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.

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.

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.

CBF-AML is defined by the presence of either t(8;21)(q22;q22)/RUNX1-RUNX1T1 or inv(16)(p13.1q22)/t(16;16)(p13.1;q22)/CBFB-MYH11. The resulting fusion genes require a "second hit" to initiate leukemogenesis. Although compared with other AML subtypes patients with CBF-AML have a relatively favorable prognosis, still almost 40% of CBF-AML patients experience relapses of their disease.

Activating mutations in BRAF are found in 50% of melanomas and although treatment with BRAF inhibitors (BRAFi) is effective, resistance often develops. We now show that recently discovered NRAS isoform 2 is up-regulated in the setting of BRAF inhibitor resistance in melanoma, in both cell lines and patient tumor tissues. When isoform 2 was overexpressed in BRAF mutant melanoma cell lines, melanoma cell proliferation and in vivo tumor growth were significantly increased in the presence of BRAFi treatment. shRNA-mediated knockdown of isoform 2 in BRAFi resistant cells restored sensitivity to BRAFi compared with controls. Signaling analysis indicated decreased mitogen-activated protein kinase (MAPK) pathway signaling and increased phosphoinositol-3-kinase (PI3K) pathway signaling in isoform 2 overexpressing cells compared with isoform 1 overexpressing cells. Immunoprecipitation of isoform 2 validated a binding affinity of this isoform to both PI3K and BRAF/RAF1. The addition of an AKT inhibitor to BRAFi treatment resulted in a partial restoration of BRAFi sensitivity in cells expressing high levels of isoform 2. NRAS isoform 2 may contribute to resistance to BRAFi by facilitating PI3K pathway activation.