LncRNA LFQR promotes cytoactivity and resistance to quizartinib via FLT3 transcriptional activation in FLT3-ITD acute myeloid leukemia

doi:10.21203/rs.3.rs-5224881/v1

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LncRNA LFQR promotes cytoactivity and resistance to quizartinib via FLT3 transcriptional activation in FLT3-ITD acute myeloid leukemia

https://doi.org/10.21203/rs.3.rs-5224881/v1

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FMS-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD) acute myeloid leukemia (AML) is an adverse subtype of leukemia associated with a poor patient prognosis. FLT3 inhibitors exhibit promising clinical activity, however, their effectiveness is limited by drug resistance. Therefore, overcoming resistance to FLT3 inhibitors remains an important clinical problem, necessitating the development of new strategies. In this study, we identified a lncRNA, AL713998.1, which was upregulated in both FLT3-ITD AML and quizartinib-resistant cells (LFQR). In vitro experiments showed that LFQR silencing significantly inhibited cell proliferation, impaired self-renewal capacity, caused cell cycle arrest and induced cell differentiation, specifically in FLT3-ITD AML. In addition, the interaction of LFQR with SFPQ was verified using RNA pull-down assays, mass spectrometry and RNA immunoprecipitation. Mechanistically, LFQR promotes SFPQ binding to the promoter region of FLT3 and facilitates transcriptional regulation, thereby increasing the expression of kinase and oncoproteins in FLT3-ITD AML cells. Notably, LFQR inhibition also impaired the cytoactivity of quizartinib cells and reversed their resistance to quizartinib. Overall, our study highlights that LFQR may serve as a potential therapeutic target in FLT3-ITD AML, and its knockdown can significantly improve the efficacy of quizartinib, thus providing a promising strategy for overcoming resistance to FLT3 inhibitors.

Biological sciences/Cancer/Haematological cancer/Leukaemia/Acute myeloid leukaemia

Health sciences/Diseases/Cancer/Cancer therapy/Targeted therapies

FLT3-ITD mutation

AML

lncRNA

AL713998.1

SFPQ

transcription

Acute myeloid leukemia (AML) is a severe and complex hematologic malignancy characterized by uncontrolled proliferation and impaired differentiation of myeloid precursor cells in the bone marrow and peripheral blood[1]. Oncogenic mutations disrupt normal hematopoiesis and promote leukemogenesis in AML[2, 3], with internal tandem duplication (ITD) within FMS-like tyrosine kinase 3 (FLT3) being one of the most frequently mutations, occurring in approximately 25% of patients[4]. The ITD mutation disrupts the autoinhibitory conformation of the FLT3 receptor and subsequently triggers the receptor tyrosine kinase and downstream signaling pathways, including the RAF/MEK/ERK, PI3K/AKT, and STAT pathways, which mediate malignant proliferation and differentiation inhibition[5, 6]. Patients with AML with FLT3-ITD mutations have poor outcomes owing to higher relapse rates and poorer overall survival (OS) than patients with wild-type FLT3[7, 8]. Recently, new treatment strategies aimed at improving the outcome of AML with FLT3-ITD by targeting FLT3 mutations have shown promising efficacy. Notably, the second generation FLT3 inhibitor quizartinib (AC220) has demonstrated a promising response rate, improved overall survival, and a favorable safety profile in the treatment of FLT3-ITD AML[9, 10]. However, some patients develop drug resistance soon after receiving the treatments due to primary or secondary mechanisms, resulting in relapse or refractoriness[11-14]. Consequently, the adverse outcomes associated with quizartinib resistance have promoted an ongoing search for new therapeutic targets for FLT3-ITD AML treatment.

Therapeutic failure in cancers can be attributed to drug resistance mediated by “Drug-Tolerant Persister” (DTP) cells present in tumors, often referred to as persistent cells. DTPs have been widely reported in different types of cancers, including leukemia, breast cancer, and lung cancer [15-17]. Notably, the presence of drug-resistant leukemic blast cells in patients with AML is considered a potential cause of FLT3 inhibitor resistance, leading to a high risk of relapse and worse survival outcomes[18, 19]. Therefore, there is an urgent need to address the issue of quizartinib resistance and improve its efficacy.

Long non-coding RNAs (lncRNAs), which are more than 200 nucleotides in length and do not encode proteins, are crucial in the development and progression of multiple cancers[20]. The molecular mechanisms of lncRNAs in cellular processes include gene transcription, translation, and chromatin structure regulation[21]. For instance, lncRNA LAMP5-AS1 promotes cell survival, self-renewal, and differentiation in mixed-lineage-leukemia (MLL) by preventing the degradation of MLL fusion proteins at the transcriptional level [22, 23]. The regulation of lncRNAs in various cell functions, such as cell cycle, apoptosis, and autophagy, havs been confirmed to be associated with the effectiveness of conventional chemotherapeutic agents in AML[24]. RUNXOR, a lncRNA that interacts with RUNX1, enhances cell sensitivity to cytarabine (Ara-C) in AML[25]. Although current studies had established the potential roles of lncRNAs in chemosensitivity, little was known about the effect of lncRNAs on drug sensitivity to FLT3 inhibitors, especially quizartinib.

In this study, we identified a lncRNA named LFQR (a lncRNA associated with quizartinib resistance in FLT3-ITD AML) upregulated in FLT3-ITD AML. LFQR knockdown specifically inhibited cell proliferation, induced differentiation, caused cell cycle arrest, and impaired the self-renewal capacity of FLT3-ITD AML and quizartinib-resistant cells. Notably, LFQR silencing enhanced the sensitivity of quizartinib-resistant cells. Mechanistically, LFQR promoted splicing factor proline/glutamine-rich (SFPQ) to bind to the promoter of the oncogene FLT3 and, in turn, modulated transcription. This finding presents not only a promising therapeutic target, but also an actionable approach to overcome quizartinib resistance in FLT3-ITD AML.

Patient sample

Bone marrow samples were collected from four patients with AML at the time of their initial diagnosis, with informed consent obtained from patients treated at the First Affiliated Hospital of Sun Yat-sen University. Ethical approval for sample collection was granted by the Hospital’s Protection of Human Subjects Committee [2020143]. Detailed clinicopathological characteristics of the patients are shown in Supplementary Table 1.

Animal model

Five-week-old male NOD-SCID mice were maintained under specific pathogen-free conditions in the Laboratory Animal Center of Sun Yat-sen University. Subsequent procedures were performed in accordance with the institutional ethical guidelines for animal experiments. The NOD-SCID mice were randomly assigned to three groups (PBS, shNC, and sh-LFQR). MOLM-13 cells or MOLM-13-RQ cells stably expressing sh-NC, sh- LFQR were tail vein injected into the mice (2 × 10⁶ cells in 150 μL PBS per mouse). For the control, 150 μL of PBS without cells was injected. Three weeks after inoculation, the mice were sacrificed for analysis. Human cells engrafted in the bone marrow, peripheral blood, or organ tissues (spleen and liver) were incubated with human CD45 (hCD45+) and evaluated using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). The differentiation status of hCD45 + cells incubated with human CD11b (BD Biosciences) or CD14 (Invitrogen) was analyzed using flow cytometry. Haematoxylin and eosin (H&E) staining was performed as previously described[26]. The remaining mice were used for the survival assays. The Laboratory Animal Center of Sun Yat-sen University approved all animal experiments [SYSU-LS-IACUC-2023-0099].

Cell lines and cell culture

Human FLT3-ITD+ cells (MOLM-13 and MV4-11), FLT3-ITD– cells (THP-1, HL-60, and K562), and 293T cells were purchased from the American Type Culture Collection (Manassas, VA, USA). MOLM-13, THP-1, and K562 cells were cultured in RPMI 1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA). HL-60 cells were cultured in RPMI 1640 medium supplemented with 20% FBS. MV4-11 cells were cultured in Iscove's modified Dulbecco's medium (IMDM) (Hyclone) supplemented with 10% FBS. Primary AML cells were cultured in IMDM supplemented with 20% FBS. 293T cells were cultured in Dulbecco's modified Eagle medium (DMEM) (Gibco) supplemented with 10% FBS. All cells were cultured at 37°C with 5% CO₂. MOLM-13-RQ cells, which can stably proliferate in a complete medium containing 40 nM quizartinib, were provided by Professor Yueqin Chen, and the established method has been described previously[27].

RNA extraction and quantitative real-time PCR (q-PCR)

Total RNA was extracted from the bone marrow and cell samples using an Invitrogen™ TRIZOL Reagent (Thermo Fischer Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. All RNA samples were stored at –80 °C before reverse transcription and quantitative RT-PCR. The RNA was reverse-transcribed using RT reagent Kit RR047A (Takara, Japan) and the following quantitative PCR (qPCR) analysis was performed using the TB Green premix ExTaq Real-Time PCR kit (Takara) on an QuantStudio™ 7 Pro Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Data were normalized to GAPDH expression as a control. The relative expression level was determined using the 2^−ΔΔCt method. The primers are listed in Supplementary Table 2.

5′ RACE and 3′ RACE

Total RNA from MOLM-13 cells was extracted using TRIzol reagent according to the manufacturer’s guidelines. The 5′ and 3′ ends of cDNA were acquired using a 5 ́ RACE System for Rapid Amplification of cDNA Ends (Invitrogen, Waltham, MA, USA) and 3 ́ RACE System for Rapid Amplification of cDNA Ends (Invitrogen), respectively, according to the manufacturer’s instructions. PCR products were obtained and then cloned into pEASY-T3 (Trans Gen Biotech, China) for further sequencing.

Subcellular fraction isolation

Nuclear and cytoplasmic fractions were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher, USA) according to the manufacturer’s instructions. Fractioned RNAs from an equal number of cells were used for reverse transcription and qRT-PCR.

Plasmid construction and lentiviral infection

For constitutive gene overexpression, AL713998.1 was PCR-amplified from MOLM-13 cDNA, and the purified PCR products were cloned into the LEGO vector (ADDgene, Watertown, MA, USA). For stable LFQR knockdown vectors, the DNA oligos encoding shRNAs were generated and cloned into the pGreenPuro™ eukaryotic expression vector, with sh-NC as the negative control. All the constructs were verified by sequencing. Lentiviruses carrying shRNAs or LEGO vectors were cultured in a 6 cm culture dish by transfecting packaging cell HEK293T with the Lentivector Expression Systems (System Biosciences, Germany) consisting of pPACKH1-GAG, pPACKH1-REV, and pVSV-G. The viruses were harvested 72 h after transfection. Lentivirus Precipitation Solution (System Biosciences) was used to precipitate the virus. A density of 3 × 10⁵ MOLM-13 cells were centrifuged and resuspended in 300 μL virus suspension, followed by incubation at 37 °C with 5% CO2 for 24 h. Subsequently, the cells were centrifuged, washed, and resuspended in fresh 1640 medium with 10% FBS containing 1 μg/mL puromycin (Sigma-Aldrich, Waltham, MO, USA). q-PCR analysis was performed to confirm the target knockdown. The shRNA primers used are listed in Supplemental Table 2.

RNA interference

Small interfering RNAs (siRNAs) were synthesized by GenePharm (Shanghai, China) to knockdown LFQR and SFPQ. Transient transfections of siRNAs were performed using the Neon™ Transfection System (Invitrogen) in 10 µL reactions according to the manufacturer’s guidelines. The siRNA sequences are listed in Supplemental Table 2.

Cell proliferation assay

Cell proliferation assays were performed using Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Rockville, MD, USA). Cells at a density of 3 × 10⁴ Cells were seeded in triplicate in 96-well plates with 100 μL of complete medium per well. CCK-8 reagent was added into each well at 0, 24, 48, and 72 h, 10 μL and incubated for 3 h prior to measurement. Absorbance was measured at 480nm and 630 nm using a VICTORTM X5 Multilabel Plate Reader (PerkinElmer, USA).

Flow cytometric analysis

For apoptosis analysis, cells were incubated with Annexin V-FITC and propidium iodide solution (Dojindo Molecular Technologies) in the dark for 15 min at room temperature. For cell differentiation analysis, cells were incubated with human CD11b (BD Biosciences) and CD14 (Invitrogen) antibodies in the dark for 30 min at 37°C. For cell cycle analysis, cell samples were harvested and incubated with propidium iodide solution (Dojindo Molecular Technologies) in the dark for 30 min at room temperature and for or an additional 30 minutes at 4°C in the dark. All the samples were immediately analyzed using a Flow Cytometer (BD Biosciences).

Colony forming unit (CFU) assay

A total of 1000 MOLM-13 cells were mixed thoroughly with 1mL methylcellulose medium (R&D Systems, Minneapolis, MN, USA) and seeded in a 35mm dish. The dishes were incubated for approximately 10 days at 37°C with 5% CO₂, and then the colonies (> 50 cells) were scanned. Each experimental group included at least two replicates.

Protein extraction and immunoblot analysis

Total protein was extracted using RIPA buffer (Beyotime Biotechnology, China) supplemented with 1 × complete ULTRA protease inhibitor (Roche, Indianapolis, IN, USA); 5 × loading buffer (Fdbio Science, China) was added to the final concentration of 1 × loading buffer and boiled at 100°C for 5 min. Protein samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (EMD Millipore, Burlington, MA, USA) using an electrophoretic system. The transferred PVDF membrane was blocked with 1 × TBST containing 5% BSA at room temperature for 1 h, followed by incubation with an appropriate primary antibody (dilution with 1 × TBST containing 1% BSA) at 4°C overnight. The membranes were then incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibodies. Membranes were visualized using an enhanced chemiluminescence detection system.

TRSA RNA pull-down assay

The LFQR sequence was cloned into the BLUNT plasmid with the tRSA tag at its 5′ end. The RNA products were transcribed in vitro using the TranscriptAid T7 High-Yield Transcription Kit (Thermo Fischer Scientific) and purified using the GeneJET RNA Purification Kit (Thermo Fischer Scientific). The RNA pull-down assay was performed using 50 pmol of RNA from each sample, in accordance with the manufacturer’s instructions, using the Pierce Magnetic RNA-Protein Pull-down Kit (Thermo Fischer Scientific). Briefly, folded tRSA and tRSA-labeled LFQR were mixed with MOLM-13 cell extract (containing 2 mg total protein) in 400 µL RIP buffer and incubated at room temperature for 1 h. Next, 50 µL of washed streptavidin magnetic beads was added to each reaction mixture and further incubated at room temperature for 1 h. The beads were washed six times with wash buffer and boiled in SDS loading buffer. Finally, the enriched proteins were resolved by SDS-PAGE and silver staining, followed by mass spectrometry (MS) identification (Wininnovate Biotechnology, China) and western blotting.

RNA immunoprecipitation (RIP)

In the RIP assay, anti-IgG and anti-SFPQ antibodies were used along with an EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (17–701; Merck Millipore, Germany) according to the manufacturer’s instructions. All proteins used for RIP were lysed with cell lysis buffer supplemented with Thermo Scientific™ Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific). To prepare antibody-coated beads, 50 μL Protein A/G magnetic beads were incubated with 3 μg antibody or control IgG in 500 μL wash buffer at 4 °C for 1 h. The beads were then washed three times and mixed with the cell lysates in new tubes. The tubes were rotated at 4 °C overnight. Finally, RNA was extracted from the beads using TRIzol reagent according to the manufacturer’s instructions. Reverse transcription and qPCR were performed as previously described.

Chromatin immunoprecipitation (ChIP)

ChIP analysis was performed on chromatin extracts from MOLM-13 cells using a Magna ChIP™ G-Chromatin Immunoprecipitation Kit (17-611; Merck Millipore) with SFPQ antibody according to the manufacturer’s standard protocol. Rabbit IgG was used as the negative control. The fold-enrichment of SFPQ was quantified using q-PCR and calculated relative to the input chromatin. The primers used for ChIP-qPCR analysis are listed in Supplemental Table 2.

RNA sequencing

si-NC and si-LFQR samples (5 × 106 cells/sample; three samples each) were obtained from MOLM-13 cells. Next-generation sequencing was performed using poly (A) RNA-seq on an Illumina NoveSeq instrument. The sequencing depth of RNA-Seq was up to 6 gites in the 150-base paired-end mode. For data analysis, paired-end reads were adapted and quality trimmed using Trim Galore (v0.6.4), and high-quality reads were aligned to the reference transcriptome (Gencode v34) and quantified using Salmon (v1.3.0) with fragment GC bias and positional bias corrections. DESeq2 was used to normalize gene expression levels and identify differentially expressed genes.

Statistical analysis

Experiments were performed at least three times, and one representative experiment is shown in the results. Data are expressed as the mean ± SEM or ± SD of three independent experiments. Statistical analysis was performed using a paired or unpaired 2-tailed Student t-test. Correlation analysis was performed using Spearman test. The Kaplan-Meier method with a log-rank test (Mantel-Cox) was used to analyze mouse survival. p < 0.05 was considered statistically significant.

2.1. Systematic screening identified the highly expressed lncRNA LFQR as a risk factor in FLT3-ITD+ AML

To identify lncRNAs that specifically involved in FLT3-ITD AML and FLT3 inhibitor resistance, we screened the lncRNAs using publicly available RNA sequencing (RNA-seq) data set GSE122435[28] and GSE116432[18]. These two datasets were based on the hematopoietic malignancies of FLT3-ITD AML cells and the mechanism of resistance to quizartinib. We overlapped these datasets to identify aberrantly expressed lncRNAs. Consequently, 43 commonly upregulated lncRNAs were observed (Figure 1A). We further analyzed the correlation between lncRNA expression levels and the overall survival of patients with AML. Among the 43 candidates identified, high expression of AL713998.1 was associated with the poorest outcome in patients with AML (Figure 1B). Therefore, we focused on this candidate lncRNA in subsequent studies. To verify the clinical relevance of AL713998.1 in AML, we first analyzed its expression pattern using a large cohort of clinical samples. A significantly higher expression level of AL713998.1 was found in the AML group than in the healthy control group (Figure 1C). Additionally, among the AML samples, AL713998.1, was remarkably upregulated in the adverse group compared to the favorable or intermediate groups (Figure 1D). Subsequently, we used another cohort of clinical samples with and without the FLT3-ITD mutation for further analysis. The FLT3-ITD+ AML group exhibited a higher expression level of AL713998.1 compared to the FLT3-ITD– group (Figure 1E). In addition, a higher expression level of AL713998.1 was observed in FLT3-ITD+ cell lines than in FLT3-ITD- cell lines (Figure 1F). Consequently, we named this lncRNA LFQR. These results prompted us to explore whether this lncRNA was essential for FLT3-ITD AML and FLT3 inhibitor resistance.

Next, we characterized the features of the LFQR. We conducted subcellular fractionation using qRT-PCR analysis and showed that LFQR was predominantly localized in the nucleus (Figure 1G). Based on data from the Ensembl database, LFQR contained three viriants: ENST00000660755.1, ENST00000666873.1, and ENST00000654364.1 (Figure 1H). To investigate the expression patterns of these three variants, qRT-PCR was performed using primers specific to each variant. Notably, the relative expression level of ENST00000654364.1 was significantly higher than that of the other variants (Figure 1I). Thus, in subsequent experiments, we primarily focused on ENST00000654364.1. To further characterize this lncRNA, we performed rapid amplification of cDNA ends (RACE) to experimentally validate its 5'- and 3'-ends in MOLM-13 cells, followed by sequencing (Figure 1J). The full-length transcript was 1276 nt. Briefly, a highly expressed lncRNA in FLT3-ITD+ AML, LFQR, has been characterized and may function in the progression of leukemia.

2.2. LFQR functions as an oncogene role specifically in FLT3-ITD AML

To investigate the oncogenic role of LFQR, specifically in FLT3-ITD AML cells, we used RNA interference (RNAi) to suppress the expression of LFQR in five myeloid cell lines (MOLM-13, MV4-11, HL-60, THP-1, and K562) and investigated the effects of LFQR on cell function (Supplemental Figure 1A). CCK-8 assays were performed to investigate their effects on cell proliferation. As shown in Figure 2A, knockdown of LFQR significantly impaired the growth of FLT3-ITD+ cell lines such as MOLM-13 and MV4-11, but had a very limited influence on FLT3-ITD-cell lines including HL60, THP-1, and K562, implying that LFQR specifically affects the functions of FLT3-ITD+ AML cells. To investigate the cellular functions and potential mechanisms of action of LFQR in FLT3-ITD AML cells, unbiased transcriptome profiling was performed using MOLM-13 cells transfected with si-NC or si-LFQR. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed marked enrichment in the cell cycle, cell differentiation, and the PI3K-Akt signaling pathway (Figure 2B). Consequently, we investigated whether knockdown of LFQR influences the cell cycle, differentiation, and the PI3K-Akt signaling pathway.

Therefore, we explored the regulation of LFQR in cell differentiation via Wright-Giemsa staining or by assessing the frequency and fluorescence intensity of the myeloid markers CD11b and CD14. Upon LFQR knockdown, the cells were induced toward monocyte/macrophage in MOLM-13 cells, but not in HL60 cells (Figure 2C and 2D). We further investigated the anti-differentiation role of LFQR in primary AML cells isolated from two patients. Consistently, LFQR depletion caused a significant increase in differentiation was observed in FLT3-ITD+ AML patient, whereas no effect was observed in FLT3-ITD- patient (Figure 2E). LFQR knockdown caused cell cycle arrest in FLT3-ITD+ cells (Figure 2F).

Next, we investigated whether LFQR affects self-renewal and found that LFQR suppression dramatically inhibited the clonal capacity of MOLM-13 cells but had minimal influence on HL60 cells (Figure 2G). Notably, when LFQR was stably overexpressed in MOLM-13 cells, differentiation was significantly blocked (Figure 2H). Taken together, these results demonstrate that LFQR knockdown substantially impairs the hematopoietic malignancy of FLT3-ITD+ cells, implying that LFQR plays an important role in the progression of FLT3-ITD AML.

2.3. Knockdown of LFQR reverses resistance to quizartinib via downregulating the oncogene in FLT3-ITD+ AML

Considering that FLT3 inhibitors such as gilteritinib and quizartinib have excellent efficacy in inducing remission, increasing attention is being placed on drug resistance, as most patients experience relapse and develop resistance after treatment. Therefore, we investigated the function of LFQR in a quizartinib-resistant MOLM-13 cell line (MOLM-13-RQ). We performed cell viability and western blot assays to identify the resistance status of MOLM-13-RQ cells, and the results showed a high level of resistance to quizartinib (Figure 3A and 3B). The knockdown of LFQR inhibited proliferation (Figure 3C). What’s more, the knockdown of LFQR greatly enhanced the ability of quizartinib to suppress the growth of MOLM-13-RQ cells (Figure 3D), implying that LFQR depletion has the potential to reverse resistance to quizartinib. Besides, the knockdown of LFQR also increased cell differentiation, and caused cell cycle arrest, consistent with wild-type MOLM-13 (Figure 3E-F). To further determine if the upregulated LFQR expression has influence on quizartinib sensitivity, we analyzed the drug sensitivity of patients with AML who were diagnosed and treated at the First Affiliated Hospital of Sun Yat-Sen University. Higher expression levels of AL713998.1 resulted in reduced inhibition of leukemia at the same dose of quizartinib among the patients, implying that LFQR plays an important role in drug resistance to quizartinib in AML (Figure 3G).

Activity of the FLT3 downstream signaling pathway is crucial for the FLT3-ITD mutation in AML progression. To elucidate the mechanism underlying FLT3-ITD downregulation induced by LFQR knockdown, we examined the protein levels of FLT3-ITD in MOLM-13, MV4-11, and MOLM13-RQ cells. As shown in Figure 3H-J, a significant decrease in the FLT3 protein levels was observed upon LFQR suppression, followed by reduced phosphorylation levels of FLT3 and STAT5 (pFLT3 and pSTAT5). In addition, we used the FLT3-ITD cell line HL60 as a control and observed no significant decrease in either FLT3 or STAT5 protein levels when LFQR was suppressed (Supplemental Figure 2A). c-MYC is a well-known target gene induced by STAT5 and functions as a positive regulator of cytokine signaling in cancer[29]. We observed that LFQR downregulation reduced the STAT5-dependent protein c-MYC in the FLT3-ITD+ cell line MV4-11 but had minimal influence on the FLT3-ITD-cell line HL60 (Supplemental Figure 2B-C). Consistently, decreased protein levels were also observed in primary FLT3-ITD+ patient samples, whereas the expression levels did not significantly decline in primary FLT3-ITD- patient samples (Figure 3K and 3L).

Additionally, we investigated the mechanism by which LFQR regulates the protein levels of FLT3. Given that mRNA transcription is a major factor that contributes to protein expression levels. Therefore, we used qRT-PCR to detect the expression of FLT3 mRNA upon LFQR knockdown in MOLM-13 and MV4-11 cells. As shown in Figure 3M and 3N, the FLT3 mRNA levels decreased remarkably when LFQR was knocked down. These results validate that the knockdown of LFQR inactivates FLT3 oncoproteins by inhibiting FLT3 mRNA transcription.

2.4. Interaction of LFQR with SFPQ regulates FLT3 expression and promotes the progression of FLT3-ITD and RQ AML

Generally, lncRNAs interact with RNA-binding proteins (RBP) to regulate cellular activities. Therefore, to further verify RBPs that may be specifically involved in the oncogenic mechanism of LFQR in FLT3-ITD AML, we performed an RNA pull-down assay[30] with a TRSA tag containing AL713998.1, and IgG as a negative control (Figure 4A and Supplemental Figure 3A). Mass spectrometry (MS) was used to identify all possible RBPs. We also performed Gene Ontology enrichment analysis to annotate the biological processes and cluster the modules of these proteins, focusing mainly on those associated with transcription (Supplemental Figure 3B). Notably, as shown in Figure 4B, SFPQ showed a stronger binding affinity for LFQR than to other proteins such as DHX9 and PTBP1. Accordingly, RIP assays showed that LFQR was significantly enriched in SFPQ (Figure 4C). These observations demonstrate that LFQR directly interacts with SFPQ in FLT3-ITD AML cells.

SFPQ, a core member of the nuclear paraspeckle family, is critical for controlling gene expression during cellular processes[31]. However, little is known regarding its function in FLT3-ITD AML. Therefore, we investigated the function of SFPQ in the FLT3-ITD AML cells. The introduction of siRNAs targeting SFPQ into MOLM-13, MV4-11 and MOLM-13-RQ cells significantly reduced proliferation (Figure 4D-F). Moreover, SFPQ silencing induces differentiation of FLT3-ITD+ cells (Figure 4G). As shown in Figure 4H, knockdown of SFPQ expression caused cell cycle arrest. In addition, inhibition of LFQR expression caused a dramatic increase in apoptosis in MOLM-13 and MV4-11 cells (Figure 4I). We investigated whether SFPQ suppression is essential in MOLM-13-RQ cells. Consistent with the phenotype observed in MOLM-13 cells, SFPQ depletion markedly increased apoptosis, induced differentiation and caused cell cycle arrest in MOLM-13-RQ cells (Figure 4J-L). These results imply that SFPQ plays an important role in the cytoactivity of quizartinib-resistant cells.

These results indicated that SFPQ is important in FLT3-ITD+ AML. Therefore, to determine whether SFPQ knockdown affected the expression of FLT3 protein, we performed a western blot assay. The results are shown in Figure 5A-C, FLT3 protein and its downstream signaling molecules significantly decreased with SFPQ depletion in MOLM-13, MV4-11, and MOLM-13-RQ cells. Additionally, the expression of FLT3 mRNA was markedly reduced upon LFQR knockdown (Figure 5D). Taken together, the effects of SFPQ downregulation were similar to those of LFQR knockdown, indicating that SFPQ had synergistic effects with LFQR in FLT3-ITD AML.

In addition, LFQR overexpression increased the expression of FLT3 protein and its downstream signaling molecules (Figure 5E).

2.5. LFQR regulates FLT3 expression via recruitment of SFPQ to the promoter region of FLT3

As the expression level of FLT3 mRNA was inhibited by either LFQR or SFPQ knockdown, we hypothesized that LFQR recruits SFPQ to bind to the FLT3 promoter region, thereby influencing the transcription of FLT3 mRNA. To validate this, we first constructed a MOLM-13 cell line with stably downregulated LFQR, sh-LFQR, and used sh-NC as a negative control (Figure 5F). Moreover, the DNA samples were fragmented as required by sonication at 4℃ (Supplemental Figure 3C). Finally, we used a CHIP-qPCR assay to determine the interactions between LFQR, SFPQ, and the FLT3 promoter. We found downregulation of SFPQ at FLT3 promoter upon LFQR depletion (Figure 5G). Chromatin immunoprecipitation (CHIP) showed successful enrichment of SFPQ protein (Figure 5H). These findings demonstrate that LFQR regulates the transcription of FLT3 by directly interacting with SFPQ to activate the oncoprotein and its downstream signaling pathways in FLT3-ITD AML.

2.6. Knockdown of LFQR restricts FLT3-ITD and RQ AML progression in vivo

NOD/SCID mice were used to investigate the role of LFQR in FLT3-ITD AML leukemogenesis in vivo. A xenograft model was established by injecting MOLM13 or MOLM-13-RQ cells transfected with LFQR short hairpin RNA (sh-LFQR) and the control (sh-NC) into the tail vein. As depicted in Figure 6A and Supplemental Figure 4A, LFQR knockdown impaired the infiltration ability, as demonstrated by decreased numbers of hCD45+ cells in the bone marrow, peripheral blood, and organs, including the liver and spleen. Notably, the characteristic differentiation block of bone marrow leukemic cells was significantly relieved, as evident by an increase in the levels of CD11b and CD14 upon LFQR suppression (Figure 6B). Consistently, hematoxylin and eosin (H&E) staining showed that LFQR knockdown decreased infiltration of leukemic cells in the bone marrow and spleen, as well as increased liver damage (Figure 6E). Moreover, the LFQR-depleted group of mice survived longer, suggesting that LFQR downregulation inhibited FLT3-ITD AML progression (Figure 6G). Consistent phenomena were observed in the xenograft model injected with MOLM-13-RQ cells (Figure 6C-D, F, H, and Supplemental Figure 4 B and D), suggesting that LFQR contributes to resistance to quizartinib in vivo. Together, these data demonstrate that LFQR is critical for FLT3-ITD AML progression, and that its downregulation inhibits AML differentiation. The proposed working model is shown in Figure 7.

FLT3-ITD AML, one of the most frequent subtypes of leukemia, is associated with increased risk of relapse and poor prognosis[4, 7, 8]. The structural characteristics of the FLT3 mutation as a kinase make it an extensively studied therapeutic target[5, 6]. Several FLT3 inhibitors with varying levels of specificity and potency, such as gilteritinib, sorafenib, and quizartinib, have been developed and evaluated in clinical trials[14]. Although these inhibitors, especially quizartinib, have demonstrated a satisfactory response in clinical settings, attention has turned to drug resistance, which is responsible for poor outcomes. Therefore, it is of utmost importance to identify new therapeutic strategies for FLT3-ITD AML and feasible ways to overcome resistance to FLT3 inhibitors. In this study, we discovered a lncRNA, LFQR, that is highly expressed in FLT3-ITD AML, and its expression was closely correlated with sensitivity to quizartinib. Furthermore, silencing LFQR specifically impaired the leukemogenesis of FLT3-ITD AML cells and quizartinib-resistant cells both in vitro and in vivo. Furthermore, we experimentally validated that LFQR directly interacted with SFPQ to regulate FLT3 transcription, thereby influencing the expression of oncoproteins and kinases in FLT3-ITD AML. Notably, we demonstrated that depletion of LFQR significantly increased the sensitivity of quizartinib-resistant cells. These findings reveal the critical role of LFQR in FLT3-ITD-mediated progression of leukemia and the mechanism of quizartinib resistance, suggesting that LFQR could serve as a potent molecular therapeutic target for the treatment of FLT3-ITD AML and provide promising insights into the reversal of FLT3 inhibitor resistance.

The FLT3-ITD mutation in AML has an oncogenic role by promoting proliferation, accelerating cell cycle progression, decreasesing apoptosis, and blocking the differentiation of hematopoietic progenitor cells[32]. The leukemic burden of the FLT3-ITD mutation largely correlates with the risk categories and prognosis of this type of AML, with elevated levels of FLT3 being associated with the mutation[33, 34]. For example, the interaction of PRMT5 with Sp1 as a miR-29b silencing complex via dimethylation of the histone 4 arginine residue H4R3 activates FLT3 transcription. This, in turn, and thus affects the survival and proliferation of AML cells[35]. At the post-translational level, the expression of FLT3 can also be regulated through the binding of MSI2 proteins to FLT3 mRNA transcripts, resulting in leukemic growth[36]. Despite its clinical relevance, the regulatory mechanisms underlying FLT3 expression remain largely unknown. In the present study, LFQR was shown to play a pivotal role in FLT3 transcription by interacting with SFPQ. Knockdown of LFQR significantly decreased the expression level of FLT3 mRNA and inactivated FLT3 downstream signaling, preventing the progression of leukemogenesis and overcoming FLT3 inhibitor resistance. Here, we present a novel finding that lncRNAs participate in regulating FLT3 expression, positioning the LFQR-SFPQ interaction upstream of that of FLT3 and thus providing an indication of how lncRNAs may influence the progression of FLT3-ITD AML.

The dysregulation of RBP expression and function via genomic mutations or transcriptional regulation facilitates cancer progression[37]. As an indispensable form of the paraspeckle, SFPQ is a multifunctional RBP involved in various cellular processes, including mRNA processing, transcription, and DNA damage[31, 38, 39]. Loss of SFPQ is responsible for pathological changes in numerous cancers, including colorectal[40], ovarian[41] and breast cancers[42]. Furthermore, a recent study showed that regulation of SFPQ expression is closely associated with cancer chemoresistance. In epithelial ovarian cancer, the downregulation of SFPQ significantly sensitizes tumor cells to platinum by modulating SRSF2 activity[41]. Accumulating evidence highlights the critical role of SFPQ in cellular function and cancer pathology; however, little is known about its influence on AML. In our study, we discovered that SFPQ knockdown remarkably inhibited cell proliferation, led to cell cycle arrest, and induced differentiation of FLT3-ITD AML cells, indicating the importance of SFPQ in hematogenic malignancy. Notably, SFPQ suppression also dramatically impaired the cytoactivity of quizzartinib-resistant cells, suggesting the potential role of SFPQ in FLT3 inhibitor resistance. Moreover, the interaction between LFQR and SFPQ on the FLT3 loci has been unveiled in our research; LFQR, promotes SFPQ binding to the promoter region of FLT3, and thus activates the transcriptional regulation of FLT3 mRNA, resulting in increased oncoprotein levels and cancer progression. Consequently, inhibiting the expression of SFPQ or disrupting the interaction between LFQR and SFPQ could represent another promising solution for curing FLT3-ITD AML. However, further studies are needed.

In summary, we identified a novel lncRNA, LFQR, which was upregulated in FLT3-ITD AML and promoted leukemia progression by interacting with SFPQ to regulate FLT3 expression. Particularly, we observed a reversal of quizartinib resistance upon LFQR knockdown in drug-resistant cells. Our results provide a promising therapeutic target for FLT3-ITD AML and a feasible approach to overcome resistance to the FLT3 inhibitor quizartinib.

Acknowledgements

This work was financially supported by the Basic and Applied Basic Research Project of Guangdong Province, China (Grant No. 2023A1515030060, 2021A1515111169), the National Natural Science Foundation of Guangdong Province (No. 2022A1515140018), the Guangzhou Science and Technology Program Projects - Basic and Applied Basic Research Topics (Grant No. 2023A04J2221), the Young Talent Support Project of Guangzhou Association for Science and Technology (Grant No. QT2024-043), the Medical Science and Technology Research Foundation of Guangdong Province, China (Grant No: A2021328, A2022443), and Fundamental Research Funds for the Central Universities, Sun Yat-sen University（Grant No. 24qnpy347.

Author Contributions

Ying-Li Liu, Xiao-Li Zhang and Xue-Qun Luo contributed equally to this work. Ying-Li Liu, Xue-Qun Luo and Yan-Lai Tang designed this study. Xue-Qun Luo supervised this study. Li-Bin Huang and Xiao-Li Zhang coordinated the sample collection. Ying-Li Liu, Jun-Yi Lian and Shi-Yao Song conducted experiments. Wen-Tao Wang, Li-Na Wang and Cong Liang provided technical support for experiments. Ying-Li Liu and Wen-Tao Wang conducted the data analysis and wrote the original manuscript. Wen-Tao Wang, Yan-Lai Tang, Li-Bin Huang, Zhi-Yong Ke and Zhong Fan reviewed and polished the manuscript. All authors approved the final manuscript.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability Statement

The data that support the findings of current study are available from the corresponding author upon reasonable request.

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LncRNA LFQR promotes cytoactivity and resistance to quizartinib via FLT3 transcriptional activation in FLT3-ITD acute myeloid leukemia

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