HOXB-AS3 Regulates Cell Cycle Progression and Interacts with the Drosophila Splicing Human Behavior (DSHB) Complex in NPM1-Mutated Acute Myeloid Leukemia

Background:The expression and prognostic significance of long non-coding RNAs (lncRNAs) in older (≥60 years) patients with cytogenetically normal acute myeloid leukemia (CN-AML) has recently been reported (Garzon et al, 2014). Among the top up-regulated lncRNAs in NPM1-mutated (NPM1mut) CN-AML, the lncRNA HOXB-AS3 wasidentified. As aberrant expression of HOX genes is associated with NPM1mut AML and HOX-related lncRNAs have been reported to regulate HOX genes (e.g. HOTAIR), we hypothesized that HOXB-AS3 could have a functional role in this disease setting.

Methods:HOXB-AS3 expression profiling was performed by qRT-PCR in AML cell lines (Kasumi-1, KG1a, MOLM-13, MV-4-11, OCI-AML3 and THP-1), 12 primary CN-AML patient samples (NPM1mut=6, NPM1-wild-type (WT)=6) and mononuclear cells from healthy donors (n=6). Correlation analysis of mRNA/lncRNA expression was performed using Spearman analysis in 71 older CN-AML patients, profiled by RNA-sequencing. Knock-down (KD) of HOXB-AS3 was performed in vitro (Amaxa-nucleoporation) and in vivo (in a patient-derived xenograft (PDX) model) using LNA-modified gapmers. Comparative proteomic analysis was conducted by applying a modified version of the RNA antisense purification (RAP) protocol (McHugh et al, 2015).

Results:Of the cell lines tested, only OCI-AML3, which harborsmutated NPM1, showed high levels of HOXB-AS3 expression. Five- and 3-prime RACE assays identified 3 previously annotated (NR_033201/NR_033203/ENST000491264) and 1 novel variant of HOXB-AS3 in OCI-AML3 cells. NPM1 mutated patient samples exhibited higher expression of HOXB-AS3 in comparison to those with WT NPM1 (P<.01) and healthy donors (P<.01). To gain insights into the function of HOXB-AS3, we derived a HOXB-AS3-related mRNA expression signature; 154 mRNA transcripts correlated positively and 122 correlated negatively with high HOXB-AS3 expression. Gene ontology analysis revealed enrichment of genes involved in the pathway of DNA repair (P<.01) in patients with high HOXB-AS3 expression.

To assess the functional impact of HOXB-AS3, HOXB-AS3 KD studies using LNA gapmers were performed. In vitro KD of HOXB-AS3 led to decreased proliferation of OCI-AML3 cells, as measured by BrdU-based cell cycle analysis (S-phase average % in control v KD: 24% v 16%, P=.02). HOXB-AS3 KD also led to a reduction in the number of formed colonies by OCI-AML3 cells (P<.01), whereas it had no significant effect on apoptosis. HOXB-AS3 KD in primary NPM1mut AML samples (n=3) led to a decrease in colony formation (P<.01). Gene set enrichment analysisperformed in HOXB-AS3 silenced OCI-AML3 cells (profiled by RNAseq) revealed activation of the DNA damage response pathway in the HOXB-AS3 expressing cells.

To assess the in vivo effects of HOXB-AS3 KD,we treated 10 NSG mice transplanted with the blasts of a NPM1mut AML patient with nanoparticle-formulated anti-HOXB-AS3 gapmers or control gapmers. We obtained RNA after 7 days of treatment and confirmed HOXB-AS3 KD in human cells (P<.01). Survival analysis is currently ongoing but when we compared the leukemia burden at 3- and 6-weeks post-transplant in mice with adequate leukemia engraftment (>30% human CD45+ cells) the percentage of circulating blasts decreased in HOXB-AS3-KD (from 43% to 30%), while it increased in controls (from 36.5% to 62.8%). Last, to identify potential HOXB-AS3-binding proteins we performed RAP experiments in OCI-AML3 cells. HOXB-AS3- and U1-specificRNA-protein complexes were isolated by hybridization with biotinylated probes. Mass spectrometry and comparative analysis of theeluates yielded 22 candidate HOXB-AS3-interacting RNA-binding proteins. These included the core components of the DSHB complex (SFPQ and NONO). RNA-immunoprecipitation experiments validated the interaction of HOXB-AS3 with NONO. The DSHB complex is a major mediator of the DNA damage response and experiments that will elucidate the importance of HOXB-AS3 interaction with NONO in this context are underway.

Conclusions: HOXB-AS3 is highly expressed in NPM1mut CN-AML and regulates cell cycle progression of AML blasts. HOXB-AS3 KD decreases cell proliferation and colony formation capacity of leukemia cells in vitro. HOXB-AS3 KD also decreases leukemia burden in an in vivo PDX model. Comparative proteomic analyses identified several binding partners of HOXB-AS3, such as the DSHB complex, which is a key mediator of the DNA repair process.