Supplementary Figures and Tables

Supplementary Methods, Supplementary References Supplementary Table S1: Inherited regions of homozygosity in >1% of 1,798 non-leukemic individuals Supplementary Table S2: Acquired uniparental disomies detected in 425 cytogenetically normal AML patients Supplementary Table S3. Associations between recurrent UPDs with pretreatment patient characteristics for patients with cytogenetically normal acute myeloid leukemia Supplementary Table S5. Variant allele fraction of gene mutations that co-occurred with UPDs Supplementary Table S6. Allelic ratio of FLT3 internal tandem duplications that co-occurred with UPDs Supplementary Table S7: Copy number gains and losses in 425 patients with cytogenetically normal acute myeloid leukemia

Mutations detected in 425 CN-AML patients

Supplementary Table 1. Linkage disequilibrium patterns of the tag-SNPs used for genotyping the 6q23.3 locus. Supplementary Table 2. Cytogenetic and molecular information on AML patients used for functional studies (n=12). Supplementary Table 3. French-American-British (FAB) classification35 and molecular information on AML patients used to determine miR-3662's abundance (n=8). Supplementary Table 4. Canonical pathway analysis of the miR-3662-associated gene expression signature. Supplementary Figure 1. Transcription factor binding according to the transcription factor chip data from ENCODE. Supplementary Figure 2. Electrophoretic mobility shift assay comparing the binding affinity of the alleles of rs66650371 and rs9483788. Supplementary Figure 3. Endogenous miR-3662 expression levels of hematopoietic progenitor (HP) cells during differentiation, total bone marrow aspirate of three non-leukemic donors (total BM 1-3), different populations of differentiated peripheral blood cells, and three AML cell lines. Supplementary Figure 4. Top panel, Macroscopic pictures of the spleens of three mice of the scramble and miR-3662-infected groups (organs harvested post-mortem). All mice had a massive splenomegaly compared to the un-injected, sacrificed control mouse. Bottom panel, images of spleen histologies (40x enlargement). Slides were stained for CD45 to proof MV4-11 origin of the leukemia. Supplementary Figure 5. Endogenous abundance of miR-3662 and IKBKB in patient samples and cell lines. Supplementary Figure 6. Comparison of the relative miR-3662 abundance of AML patient blasts and AML cell lines before (black) and after (red) forced miR-3662 expression with the lentiviral expression construct.

Abstract Clinical outcome of patients with acute myeloid leukemia (AML) is associated with cytogenetic and molecular factors and patient demographics (e.g., age and race). We compared survival of 25,523 non-Hispanic Black and White adults with AML using Surveillance Epidemiology and End Results (SEER) Program data and performed mutational profiling of 1,339 patients with AML treated on frontline Alliance for Clinical Trials in Oncology (Alliance) protocols. Black patients had shorter survival than White patients, both in SEER and in the setting of Alliance clinical trials. The disparity was especially pronounced in Black patients <60 years, after adjustment for socioeconomic (SEER) and molecular (Alliance) factors. Black race was an independent prognosticator of poor survival. Gene mutation profiles showed fewer NPM1 and more IDH2 mutations in younger Black patients. Overall survival of younger Black patients was adversely affected by IDH2 mutations and FLT3-ITD, but, in contrast to White patients, was not improved by NPM1 mutations. Significance: We show that young Black patients have not benefited as much as White patients from recent progress in AML treatment in the United States. Our data suggest that both socioeconomic factors and differences in disease biology contribute to the survival disparity and need to be urgently addressed. See related commentary by Vyas, p. 540. This article is highlighted in the In This Issue feature, p. 521

AbstractPurpose:Uniparental disomy (UPD) is a way cancer cells duplicate a mutated gene, causing loss of heterozygosity (LOH). Patients with cytogenetically normal acute myeloid leukemia (CN-AML) do not have microscopically detectable chromosome abnormalities, but can harbor UPDs. We examined the prognostic significance of UPDs and frequency of LOH in patients with CN-AML.Experimental Design: We examined the frequency and prognostic significance of UPDs in a set of 425 adult patients with de novo CN-AML who were previously sequenced for 81 genes typically mutated in cancer. Associations of UPDs with outcome were analyzed in the 315 patients with CN-AML younger than 60 years.Results:We detected 127 UPDs in 109 patients. Most UPDs were large and typically encompassed all or most of the affected chromosome arm. The most common UPDs occurred on chromosome arms 13q (7.5% of patients), 6p (2.8%), and 11p (2.8%). Many UPDs significantly cooccurred with mutations in genes they encompassed, including 13q UPD with FLT3-internal tandem duplication (FLT3-ITD; P < 0.001), and 11p UPD with WT1 mutations (P = 0.02). Among patients younger than 60 years, UPD of 11p was associated with longer overall survival (OS) and 13q UPD with shorter disease-free survival (DFS) and OS. In multivariable models that accounted for known prognostic markers, including FLT3-ITD and WT1 mutations, UPD of 13q maintained association with shorter DFS, and UPD of 11p maintained association with longer OS.Conclusions:LOH mediated by UPD is a recurrent feature of CN-AML. Detection of UPDs of 13q and 11p might be useful for genetic risk stratification of patients with CN-AML.

Introduction: The vast majority of solid tumors and half of leukemias and lymphomas possess an abnormal number of chromosomes. Individuals with congenital trisomy 21 have an increased risk of developing leukemia and respond differently to treatment when compared to leukemic individuals without congenital aneuploidies. Curiously, the chromosomes that most frequently undergo non-disjunction in malignancy are similar to the chromosomes that occur as trisomies in utero. We aimed to characterize the molecular landscapes of individuals with congenital trisomies to elucidate what changes may lead to an increased risk of cancer development in these individuals. Methods: We extracted DNA and RNA from fibroblasts of 14 individuals with congenital trisomies, including trisomy 8, 9, 13, 18 and 21 (obtained from Coriell Biorepository). DNA and RNA sequencing were performed with AmpliSeq for Illumina Comprehensive Panel v3. Variants were detected in the DNA using VarScan and annotated with SnpEff, while Kallisto was used for mRNA expression analysis. We applied mRNA context mapping methodology developed by the Computational Cancer Analysis Lab (University of California, San Diego). We used quantitative PCR and Western blotting to validate RB1 and MET expression. Results: Identification of transcripts highly expressed in multiple trisomies was performed. AXL was the only transcript that was highly expressed in all five trisomy groups, while ACBD5 and CCDC6 were each highly expressed in four. Interestingly, the number of unique highly expressed transcripts differed drastically by trisomy, ranging from n=0 for trisomy 9 to n= 111 for trisomy 13. We then generated a context map of the samples and observed that they cluster together by trisomy, with the exception of two samples. RB1 and MET were up-regulated in eleven samples, and down-regulated in the remaining three samples. We were able to validate these findings at the mRNA and protein level. Finally, we are in the process of identifying rare genetic variants that are unique to or enriched in this population. Conclusion: Our data indicate that several genes with well-described roles in cancer might also be differentially expressed in individuals with trisomies. RB1 is a tumor suppressor and previous studies have shown that pRB loss leads to centromere dysfunction and chromosomal instability, which could imply dysregulated RB1 plays a role in trisomy acquisition. MET is a proto-oncogene and is implicated as the major driver of oncogenesis in papillary renal carcinomas with chromosomal gain. Further studies that compare congenital trisomy with malignancy-associated trisomy may improve our understanding of non-disjunction and mechanisms of oncogenesis that occur in congenital trisomy patients. Citation Format: Maryam A. Bainazar, Sophia E. Maharry, Christopher J. Walker, Luke K. Genutis, Albert de la Chapelle, Ann-Kathrin Eisfeld. Genomic and transcriptomic characterization of congenital trisomy reveal possible role for RB1 and MET [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1753.

AML is a disease of the elderly. Patients aged ≥60 years (y) account for ~65% of all AML patients. However, while 40% of younger AML patients achieve long term survival, only 5-15% of older patients do. Especially given increasing availability of alternative treatment strategies the identification of patients who benefit from standard induction chemotherapy and those who do not is of crucial importance. The 2017 European LeukemiaNet Genetic Risk Classification (ELN GRC) provides guidance on genetic risk stratification of AML patients and is currently routinely used in clinical practice for both younger and older AML patients. The goal of our study was to test the performance of the 2017 ELN GRC in our cohort of 399 de novo AML patients aged ≥60 y (median: 69 y) which were similarly treated with cytarabine/daunorubicin-based chemotherapy on Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology protocols. Our cohort did not contain any patients with core-binding factor AML [i.e., with inv(16)/t(16;16) or t(8;21)]. We also aimed to identify additional molecular prognosticators in older AML patients. Utilizing the 2017 ELN GRC, 31%, 25% and 44% of patients were classified as Favorable (Fav), Intermediate (Int) and Adverse (Adv) risk, respectively. The median CR rates of 81% for Fav risk, 55% for Int risk and 39% for Adv risk separated patients in accordance with the ELN GRC groups (P<.001). However, there was no difference in disease-free (DFS) and overall survival (OS) rates for Int and Adv risk patients (3-year [3y] DFS, Int risk, 6%, Adv risk, 3%, P=.91; 3y OS; Int risk, 11%, Adv risk, 6%, P=.46). Fav risk patients had superior DFS and OS compared to both Int and Adv risk patients, but with 3y rates for DFS of 25% and OS of 29%, outcomes were still relatively poor. We next tested the prognostic impact of each 2017 ELN GRC marker in our patient cohort. Fav risk group patients with NPM1 mutations (NPM1mut) and either no FLT3-ITD or low FLT3-ITD allelic ratio (FLT3-ITDno/low) had a CR rate of 84%, while patients with biallelic CEBPAmut had a CR rate of only 54% (P=.02, Table 1). While 3y OS of NPM1mut/FLT3-ITDno/low was 31%, it was only 15% for patients with biallelic CEBPAmut (P=.13). Within Int risk patients the outcomes of NPM1mut patients with high FLT3-ITD allelic ratio (FLT3-ITDhigh) and NPM1 wildtype (NPM1wt)/FLT3-ITDno/low patients were equally dismal, and actually resembled those of NPM1wt/FLT3-ITDhigh patients (Table 1). All other patients with gene mutations classified as ELN Adv risk had poor outcomes (Table 1). Thus, the only genetic classification in 2017 ELN GRC that associated with favorable outcome in AML patients ≥60 years was NPM1mut/FLT3-ITDno/low, while biallelic CEBPAmut patients resembled more Int risk patients. NPM1mut/FLT3-ITDhigh, NPM1wt/FLT3-ITDno/low andNPM1wt/FLT3-ITDhighpatients all had CR rates in line with Int risk, but very poor DFS and OS. To potentially refine criteria used to classify patients, we performed outcome analyses of mutations co-occurring with NPM1mut on CR, DFS and OS. In addition to the known adverse impact of FLT3-ITDhigh, presence of DNMT3Amut was also associated with lower CR and OS in NPM1mut (CR, 85 vs 32%, P<.001; 3-yr OS rates, 15 vs 7%, P<.001) vs DNMT3Awt patients, resembling the outcomes of Adv risk patients. In contrast, NPM1mut patients that also harbored mutations in SRSF2 (n=18) had longer DFS and OS compared to NPM1mut/SRSF2wt patients (3-yr DFS rates, 44 vs 16%, P=.01; 3-yr OS rates, 50 vs 21%, P=.03). Thus, with the exception of the small subsets of NPM1mut/FLT3-ITDno/low and patients harboring both NPM1mut/SRSF2mut all evaluated subsets of AML patients had 3-y DFS and OS rates of <20%, indicating the need for early additional interventions in patients that achieve a CR. In summary, our data demonstrate the very poor DFS and OS of older AML patients treated with standard chemotherapy, despite the relatively high probability of achieving a CR. We show that the 2017 ELN GRC performs suboptimally to risk-stratify AML patients aged ≥60 years. NPM1mut/FLT3-ITDno/low status was the only current 2017 ELN GRC prognostic factor associated with improved outcomes in older AML patients. We identified NPM1/DNMT3A (which associated with shorter DFS+OS) and NPM1/SRSF2 (which associated with longer DFS+OS) as additional mutation combinations that might be useful for further group refinement. Disclosures Mims: Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees. Stone:Cornerstone Pharmaceuticals: Consultancy; Pfizer: Consultancy; Takeda: Other: Fees for serving on a data and safety monitoring board ; Astellas Pharma: Membership on an entity's Board of Directors or advisory committees; Stemline Therapeutics: Consultancy; Agios: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Fujifilm: Consultancy; Roche: Consultancy; Celator Pharmaceuticals: Consultancy; Ono Pharmaceutical-Theradex Oncology: Consultancy; Orsenix: Consultancy; Actinium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Arog Pharmaceuticals: Consultancy, Honoraria, Research Funding; Otsuka-Astex Pharmaceuticals: Consultancy; Daiichi Sankyo: Consultancy; Celgene: Consultancy, Other: Fees for serving on a steering committee, and fees for serving on a data and safety monitoring board; Novartis: Consultancy, Research Funding; Argenx: Other: Fees for serving on a data and safety monitoring board ; AstraZeneca: Consultancy; MacroGenics: Consultancy; Jazz Pharmaceuticals: Consultancy. Powell:Rafael Pharmaceuticals: Consultancy, Research Funding; Novartis: Consultancy, Speakers Bureau; Janssen: Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Research Funding. Wang:Pfizer: Other: Advisory role, Speakers Bureau; Stemline: Other: Advisory role, Speakers Bureau; Jazz: Other: Advisory role; Astellas: Other: Advisory role, Speakers Bureau; celyad: Other: Advisory role; Abbvie: Other: Advisory role; Kite: Other: Advisory role; Daiichi: Other: Advisory role; Amgen: Other: Advisory role; Agios: Other: Advisory role. Kolitz:Boeringer-Ingelheim: Research Funding; Roche: Research Funding; Astellas: Research Funding. Byrd:Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Acerta: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding.

Context Previous genome-wide association studies have shown that single-nucleotide polymorphism (SNP) rs2439302 in chromosome 8p12 is significantly associated with papillary thyroid carcinoma (PTC) risk and dysregulated NRG1 expression. The underlying mechanisms remain to be discovered. Objective To evaluate the expression of NRG1 isoforms, candidate functional variants, and potential genes downstream of NRG1 in thyroid tissue. Methods Quantitative reverse transcription polymerase chain reaction was applied for gene expression analysis. SNaPshot assay, haplotype, and computer analyses were performed to evaluate candidate functional variants. Other functional assays [chromatin immunoprecipitation (ChIP) assay, luciferase assay, small interfering RNA knockdown, and RNA sequencing] were performed. Results Three NRG1 isoforms (NM_004495, NM_013958, and NM_001160008) tested were highly expressed in thyroid tissue. The expression levels of the three isoforms were significantly correlated with the genotypes of rs2439302. A DNA block of ~32 kb containing the risk G allele of rs2439302 was revealed, harboring multiple candidate functional variants. ChIP assay for active chromatin markers indicated at least nine regions in the DNA block showing strong H3Kme1 and H3K27Ac signals in thyroid tissue. Luciferase reporter assays revealed differential allelic activities associated with seven SNPs. Knocking down NRG1 in primary thyroid cells revealed downstream or interacting genes related to NRG1. Conclusions Our data suggest a role for transcriptional regulation of NRG1 in the predisposition to PTC.

Targeted mutation assessment of 81 genes in 1021 adults with de novo acute myeloid leukemia (AML) identified recurrent mutations in the neurofibromin 1 (NF1) gene in 52 (5.1%) patients, including 36 (5.2%) younger and 16 (4.8%) older patients, which suggests NF1 belongs to the 20 most frequently mutated genes in adult AML. NF1 mutations were found throughout the gene, and comprised missense, frameshift, and nonsense mutations. One mutation hotspot, at amino acid threonine 676 (Thr676), was found in 27% of AML patients with NF1 mutations. NF1-mutated patients belonged more often to the adverse European LeukemiaNet (ELN) risk category than NF1 wild-type patients. Among patients aged <60 years, the presence of NF1 Thr676 mutations was associated with lower complete remission (CR) rates (P = 0.04) and shorter overall survival (OS; P = 0.01), as was the presence of any NF1 mutation in patients in the adverse ELN risk category (CR, P = 0.05; OS, P < 0.001). CR rates were also lower in NF1-mutated patients aged ≥60 years compared with NF1 wild-type patients (P = 0.001). In summary, our findings provide novel insights into the frequency of NF1 mutations in AML, and are suggestive of an adverse prognostic impact in patients treated with standard chemotherapy.

Thus far, only 5–15% of AML patients aged ≥60 years are cured with chemotherapy. Identification of patients who are less (more) likely to respond to standard chemotherapy might enable early risk stratification toward alternative treatment regimens. We used a next-generation sequencing panel of 80 cancer- and/or leukemia-associated genes to profile molecularly 423 older patients with de novo AML. Using variables identified in multivariable models and co-occurring mutations in NPM1 -mutated AML, we classified the patients into good-, intermediate-, and poor-risk groups for complete remission (CR) attainment, disease-free (DFS), and overall survival (OS). Whereas 81% of good-risk patients (comprising NPM1 -mutated patients harboring mutations in chromatin remodeling, cohesin complex, methylation-related, spliceosome, and/or RAS pathway genes, FLT3 -TKD, and/or patients without FLT3 -ITD) achieved a CR, only 32% of poor-risk patients (with U2AF1 , WT1 mutations and/or complex karyotype) did. Intermediate-risk patients had a 50% CR rate. Similarly, using NPM1 co-mutation patterns and SF1 mutation status, we identified patients with favorable DFS and OS 3-year rates of 46% and 45%, respectively. Patients with adverse genetic features had DFS and OS rates of only 2% and 4%. We show that application of our proposed criteria may refine the 2017 European LeukemiaNet classification for older patients treated with chemotherapy.

Significance This study indicates that one of the isoforms of NRAS, specifically NRAS isoform 2, plays a role in BRAF inhibitor resistance by facilitating alternative survival signaling through the PI3K pathway in the presence of MAPK pathway inhibition. Targeting NRAS isoform 2 may be a beneficial treatment strategy in the prevention and management of BRAF inhibitor resistance in melanoma.

Abstract Chromosomal aberrations and multiple genome-wide association studies (GWAS) have established a major hematopoietic quantitative trait locus in chromosome 6q23.3. The locus comprises an active enhancer region, in which some of the associated SNPs alter transcription factor binding. We now identify miR-3662 as a new functional driver contributing to the associated phenotypes. The GWAS SNPs are strongly associated with higher miR-3662 expression. Genome editing of rs66650371, a three-base-pair deletion, suggests a functional link between the SNP genotype and the abundance of miR-3662. Increasing miR-3662′s abundance increases colony formation in hematopoietic progenitor cells, particularly the erythroid lineage. In contrast, miR-3662 is not expressed in acute myeloid leukemia cells, and its overexpression has potent antileukemic effects in vitro and in vivo. Mechanistically, miR-3662 directly targets NF-κB–mediated transcription. Thus, miR-3662 is a new player of the hematopoietic 6q23.3 locus. Significance: The characterization of miR-3662 has identified a new actor in the prominent hematopoietic quantitative trait locus in chromosome 6q23.3. The mechanistic insights into miR-3662′s function may reveal novel or only partially known pathways for normal and malignant hematopoietic cell proliferation. Cancer Discov; 6(9); 1036–51. ©2016 AACR. This article is highlighted in the In This Issue feature, p. 932

The B-Raf proto-oncogene serine/threonine kinase (BRAF) gene is the most frequently mutated gene in malignant melanoma (MM) and papillary thyroid cancer (PTC) and is causally involved in malignant cell transformation. Mutated BRAF is associated with an aggressive disease phenotype, thus making it a top candidate for targeted treatment strategies in MM and PTC. We show that BRAF mutations in both MM and PTC drive increased expression of oncomiR-3151, which is coactivated by the SP1/NF-κB complex. Knockdown of microRNA-3151 (miR-3151) with short hairpin RNAs reduces cell proliferation and increases apoptosis of MM and PTC cells. Using a targeted RNA sequencing approach, we mechanistically determined that miR-3151 directly targets TP53 and other members of the TP53 pathway. Reducing miR-3151's abundance increases TP53's mRNA and protein expression and favors its nuclear localization. Consequently, knockdown of miR-3151 also leads to caspase-3-dependent apoptosis. Simultaneous inhibition of aberrantly activated BRAF and knockdown of miR-3151 potentiates the effects of sole BRAF inhibition with the BRAF inhibitor vemurafenib and may provide a novel targeted therapeutic approach in BRAF-mutated MM and PTC patients. In conclusion, we identify miR-3151 as a previously unidentified player in MM and PTC pathogenesis, which is driven by BRAF-dependent and BRAF-independent mechanisms. Characterization of TP53 as a downstream effector of miR-3151 provides evidence for a causal link between BRAF mutations and TP53 inactivation.

Background: Overcoming the block of differentiation is a major effort in Acute Myeloid Leukemia (AML) research. We visited the major quantitative trait locus (QTL) for hematopoietic differentiation on chromosome 6q23 to identify genes involved in hematopoietic development that are possibly deregulated during leukemogenesis. We noticed a novel microRNA within the QTL: miR-3662. Aims: The goal was to elucidate a possible causal involvement of miR-3662 in the association of the 6q23 QTL with hematopoietic differentiation and/or leukemogenesis. Results: Monitoring of miR-3662 expression during cytokine stimulated differentiation of CD34+ hematopoietic progenitor cells (HPCs) revealed increasing miR-3662 abundance during differentiation, with the highest expression found in terminally differentiated cells. In contrast, miR-3662 had very low expression in AML cell lines (KG1a, MV4-11, OCI-AML3, THP1) and primary AML patient blasts (n=12). We stably introduced miR-3662 into HPCs of 3 non-leukemic donors, and differentiated the cells into erythroid, megakaryocytic and granulocytic lineages. Forced expression of miR-3662 led to significant increase of colony formation, most pronounced in the erythroid lineage. In contrast, targeted knock-down of miR-3662 significantly reduced colony formation (Figure 1). To test if the observed downregulation of miR-3662 in leukemic cells is favorable to leukemic cells, we stably introduced miR-3662 in AML cell lines (MV4-11, KG1a) and AML patient blasts (n=12). Increasing the abundance of miR-3662 led to reduction of cell viability, increased cell death and reduced colony formation. To find the downstream targets of miR-3662, we performed a comprehensive targeted RNA sequencing approach with a panel of 361 genes (TruSeq platform, Illumina; followed by Ingenuity Pathway analysis), using RNA from two AML patient blasts infected with miR-3662 vs. scramble. miR-3662 was associated with >20% reduction in the mRNA expression of 34 genes. Among these, 6 genes harbored predicted miR-3662 binding sites in their 3′-UTRs, qualifying as potential direct miR-3662 targets. Of these, the inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKBKB) had the highest affinity score for miR-3662, and was thus chosen as the primary candidate. Determination of endogenous miR-3662 and IKBKB expression in primary AML patient blasts (n=12) showed a significant association of higher miR-3662 with lower IKBKB abundance. qPCR and Western blotting confirmed miR-3662 mediated downregulation of IKBKB in HPCs, KG1a and MV4-11 cells and primary patient blasts. Because IKBKB leads to reduced phosphorylation of the inhibitor in the inhibitor/NF-ĸB complex (IKBa), it limits NF-ĸB9s nuclear localization and consequent transcriptional activation. Increasing miR-3662 should thus decrease NF-ĸB9s nuclear localization. Western blotting showed reduced phosphorylation of IKBa and confocal imaging showed less NF-ĸB in the nucleus in miR-3662 expressing MV4-11 and HPC cells (Figure 2). Luciferase assay validated a direct interaction of miR-3662 with the 3′-UTR of IKBKB. Finally, we attempted to gain insights into the upstream regulation of miR-3662. We identified a possible transcription start site for miR-3662 (TSS-3662), whose activation potential was validated by both luciferase assays and enrichment of RNA polymerase II (Pol II), histone H3 methylated Lys (H3K4), and total histone H3 CHIP assays of TSS-3662. Testing of predicted activating transcription factors showed GATA1 and CEBPA to have an activating potential (as shown by luciferase assays) and binding affinity (as shown by electrophoretic mobility shift assays) to TSS-3662. Overexpression of GATA1 and CEBPA significantly increased miR-36629s abundance. Conclusion: We have identified miR-3662, located in the major hematopoietic QTL on chromosome 6q23, as a novel regulator of hematopoietic differentiation that acts by regulating the NF-ĸB pathway. Reduced abundance of miR-3662 in leukemic cells potentially contributes to the de-differentiated phenotype of leukemic cells and to increased cell growth by increasing nuclear localization of NF-ĸB. Disclosures Walker:Karyopharm Therapeutics Inc.: Research Funding.

Abstract Background: Despite all progress, the prognosis of many acute myeloid leukemia (AML) patients remains poor. Identification of candidate genes for targeted treatment strategies may be beneficial for patient survival. Using online prediction programs, we identified miR-3662 as a novel tumor suppressor candidate in AML. Its predicted targets include numerous genes involved in hematopoietic differentiation and leukemogenesis, such as the oncogene BAALC. So far, miR-3662 has not been implicated in cancer biology. Aims: First, we strived to elucidate the role of miR-3662 in hematopoietic differentiation and leukemogenesis. Second, we aimed to experimentally validate BAALC as a miR-3662 target to explore the downstream biology of the tumor suppressor candidate. Methods & Results: As tumor suppressor genes are often downregulated in cancer, we determined the expression of miR-3662 in peripheral blood blasts from cytogenetically normal (CN-) AML patients (n=10) and peripheral blood samples from non-cancer controls (n=20). Indeed, the non-cancer controls had an average 97-fold (P≤.001) higher miR-3662 expression compared to CN-AML patients, suggesting a downregulation of miR-3662 in leukemic cells. Next, we assessed the effects of forced miR-3662 expression and knock-down of miR-3662 on the growth and colony formation of hematopoietic progenitor cells (CD34+) from three healthy donors. While miR-3662 increased cell growth and colony formation, antagomiR-3662 reduced both parameters (miR-3662 vs. scramble: P≤.001; antagomiR-3662 vs. scramble: P≤.001). In contrast, colony forming assays with leukemia cell lines (OCI-AML3, KG1a) after stable infection with miR-3662 versus scramble reduced the cell growth and colony formation (miR-3662 vs. scramble: both P≤.001). To assess the effect of miR-3662 on leukemia cell survival, we performed caspase-3/7 assays with the leukemic blasts of five CN-AML patients infected with miR-3662 or scramble. miR-3662 increased caspase-3/7 activity (miR-3662 vs. scramble, P=.004), indicating miR-3662-mediated increased apoptosis. Thus, miR-3662 has tumor suppressive potential. Next we tested the effects of miR-3662 on the expression of its predicted target gene BAALC by overexpressing miR-3662 in two AML cell lines (KG1a, MV4-11) and blasts of CN-AML patients (n=10). miR-3662 reduced BAALC expression in both AML cell lines (KG1a: 70% reduction, MV4-11: 75% reduction, both P≤.001) and in the CN-AML patients (average reduction: 38.6%, P=.007). Finally, we performed luciferase assays with a BAALC 3′-UTR construct. Co-transfection with miR-3662 led to a 38% reduction of luciferase activity (miR-3662 vs. scramble, P≤.001), indicating that miR-3662 directly targets BAALC. Conclusion: miR-3662 is a novel tumor suppressor miR that has potential implications for leukemogenesis, as it increased colony formation and differentiation in CD34+ cells and decreased growth of leukemic cells. Citation Format: Sophia Maharry, Sujay Mehta, Sandya Liyanarachchi, Guido Marcucci, Clara D. Bloomfield, Albert de la Chapelle, Ann-Kathrin Eisfeld. Identification of microRNA-3662 as a novel tumor suppressor miR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5235. doi:10.1158/1538-7445.AM2014-5235