Abstract Introduction: Human bone marrow aging is typified by decreased cellularity, stem cell exhaustion and myeloid lineage bias that may set the stage for development of myeloid malignancies. Secondary AML (sAML) is a malignancy that has been associated with alterations in RNA processing genes and currently has few effective treatment options available. A central goal of future therapeutic strategies is to prevent disease relapse and therapeutic resistance by selectively targeting unique gene products that are essential to LSC but not normal HSC function. Therefore, we established whole gene, long non-coding RNA (lncRNA), splice isoform, and RNA editing signatures of benign versus malignant bone marrow progenitor cell aging, and evaluated the therapeutic efficacy of splicing-targeted agents in pre-clinical humanized in vitro and in vivo model systems. Methods: Whole transcriptome sequencing (RNA-Seq) was performed on FACS-purified hematopoietic stem (CD34+CD38-Lin-) and progenitor cells (CD34+CD38+Lin-) from aged (average age = 65.9 ± 6.8 years old) versus young (average age = 25.8 ± 3.0 years old) adult healthy bone marrow samples, and in leukemia stem cells (LSC) from patients with sAML (average age = 71.4 ± 7.9 years old). Comparative gene set enrichment analyses (GSEA), splice isoform, lncRNA, and RNA editing profiles were identified for normal and malignant progenitor cell aging. Then, we evaluated the spliceosome modulatory agent 17S-FD-895 in splicing reporter activity, PCR, and functional in vitro hematopoietic progenitor and in vivo LSC primagraft assays. Results: Disruption of pre-mRNA splicing activity has recently been implicated as a therapeutic vulnerability in some types of cancer. Comparative whole transcriptome RNA sequencing (RNA-seq) analyses revealed pre-mRNA splicing factor gene expression was significantly disrupted in human AML LSC compared with age-matched normal progenitors. Comparative splice isoform RNA-seq and qRT-PCR validation revealed recurrent intron retention and exon skipping in expressed transcripts, such as PTK2B and several protein phosphatase gene products. Notably, transcription factor profiling of AML LSC demonstrated downregulation of key tumor suppressor genes, such as IRF8 and TP53. We then investigated the LSC inhibitory efficacy of a stable and potent splicing modulatory agent, 17S-FD-895, in humanized stromal co-culture and AML LSC primagraft assays. Pharmacological spliceosome modulation disrupted AML LSC maintenance in vivo by altering splicing of stem cell survival and AML-associated transcripts at doses that spared normal hematopoietic progenitors. Conclusions: Detection and targeted modulation of aberrant RNA processing provides an innovative strategy for AML LSC eradication with implications for treatment of a variety of human malignancies and other age-related disorders. Citation Format: Leslie A. Crews, Larisa Balaian, Heather S. Leu, Nathaniel P. Delos Santos, Angela C. Court, Anil Sadarangani, Maria A. Zipeto, James J. La Clair, Reymundo Villa, Sheldon R. Morris, Rainer Storb, Anna Kulidjian, Edward D. Ball, Michael D. Burkart, Catriona H.M. Jamieson. RNA processing signatures of normal versus malignant progenitor cell aging predict leukemia stem cell sensitivity to RNA splicing modulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 915.