Abstract 519: The long non-coding RNA (lncRNA) HOXB-AS3 regulates transcription of ribosomal RNA (rRNA) in NPM1-mutated (NPM1mut) acute myeloid leukemia (AML)
Authors: Papaioannou Dimitrios , Petri Andreas , Terreri Sara , Thrue Charlotte A. , Nicolet Deedra , Collins Frances A. , Woodward Lauren A. , Kumchala Prasanthi , Karunasiri Malith , Pepe Felice , Bill Marius , Zitzer Nina , Singh Guramrit , Kaupinnen Sakari , Bloomfield Clara D. , Dorrance Adrienne M. , Garzon Ramiro 
Published: August 2018

Background: A HOXB-locus-embedded lncRNA, named HOXB-AS3 significantly associates with NPM1 mutations in AML. Herein, we evaluate the functional role of HOXB-AS3 expression in NPM1mut AML.

Methods: HOXB-AS3 expression was measured by real-time PCR. Knock-down (KD) of HOXB-AS3 was performed in vitro and in vivo with locked nucleic acid-modified gapmers. RNA antisense purification (RAP), RNA-immunoprecipitation (RIP), and Chromatin-immunoprecipitation (ChIP) experiments were performed according to published protocols.

Results: Among 7 AML cell lines tested, only OCI-AML3 cells, which harbor NPM1mut, showed detectable HOXB-AS3 expression. HOXB-AS3 was more abundant in NPM1mut AML patient (pt) blasts than blasts of AML pts with wild-type NPM1 (P=.001) and bone marrow samples from healthy donors(P=.001). HOXB-AS3 localized in the nucleus and did not associate with isolated polysomes of OCI-AML3 cells.

In vitro HOXB-AS3 KD in OCI-AML3 cells decreased the cells in S phase (P&lt;.001) and increased those in G2/M phase (P=.006). HOXB-AS3 KD reduced the number of formed colonies by OCI-AML3 cells (P=.02). In contrast, overexpression of HOXB-AS3 in K562 cells increased the cells in S phase (P=.02) and decreased those in G0/G1 phase (P=.008). HOXB-AS3 KD in blasts of 3 NPM1mut AML pts decreased the number of formed colonies (P=.03, P=.02, and P&lt;.001). In vivo HOXB-AS3 KD in murine patient-derived xenografts of 2 NPM1mut AML pts prolonged their overall survival (P&lt;.001 and P=.03). RAP-based isolation of HOXB-AS3 and comparative proteomic analyses identified 23 candidate HOXB-AS3-binding proteins. EBP1 was validated as the most avid HOXB-AS3 interactor (P&lt;.001) by RIP experiments. Manipulations of HOXB-AS3 impacted on the (previously reported) EBP1 interaction with NPM1; HOXB-AS3 KD reduced, whereas overexpression of HOXB-AS3 increased the EBP1-NPM1 complex formation. Consequently, HOXB-AS3 KD reduced transcription of rRNA and de novo protein synthesis in OCI-AML3 cells (P&lt;.001 and P=.002) and AML pt blasts (P&lt;.001 and P=.03, respectively). Overexpression of HOXB-AS3 increased rRNA transcription (P&lt;.001), de novo protein synthesis (P=.001). ribosomal DNA (rDNA) promoter occupancy by RNA-Polymerase I (P=.001), and activity of an rDNA promoter-containing luciferase reporter (P=.002) in K562 cells. We hypothesized that HOXB-AS3 guides EBP1 to the rDNA locus. RAP-DNA experiments validated the interaction of HOXB-AS3 with rDNA chromatin (P=.001) and HOXB-AS3 KD decreased the occupancy of the rDNA promoter by EBP1 (P=.002), as shown by ChIP assays.

Conclusions: We describe the function of the HOXB-AS3 lncRNA as a compensatory mechanism, which mediates increased rRNA transcription and adequate protein production, in

NPM1mut AML. From a therapeutic standpoint, we show that HOXB-AS3-targeting yields anti-leukemic activity in pre-clinical models.

Citation Format: Dimitrios Papaioannou, Andreas Petri, Sara Terreri, Charlotte A. Thrue, Deedra Nicolet, Frances A. Collins, Lauren A. Woodward, Prasanthi Kumchala, Malith Karunasiri, Felice Pepe, Marius Bill, Nina Zitzer, Guramrit Singh, Sakari Kaupinnen, Clara D. Bloomfield, Adrienne M. Dorrance, Ramiro Garzon. The long non-coding RNA (lncRNA) HOXB-AS3 regulates transcription of ribosomal RNA (rRNA) in NPM1-mutated (NPM1mut) acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 519.