Refractory and relapsed disease is the greatest challenge in acute myeloid leukemia (AML), and we have shown that blood vessels serve as sanctuary sites for AML. Using a high throughput screening assay mimicking AML in the vascular niche, we screened 31 million compounds and identified a hit compound 2470-51 that selectively killed AML cells and CD34+CD38-CD123+ AML stem cells, while sparing bone marrow-derived endothelial cells, normal hematopoietic stem and progenitor cells (HSPC) as well as CD4+ T lymphocytes from healthy volunteers. In vivo AML patient-derived xenograft modeling further validated the efficacy of 2470-51 as a selective anti-leukemic agent compared to cytarabine (conventional control). We performed quantitative proteomics combining ITRAQ differential protein expression analysis, label-free shotgun analysis and SPR imaging to identify heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC) as the target of 2470-51. HNRNPC depletion in AML cell lines THP1, K562, MV411 and HL-60 significantly decreased cell proliferation, viability and clonogenic capacity of the AML cells. In contrast, normal mesenchymal/fibroblastic cells, endothelial cells and normal HSPCs from human umbilical cord blood specimens were unaffected by HNRNPC depletion, indicating the selective dependence of AML cells on hnRNPC. Furthermore, clinical studies using TCGA AML datasets showed significant survival advantage associated with lower HNRNPC expression. RNA-seq analyses revealed a distinct gene expression pattern suggesting widespread inhibition of Myc transcriptional targets. Using differential alternative splicing and differential transcript expression analyses, we discovered significant alternative splicing of MAX after HNRNPC depletion, resulting in MAX transcript isoforms that lacked Myc-interacting domains (“inactive” MAX). These Myc-interacting domains are necessary for the obligate heterodimerization of Myc and Max and are critical for Myc transcriptional activation. We further analyzed splicing landscapes of 578 AML patients and 33 healthy controls and identified substantial mis-splicing of mRNA in AML patients, despite the absence of somatic gene mutations of splicing factors, when compared to the healthy controls. Moreover, we found significant overexpression of “inactive” MAX isoforms in the healthy controls, while AML patients overexpressed fully functional MAX isoforms, suggesting that MAX splicing may have an important functional role in AML. Collectively, our studies show significant RNA splicing changes in AML and an essential role of HNRNPC in AML in contrast to normal hematopoietic and stromal cells where HNRNPC is dispensable. We present a pharmacologic agent for targeting HNRNPC and Myc-Max as a molecular mechanism of action. Our data indicate that hnRNPC is a critical factor in AML and inhibiting this splicing repressor may represent a new therapeutic strategy.

Citation Format: Vindhya Vijay, Amy Meacham, Lauren Katzell, Aaron Winer, Jesse Terrell, Vincent Archibald, Jodi Bubenik, Alberto Riva, Jon Boatwright, Cristina Tognon, Jeffrey Tyner, Brian Druker, Christopher Cogle. Splicing repressor HNRNPC is an indispensable and 'druggable' target in acute myeloid leukemia [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 3042.