 hsa-mir-607 2.3239 0.0014 0.0405 -4.1551 hsa-mir-7976 -1.1299 0.0020 0.0541 -2.0771 hsa-mir-382 -1.9266 0.0027 0.0623 2.4148 hsa-mir-3912 1.0942 0.0031 0.0677 2.0188 hsa-mir-6718 -1.2294 0.0045 0.0841 -2.2291 hsa-mir-3911 2.7209 0.0047 0.0851 -2.9072 hsa-mir-5087 1.8014 0.0081 0.1154 2.4148 hsa-mir-4786 1.0669 0.0088 0.1203 -3.3666 hsa-mir-450a-1 1.4821 0.0134 0.1545 2.2221 hsa-mir-215 0.9579 0.0189 0.1765 2.8298 hsa-mir-6747 1.2568 0.0339 0.2443 -2.4921 hsa-mir-449a 1.9596 0.0536 0.3393 -2.2608  Common miRNA between in-silico and in-vitro datasets of AML with NPM1 mutation compared to NPM1 wildtype AML miRNA Mature miRNA Genes (mRNA) Log2 FC (in-silico dataset) p value (in-silico da- taset) Log2 FC (in-vitro da- taset) hsa-miR-363 hsa-miR-363-3p NRAS 0.0614 0.7581 0.3042 hsa-mir-607 hsa-mir-607 NPM1 0.2240 0.1686 0.4103 PTEN -0.3414 0.0846 0.1482 SRSF2 -0.0263 0.8232 0.0504 hsa-mir-382 hsa-miR-382-5p PTEN -0.3414 0.0846 0.1482 SF3B1 -0.1508 0.4729 0.0647 SMC3 -0.1740 0.3946 0.2259 hsa-miR-215 hsa-miR-215-3p SMC1A -0.4816 0.0464 0.7043 hsa-miR-215-5p BCOR 0.1747 0.3525 0.5026 CDKN2A 0.6033 0.1625 0.5748 PHF6 -0.2824 0.3182 0.5899 RAD21 -0.1296 0.4879 0.2637 WT1 0.0509 0.9041 -0.0557 hsa-mir-449a hsa-mir-449a NOTCH1 -0.0187 0.9394 0.3419  miRNA-mRNA interactive pairs derived from in-silico and in-vitro analyses of NPM1 mutated AML  SMC1A median expression* versus survival outcome A) NPM1 non-mutated (N=160); B) NPM1 mutated AML patients (N=20) OS at 3 yrs (high vs low) 22% vs 58%, p=0.025 High SMC1A expression Low SMC1A expression OS at 3 yrs (high vs low) 73% vs 52%, p=0.36 High SMC1A expression Low SMC1A expression * SMC1A expression was normalized with the ABL1 expression (A) (B) OS (days) OS (days) OS at 6 yrs (high vs low) 10% vs 48.7%, p<0.0001 High SMC1A expression Low SMC1A expression OS at 6 yrs (high vs low) 10% vs 48.7%, p<0.0001 High SMC1A expression Low SMC1A expression Cumulative survival Cumulative survival * SMC1A expression was normalized with the ABL1 expression (A) (B)  SMC1A median expression* versus survival outcome in an independent cohort of AML patients A) NPM1 wild type (N=40) B) NPM1 mutated patients (N=17) CONCLUSION The down - regulation of SMC1A mediated by miR - 215 suggesting its role behind the better prognosis of NPM1 mutated AML as compared to AML with wildtype NPM1. Considering the small cohort patients in the present study, the future studies are warranted to confirm these findings in a larger cohort of AML patients. ACKNOWLEDGEMENTS The authors are grateful to Prof. F. Lo-Coco from Rome, Italy, who has provided NPM1 mutation specific antibody and IEC, and Tata Memorial Centre Mumbai for funding. Validation of SMC1A Expression in an Independent Cohort of AML patients  Seventeen patients were positive for NPM1 type A mutation while 50 cases were negative for the mutation. The median age of the cohort was 27 years (range 15-58 yrs). Out of 67 patients, three patients died during induction, five were refractory to the treatment while the follow up data was not available for two patients and hence these cases were excluded from OS analysis. The OS of 57 patients was 28.5% at 3 years.