Abstract
Background
Acute megakaryoblastic leukemia (AMKL) is a rare hematological
malignancy in adults but children. Alternative splicing (AS) has been
shown to affect hematological cancer progression, making splicing
factors promising targets. Our research aims to investigate the
efficacy of the molecular glue degrader indisulam, which targets the
splicing factor RNA binding motif protein 39 (RBM39) in AMKL models.
Results
Public drug sensitivity data analysis revealed that AMKL cell lines
exhibited the highest sensitivity to indisulam compared with other
tumor types. Then we confirmed that RBM39 depletion by indisulam
treatment induced AMKL cell cycle arrest and apoptosis. In AMKL mouse
model, indisulam treatment significantly reduced the leukemic burden
and prolonged the lifetime of AMKL mice. Mechanically, integration of
transcriptomic and proteomic analyses revealed that indisulam-mediated
RBM39 degradation resulted in AS of the transcription factor zinc
finger MYND-type containing 8 (ZMYND8), an AMKL cell growth regulator.
Finally, the effectiveness of indisulam depended on DDB1- and Cul4-
Associated Factor 15 (DCAF15) expression because knockout of DCAF15
rescued the indisulam-induced RBM39 degradation and mis-splicing of
ZMYND8.
Conclusion
Indisulam is a promising therapeutic candidate for AMKL and the
RBM39-mediated ZMYND8 splicing plays an important role in promoting the
development of AMKL.
Supplementary Information
The online version contains supplementary material available at
10.1186/s13578-025-01380-3.
Keywords: Acute megakaryoblastic leukemia, Indisulam, RBM39,
Alternative splicing, ZMYND8
Background
Acute megakaryocytic leukemia (AMKL) is a heterogeneous subtype of
acute myeloid leukemia (AML) based on the French American British (FAB)
classification (AML M7) [[83]1]. AMKL accounts for 4 to 15% of
pediatric AML cases [[84]2], with a significantly lower 5-year survival
rate than other AML subtypes [[85]3, [86]4]. Despite advances in
treatment including intensive treatment regimens and allogeneic
hematopoietic stem cell transplantation, there are still no
individualized treatment approaches for AMKL which calls for the
identification of novel treatment strategies [[87]5].
Splicing factors cause splicing regulatory changes and errors closely
associated with diseases [[88]6]. Studies have shown that abnormal
splicing is a common feature of AML and provides potential targets for
AML [[89]7–[90]9]. Aberration in splicing contributes to the
megakaryocytic malignancies. An early study demonstrated that an AS
product of GATA Binding Protein 1 (GATA1) isoform which lacks the
transcriptional activation domain, triggers excessive megakaryocyte
proliferation [[91]10]. More recently, it was reported that c-Mpl-del,
an AS isoform of the thrombopoietin receptor c-Mpl, is upregulated in
AMKL patients. This mis-spliced product promotes chemoresistance and
proliferation of AMKL cells [[92]11]. These studies indicate that the
abnormalities in splicing contribute to the pathogenesis of AMKL and
targeting the splicing process is a potential therapeutic strategy for
AMKL treatment.
RBM39 is an RNA-binding protein (RBP), as well as a pre-mRNA splicing
factor and transcription coactivator [[93]12–[94]14]. When working as
components of splicesome, RBM39 interacts with other splicing factors,
including U2AF65, SF3b155 et al., to regulate pre-mRNA alternative
splicing [[95]15–[96]17]. Increasing evidence supports that splicing
homeostasis maintained by RBM39 was essential in tumor progression,
which makes RBM39 a promising target of cancer [[97]18–[98]21]. The
aryl sulfonamide drug indisulam (also known as E7070) selectively
targeting RBM39 is initially identified by Eisai during screening for
small molecule inhibitors that target cell cycle progression [[99]22,
[100]23]. Mechanistically, indisulam promotes the interaction between
RBM39 and DCAF15 E3 ligase substrate receptors, leading to the
ubiquitination and proteasome-mediated degradation of RBM39 [[101]24],
which belongs to the U2AF-like RBP family and participates in
tumorigenesis and developmental processes [[102]25–[103]27] by
regulating AS, transcription, and translation [[104]28]. Its efficacy
and safety have been demonstrated in multiple phase I [[105]29–[106]31]
and phase II clinical trials [[107]32–[108]34] in patients with
advanced cancers. However, the efficacy of indisulam has never been
tested in AMKL.
In this study, we investigated the efficacy of RBM39 inhibitor
indisulam and analyzed the role of RBM39 in cell survival in AMKL.
Using RNA-seq and proteomics analysis, we demonstrated the role of
RBM39 in maintaining the precise splicing in AMKL cells. Together, our
study provided new clues of using RBM39 inhibitor for AMKL treatment.
Materials and methods
Cell lines and culture
The human tumor cell lines and 293FT cells were obtained from the Cell
Bank of the Chinese Academy of Sciences. CMK, MEG01, M07e, U937, and
K562 cells were cultured in RPMI medium (Vivacell, C3001-0500,
Shanghai, China) supplemented with 1% penicillin/streptomycin (P/S,
Beyotime, C0222 ) and 10% fetal bovine serum (FBS, Vivacell,
C04001-050). In addtion, M07e cells were treated with 10 ng/ml
recombinant human GM-CSF (E.coli) (Novoprotein, C003, Suzhou, China).
293FT cells were cultured in high-glucose DMEM medium (Vivacell,
C3113-0500) supplemented with 1% P/S and 10% FBS. All the cell lines
were maintained in a humidified atmosphere (37℃, 5%CO[2]). All the cell
lines were subjected to short tandem repeat (STR) identification, and
the identification results are shown in Supplementary Material 1.
Cell counting kit-8 (CCK-8) assay
To assess the effect of indisulam on cell viability, CMK, MEG01, M07e,
U937, and K562 cells were seeded in 96-well plates at a density of
1 × 10^4 cells/well. Indisulam (MedChemExpress, HY-13650, NJ, USA) was
dissolved and diluted with dimethyl sulfoxide (DMSO, Sigma-Aldrich,
D2650, MO, USA). After indisulam treatment for 72 h, cell proliferation
ability and viability were detected via a CCK-8 kit (APExBIO, K1018,
TX, USA) according to the manufacturer's instructions. Each
concentration was tested in three independent experiments. The short
hairpin RNA (shRNA)-treated cells were seeded in 96-well plates at a
density of 1 × 10^3 cells/well and were tested every two days for a
total of one week after puromycin selection. The absorbance at 450 nm
was measured with a spectrometer (Thermo, MA, USA), and the ability of
proliferation and viability were quantified via Graph Prism 9.0
(GraphPad Software Inc., San Diego, CA, USA). Three technical
replicates were performed for each experiment.
Soft agar colony formation
To detect the proliferative ability and invasiveness, the cells were
treated or transduced as described above and 1 × 10^4 cells/well were
inoculated in a soft agar medium seeded into 6-well plates. Every 1 to
2 days, 100 to 200 μl complete medium was added to support nutrients
and prevent gel cracking. After approximately 2 to 3 weeks, the cells
were fixed overnight with 4% paraformaldehyde (Beyotime, P0099). The
samples were subsequently stained with Giemsa solution (Beyotime,
C0131) and photographed. Finally, the number of colonies in each group
was calculated as the number of cells that formed colonies
[MATH: ÷ :MATH]
the total number of cells × 100%. The data are representative of 3
technical replicates.
Lentivirus preparation and infection
The shRNAs targeting RBM39 and ZMYND8 were inserted into the
pLKO.1-puro lentiviral vector (IGE Biotechnology Ltd., Guangzhou,
China) containing puromycin resistance. To prepare lentiviruses, we
purchased the envelope plasmid and packaging plasmid from Addgene
(pMD2. G: #12259; psPAX2: #12260; Cambridge, MA, USA). Then, we
transfected the purified plasmids together with pMD2. G and psPAX2 into
293FT cells via polyethyleneimine (PEI) (Sigma-Aldrich, 49553-93-7) at
a ratio of 4:1:3 according to the manufacturer’s protocol. After 6 h of
transfection, replace the medium with fresh medium. The supernatants
were collected at 48 h and 72 h after transfection and filtered through
a 0.45 μm filter. To concentrate the lentivirus, a quarter volume of
PEG8000 (Sigma-Aldrich, P5413) was then mixed with lentivirus and
incubated on a shaker at 4 °C. After 24 h, the samples were centrifuged
at 4000 g for 20 min. The resulting pellet was finally resuspended by
small volume of Phosphate-buffered saline (PBS). When performing
lentivirus transfection, AMKL cells were incubated with concentrated
lentivirus in the presence of 1 μg/mL hexadimethrine bromide
(Polybrene) (Sigma-Aldrich, H9268) for 24 h. After transfection, stable
cell lines were selected by puromycin (Beyotime, ST551) for 3 days. The
sequences of shRNA are listed in Supplementary Table S1.
Real-time quantitative PCR (RT-qPCR)
To detect the mRNA expression level, RNA was extracted via a Total RNA
Isolation Kit (Vazyme, RC112-01; Nanjing, China) according to the
manufacturer's instructions. Total RNA was then reverse transcribed
into cDNA via a reverse transcription kit (Applied Biosystems, 94404,
CA, USA). RT-qPCR was performed via LightCycler 480 SYBR Green I Master
Mix (Roche, 4887352001, Basel, Switzerland) in a LightCycler 480
Real-Time System (Roche). The relative mRNA expression of the target
genes was subsequently calculated via the
[MATH: 2ΔΔCt :MATH]
method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
expression used as a control. Three technical replicates were performed
for each experiment. The primers used in this study are shown in
Supplementary Table S2.
RNA splicing analysis and verification
Following the alignment of the RNA-seq data, rMATS (4.1.2) software was
used to analyze alternative splicing events, including skipped exons
(SEs), retained introns (RIs), mutually exclusive exons (MXEs),
alternative 5' splice sites (A5SSs), and alternative 3' splice sites
(A3SSs). The splicing events were visualized via the IGV Genome
browser (version 2.19.2). Significant splicing events were subsequently
chosen based on the criteria of a false discovery rate (FDR) < 0.05 and
an absolute difference in inclusion level (IncLevel) > 0.2. Total RNA
from vehicle- or indisulam- treated cells were extracted by fastPure
Cell/Tissue Total RNA Isolution Kit V2 (Vazyme, RC112-01; Nanjing,
China). Then, cDNA was synthesized from 1000 ng of total RNA using the
reverse transcription kit (Applied Biosystems, 94404, CA, USA) in a
25 μl reaction on an ABI PCR instrument (Thermo Fisher, Applied
Biosystems). To confirm the RNA splicing events, PCR was performed to
detect SE of EZH2 and MXE of ZMYND8. cDNA was amplified using 2 × Taq
Plus Master Mix II (Vazyme, P213) and subjected to electrophoresis on
1.2% agarose gel at 130 V for 30 min. Finally, the gel was visualized
using a gel imager (UVITECT, FireReader, UK) under ultraviolet light.
The PCR primers used are listed in Supplementary Table S2.
Western blot analysis
On the first day, AMKL cells were collected after the corresponding
treatment and washed with PBS before lysis using RIPA lysis buffer
(Beyotime, P0013B). The extracted protein concentration was quantified
to be 10 mg/ml. Proteins of the same mass were separated via
electrophoresis, transferred to polyvinylidene fluoride (PVDF)
membranes (GVS, 1212639, Zola Predosa, Italy), and blocked with 5% skim
milk. Then, primary antibodies were added, and the samples were
incubated overnight at 4 °C with shaking. On the second day, the PVDF
membranes were incubated with secondary antibodies at room temperature
for 1 h. After the PVDF membranes were wetted with ECL luminescence
solution (Millipore, WBKLS0500, USA), the membranes were visualized
with an AI600 image gel imaging analyzer (GE, MA, USA). GAPDH was used
as a loading control. The raw images of western blots are provided in
the supplementary material. The antibody details are shown in
Supplementary Table S3.
RNA sequencing (RNA-seq)
To detect the mechanically induced effects of indisulam on AMKL, CMK
cells treated with indisulam were collected and sent to Novogene
Bioinformatics Technology Co., Ltd. (Beijing, China) for sequencing.
Libraries were constructed from total RNA, and the resulting mRNA
fragments were used as templates to synthesize the first strand of cDNA
via the M-MuLV reverse transcriptase system, followed by the second
strand of cDNA from dNTPs. The purified and screened cDNAs were
subjected to PCR amplification, and the resulting products were
subsequently purified to obtain a library. DESeq2 software
(version 1.40.0) was used for differential expression analysis between
the two comparison combinations. The sequencing results are shown in
Supplementary Table S4.
Quantitative proteomics
Protein extraction
Proteomic analysis was performed by Jingjie Biotechnology Co., Ltd.
(Hangzhou, China). Samples were suspended in lysis buffer (8 M urea, 1%
protease inhibitor) and lysed by ultrasonic disruption. The total
protein was collected by centrifuge at 4 °C, 12,000 g for 10 min and
proceed to concentration determination using BCA assay.
Trypsin digestion
For digestion, equal amounts of protein were taken from each sample and
adjusted to the same volume with lysis buffer. Trichloroacetic Acid was
added to a final concentration of 20%, vortexed, and incubated at 4 °C
for 2 h to precipitate proteins. After centrifugation (4500g, 5 min),
the supernatant was discarded, and the pellet was washed 2–3 times with
cold acetone. After drying the precipitate, 200 mM Triethylammonium
Bicarbonate was added to reach the final concentration. The precipitate
was sonicated to disperse, and trypsin was added at a 1:50
(protease:protein, m/m) ratio for overnight digestion. Dithiothreitol
was added to a final concentration of 5 mM, and reduction was carried
out at 56 °C for 30 min. Iodoacetamide was then added to a final
concentration of 11 mM, and the mixture was incubated at room
temperature in the dark for 15 min.
LC–MS/MS analysis
The peptides were dissolved in mobile phase A of the liquid
chromatography system and loaded onto the Evotip according to the
manufacturer's instructions. The peptides were then separated using the
Evosep One ultra-high-performance liquid chromatography system,
utilizing the pre-set 60 SPD method. After separation, the peptides
were ionized in the Capillary ion source and injected into the timsTOF
Pro 2 mass spectrometer for data acquisition. The data were acquired
using the data-independent parallel accumulation serial fragmentation
mode. FDR was adjusted to < 1%. The proteins with a P-value < 0.01 and
a log2-fold change between the two groups, either < − 0.9 or > 1.11,
were considered significantly different in expression. These
significant proteins were then selected and verified via Western blot
analysis.
Apoptosis and cell cycle assays
We collected AMKL cells and washed them with precooled PBS. Then, the
cells were suspended in 1 × binding buffer and stained with fluorescein
isothiocyanate (FITC)-Annexin V antibody (BD Biosciences, 556420, NJ,
USA) and propidium iodide (PI) solution (BD Biosciences, 556463) for
apoptosis analysis. For cell cycle analysis, the cells were harvested,
washed with precooled PBS, and fixed in 75% ethanol for 24 h. The cells
were processed via the Cell Cycle and Apoptosis Assay Kit (Beyotime,
C1052) following the manufacturer's instructions. Both the apoptosis
and cell cycle data were processed via flow cytometry (Beckman Gallios™
Flow Cytometer; Beckman, USA). Each experiment was performed three
technical replicates.
Generation of CRISPR–Cas9 DCAF15 knockout
The Cas9 gene was introduced into CMK and MEG01 cell lines through
lentiviral transduction and selected with 500 μg/mL geneticin
(MedChemExpress, HY-108718) for one week to establish stable
Cas9-expressing cell lines. Small guide oligos (sgRNAs) targeting
DCAF15 were then cloned and inserted into the Lenti-CRISPR plasmid,
which was purchased from GENECHEM (Shanghai, China). Cells expressing
the Cas9 gene were transduced with either sgRNA-DCAF15 or nontargeting
control sgRNA (sgNC) lentiviral particles. Lentivirus preparation was
conducted by GENECHEM via previously established protocols. The
sequences of the sgRNAs are listed in Supplementary Table S1.
In vivo experiments
All experiments related to animals were approved by the ethics
committee of the Animal Care Committee of Soochow University
(CAM-SU-AP#: JP-2018-1). NSG female mice aged 4–6 weeks were randomly
divided into two or three groups. One hundred microliters of a cell
suspension prepared in PBS (1 × 10^6 CMK cells expressing firefly
luciferase) was injected into each mouse via the tail vein. For drug
treatment, indisulam was dissolved in 20% SBE-β-CD (MedChemExpress,
HY-17031) in saline and administered via intraperitoneal injection
(12.5 mg/kg/day) or vehicle for 7 days. After 10 days of injection, in
vivo imaging was conducted by an imaging system (Berthold, NightOWL II
LB 983, Germany) every 2 to 3 days. The fluorescence intensity, body
weight, adverse skin events, hair condition, and incidence of diarrhea
were monitored during the treatment period. At the end of the study,
the paraffin-embedded tissue blocks from the liver, spleen, and hind
limbs were prepared for hematoxylin and eosin (HE) staining and
immunohistochemistry (IHC) with a Ki67-antihuman antibody (Servicebio,
GB12114, China). All mice were euthanized before reaching a 20% body
weight loss.
Statistical analysis
Statistical analysis was performed using GraphPad Prism software
(version 9.0) and R (version 4.2.2). The Mann–Whitney U test was used
to compare the two groups' differences, and the Pearson method was used
for correlation analysis. In the case of three groups, one-way ANOVA
was performed. The survival time was analyzed using the Log-Rank test
with P < 0.05 considered statistically significant. The following
symbols indicated the data significance: * P< 0.05, ** P < 0.01, *** P
< 0.001, and **** P < 0.0001.
Results
Indisulam effectively inhibits survival of AMKL cells
Nijhuis et al. have shown that leukemia cells exhibited excellent
sensitivity to indisulam [[109]35]. We focused on the AMKL subtype of
leukemia and found AMKL showed the greatest sensitivity among 745 tumor
cell lines (Fig. [110]1A, B). The half-inhibitory concentration (IC50)
values of indisulam in AMKL cells (CMK, MEG01, and M07e) were
significantly lower than those in non-AMKL cells (U937 and K562)
(Fig. [111]1C, D). Moreover, indisulam treatment inhibited the growth
of AMKL cells and reduced cell survival (Fig. S1). The proportion of
apoptotic cells increased in a dose-dependent manner (Fig. [112]1E, G).
Cell cycle analysis revealed that AMKL cells exhibited a decrease in
the number of cells in the G1 phase and an increase in the number of
cells in the G2 phase after indisulam treatment (Fig. [113]1F, H).
Finally, we confirmed that indisulam treatment led to degradation of
the RBM39 protein, cleavage of the classical apoptotic protein poly
ADP-ribose polymerase (PARP), and decreased expression levels of c-MYC
and the classical cell cycle protein Cyclin-dependent kinase 4 (CDK4)
(Fig. [114]1I). These findings confirmed that AMKL is highly sensitive
to indisulam. Indisulam-induced RBM39 degradation leads to cell cycle
arrest and apoptosis in AMKL cells.
Fig. 1.
[115]Fig. 1
[116]Open in a new tab
AMKL cells are highly sensitive to indisulam. A Median area under the
curve (AUC) of mortality in 25 types of tumors. B AUC comparisons
between AMKL cell lines (CMK and M07e cell lines, n = 2) and non-AMKL
cell lines (all other tumor cell lines, n = 743). Data were acquired
from the Cancer Target Discovery and Development (CTD^2) Network, each
dot represents a cell line. C Dose–response curves of AMKL (CMK, MEG01,
M07e) and other AML subtypes (U937 and K562) treated with indisulam.
Cell viability was measured by a CCK-8 assay. D IC50 values and 95%
confidence intervals (95% CIs) of indisulam in each AML cell line. E
Flow cytometry analysis of Annexin V + cells after indisulam treatment
at different concentrations. F Flow cytometry analysis of the cell
cycle after indisulam treatment at different concentrations. G
Statistical plots of the proportion of cells with indisulam-induced
apoptosis. H Compared with DMSO treatment, indisulam treatment resulted
in strong G2 phase arrest in AMKL cells. I Western blot detection of
PARP, c-MYC, and CDK4 expression after indisulam treatment in AMKL cell
lines. The error bars denote the Standard Deviation (SD). P values were
determined via Mann–Whitney U test and are indicated as *P < 0.05, **P
< 0.01, ***P < 0.001, and ****P < 0.0001. "ns" signifies not
significant. VC = vehicle control, 0.1% DMSO. Each experiment was
performed with three technical replicates
Knockdown of RBM39 recapitulates the effects of indisulam
As the target of indisulam, the role of RBM39 in AMKL has not yet been
clarified. DEMETER2 is a framework that evaluates gene dependencies
across 712 cancer cell lines and was examined in three distinct
comprehensive pooled RNA interference screens. By using this platform,
we revealed a high dependency of RBM39 in 2 AMKL cell lines (CMK and
M07e), underscoring an indispensable role of RBM39 in AMKL cell
survival (Fig. [117]2A, B). We then investigated RBM39 expression
patterns in AML patients via the Sangerbox web tool (a bioinformatics
data analysis platform) via The Cancer Genome Atlas (TCGA) database,
which revealed increased RBM39 expression in AML samples compared with
normal samples (Fig. S2A, B), and high RBM39 expression was correlated
with an unfavorable prognosis in AML patients (Fig. S2C). Additionally,
our experimental results confirmed that RBM39 was highly expressed in
AMKL cell lines (Fig. [118]2C, S2D). The shRNA-mediated knockdown of
RBM39 expression in AMKL (Fig. S2E) significantly reduced the cell
proliferation rate (Fig. [119]2E–G), promoted apoptosis (Fig. [120]2H,
S2F), and disrupted cell cycle progression (Fig. [121]2I, S2G).
Finally, knockdown of RBM39 induced cleavage of PARP, decreased c-MYC,
and CDK4 expression, confirming the role of RBM39 in regulating
proliferation, apoptosis, and the cell cycle in AMKL cells
(Fig. [122]2J). Taken together, our results indicate that RBM39 is
essential for the growth and survival of AMKL cells.
Fig. 2.
[123]Fig. 2
[124]Open in a new tab
RBM39 is highly expressed in AMKL and promotes AMKL cell survival. A, B
CRISPR screen data indicates the gene dependency of RBM39 in CMK and
M07e cells. C Western blot analysis showing the protein expression of
RBM39 in AML cells. D Western blot analysis showing the knockdown
efficiency of RBM39 in AMKL cells. E–G CCK8 assay showing the
proliferation curves of CMK, MEG01, and M07e cells transduced with
shRBM39#1, shRBM39#2 or shNC. H Quantification of apoptotic AMKL cells
after RBM39 knockdown using AnnexinV/PI dual staining. I The bar graphs
show the proportion of AMKL cells at each cell cycle phase after RBM39
knockdown. J Western blot analysis of the PARP, c-MYC, and CDK4
proteins after RBM39 knockdown in AMKL cell lines. The error bars
denote the SD. P values were determined via Mann–Whitney U test and are
indicated as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
"ns" signifies not significant. Each experiment was performed with
three technical replicates
RBM39 knockdown delays AMKL development in vivo
To evaluate the role of RBM39 in vivo, we intravenously injected
CMK-luciferase-shNC cells (negative control group) or
CMK-luciferase-shRBM39 cells (knockdown group) into NSG mice
(Fig. [125]3A). The knockdown group presented a significantly reduced
overall leukemia burden (Fig. [126]3B–D, S3A, B). There were no
significant changes in body weight (Fig. S3C) or pathological changes
in the appearance of the liver or spleen (Fig. S3D). Histological
examination with HE staining (Fig. S3E) and IHC confirmed reduced
leukemia cell infiltration in the bone marrow, liver, and spleen in the
RBM39-knockdown group (Fig. [127]3E, F, S3F). Overall survival was
significantly prolonged in the knockdown group (Fig. [128]3G). These
findings demonstrated that RMB39 promoted AMKL development in vivo.
Fig. 3.
[129]Fig. 3
[130]Open in a new tab
Knockdown of RBM39 expression delayed the leukemia development in the
AMKL mouse model. A Scheme of the mouse xenograft experiment using
CMK-luciferase cells transduced with shNC (n = 11) or shRBM39
lentivirus (n = 10). "n" refers to biological replicates. B
Representative bioluminescence images of NSG mice transplanted with
CMK-luciferase-shNC or CMK-luciferase-shRBM39 cells. C The tumor
fluorescence signal intensity in the liver (upper), spleen (middle),
femur and tibia (lower). D Statistical analysis of tumor fluorescence
signal intensity. E IHC (Ki67) staining of the bone marrow, spleen, and
liver tissue sections. Ki67-positive cells are indicated by red arrows.
F Line graph showing the tumor burden based on bioluminescence imaging.
"n" refers to biological replicates. G Kaplan–Meier analysis of CMK
xenografts suggested that the shRBM39 group had a significantly
prolonged survival time compared with the shNC group. "n" refers to
biological replicates. The error bars denote the SD. P values were
determined via Mann–Whitney U test and are indicated as **P < 0.01 and
***P < 0.001. "ns" signifies not significant. The survival time was
analyzed by the Log-Rank test, and P < 0.05 was considered
statistically significant
RBM39 degradation leads to AS defects in AMKL cells
RBM39 is involved in transcription regulation and RNA splicing
[[131]25], and we then investigated the mechanism by which RBM39
regulates the growth of AMKL cells. In AMKL cells, the RBM39 protein
was almost completely degraded within 24 h when 5 μM indisulam was
administered (Fig. [132]4A, B). Therefore, 5 μM indisulam-treated CMK
cells were collected and subjected to proteomic assays (Fig. [133]4C).
Transcriptome analysis revealed increased RNA splicing events,
including primarily SE events and RI events, in the indisulam-treated
group than in the control group (Fig. S4A, B). Among these genes, 907
mis-spliced genes were downregulated (Fig. [134]4D). Kyoto Encyclopedia
of Genes and Genomes (KEGG) pathway enrichment analysis suggested that
these 907 affected genes were enriched in ubiquitin-mediated
proteolysis and cell cycle pathways (Fig. [135]4D). EZH2 has been
identified as a critical target with splicing process defects
[[136]26]. In this study, EZH2 exhibited SE events at exon 14 in
indisulam-treated CMK cells (RNA-seq read counts, IGVs; Fig. [137]4E).
We validated the RNA splicing (Fig. [138]4F) and lack of protein
(Fig. [139]4G) of EZH2, suggesting that the mis-splicing of EZH2
resulted in a defect in translation. Additionally, the knockdown of
RBM39 by shRNA resulted in the same pattern of mis-splicing of EZH2 at
the mRNA level and a reduction in the EZH2 protein level (Fig. [140]4H,
I). Therefore, RBM39 deficiency leads to extensive splicing
dysregulation in AMKL.
Fig. 4.
[141]Fig. 4
[142]Open in a new tab
RBM39 degradation by indisulam results in the overall mis-splicing in
AMKL cells. A, B Western blot analysis of RBM39 in CMK cells and M07e
cells treated with indisulam at the indicated time points. C Volcano
plot of the proteomic analysis of CMK cells treated with indisulam
(5 µM) or VC for 24 h. D Venn diagram of mis-spliced genes versus
upregulated proteins (left) and KEGG pathway analysis of the
intersection between mis-spliced genes and downregulated protein genes
(right). E The Sashimi plot depicts the SE event of the EZH2 gene
region in CMK cells following indisulam treatment. The black arrows
highlight the skipping of exon 14. F PCR analysis of EZH2 (exons 12–15)
in CMK and MEG01 cells treated with VC or indisulam for 24–72 h. G
Western blot analysis of RBM39 and EZH2 expression after indisulam
treatment in AMKL cells. H PCR analysis of EZH2 in CMK cells with RBM39
knockdown. I Western blot analysis of EZH2 in CMK cells with RBM39
knockdown
ZMYND8 is a downstream target of RBM39 in AMKL cells
To identify genes targeted by RBM39 in AMKL, we performed combined
RNA-seq and proteomics analyses of indisulam-treated cells (Figs.
[143]5A, S5A). We identified 22 key genes that underwent multiple types
of AS events (Fig. [144]5B). Among these genes, N-Acylsphingosine
Amidohydrolase 1 (ASAH1), Enoyl-CoA Delta Isomerase 2 (ECI2, or PECI),
Selenium Binding Protein 1 (SELENBP1), and ZMYND8 presented complex
splicing patterns, generating four or five differential splicing events
(Fig. [145]5C–F, S5B–E). Furthermore, we compared the mRNA expression
levels of these genes across different AML FAB subtypes in the TCGA
database and revealed that the expression levels of these genes are
relatively higher in M6 and M7 (Fig. S5F–I) than in other subtypes,
suggesting that RBM39 maintains the precise splicing of genes crucial
for AMKL. One previous study revealed that ZMYND8 is essential for AML
proliferation [[146]36]. Therefore, we investigated the function of
ZMYND8 in AMKL. First, we found that ZMYND8 exhibited significant
splicing dysregulation after indisulam treatment or RBM39 knockdown
(Fig. [147]6A–C, S6D), leading to a reduction in the protein level
(Fig. [148]6D, E). Second, by examining the TCGA database via the
Sangerbox web tool, we observed that ZMYND8 expression was higher in
AML patients than in normal controls and that high ZMYND8 expression
was associated with poor prognosis (Fig. S6A, B). Notably, a positive
correlation was found between the mRNA expression of ZMYND8 and RBM39
in AML patients (Fig. S6C). We subsequently explored the expression of
ZMYND8 in various AML cell lines and found that it is highly expressed
in AMKL cells (Fig. [149]6F, S6E). Furthermore, the proliferation rate
of AMKL cells significantly decreased after ZMYND8 knockdown
(Fig. [150]6H–K). Finally, western blot analysis revealed increased
cleaved-PARP and decreased c-MYC after the depletion of ZMYND8
(Fig. [151]6L). Together, these findings indicate that ZMYND8 is a
direct splicing target of RBM39 and is required for AMKL survival.
Fig. 5.
[152]Fig. 5
[153]Open in a new tab
Indisulam causes aberrant splicing of important AMKL genes. A Schematic
representation of CMK cells subjected to RNA-seq and proteomic
analyses. B Venn diagram of mis-spliced genes (≥ 4 AS types) and
downregulated proteins (P value < 0.01 and the ratio between the two
groups < 0.9). C–F Sashimi diagram demonstrating the SE events of ASAH1
(C), ECI2 (D), SELENBP1 (E), and ZMYND8 (F) in indisulam-treated CMK
cells
Fig. 6.
[154]Fig. 6
[155]Open in a new tab
ZMYND8 is a downstream target of RBM39 in AMKL cells. A Sashimi plot
generated via the IGV program showing the splicing changes of ZMYND8
gene in CMK cells after indisulam treatment. B, C PCR testing of ZMYND8
AS after indisulam treatment. D Western blot analysis of the expression
of ZMYND8 after indisulam treatment. E Western blot analysis of ZMYND8
after RBM39 knockdown. F Western blot analysis of the expression levels
of ZMYND8 in AML cell lines. G Western blot analysis of the knockdown
efficiency of ZMYND8. H–J Knockdown of ZMYND8 inhibited the
proliferative capacity of AMKL cells. K Colony formation ability of
AMKL cells transduced with lentiviral shNC or shZMYND8. L The protein
expression of cleaved PARP and c-MYC following ZMYND8 knockdown. The
error bars denote the SD. P values were determined via Mann–Whitney U
test and are indicated as **P < 0.01, ***P < 0.001. "ns" signifies not
significant. Each experiment was performed with three technical
replicates
The anti-AMKL effects of indisulam are DCAF15-dependent
To confirm whether DCAF15 is a key factor in the sensitivity of AMKL to
indisulam, we explored DCAF15 mRNA expression levels in AML patients
from the TCGA database. The results showed that the DCAF15 expression
levels in AMKL (M7) were greater than those in other AML subtypes (Fig.
S7A). Furthermore, we employed the CRISPR/Cas9 system to knock out
DCAF15 in the CMK and MEG01 cell lines. The knockout of DCAF15 greatly
reduced the cytotoxicity of indisulam to AMKL cells (Fig. [156]7A, B),
as there was no effect on cell viability (Fig. [157]7C, D) or apoptosis
(Fig. [158]7E, F, S7B, C). In addition, indisulam treatment did not
induce the AS of ZMYND8 (Fig. [159]7G, H) or EZH2 (Fig. S7D) in the
absence of DCAF15. Similarly, western blot analysis verified these
findings; in the absence of DCAF15, indisulam could not induce the
degradation of RBM39. Consequently, the ZMYND8, PARP, c-MYC, CDK4, and
EZH2 proteins did not change (Fig. [160]7I, S7E). Collectively, these
data indicate that indisulam exerts its anti-AMKL effect through
DCAF15.
Fig. 7.
[161]Fig. 7
[162]Open in a new tab
Indisulam-induced RBM39 degradation and RNA mis-splicing are
DCAF15-dependent. A, B Representative images of DCAF15^WT or DCAF15^KO
cells after indisulam treatment. The scale bar is 50 µm. C, D
Dose–response curve of cell viability following treatment with
indisulam for 72 h. E, F Comparison of apoptosis between DCAF15^WT and
DCAF15^KO cells. G, H PCR analysis of ZMYND8 mis-splicing in DCAF15^WT
and DCAF15^KO cells. I Western blot analysis of the protein expression
of RBM39, ZMYND8, cleaved-PARP, c-MYC, and CDK4. The error bars denote
the SD. P values were determined via Mann–Whitney U test and are
indicated as **P < 0.01, ***P < 0.001. "ns" signifies not significant.
Each experiment was performed with three technical replicates
Indisulam impairs tumor growth in vivo
To evaluate the anti-AMKL efficacy of indisulam in NSG mice, we
constructed a mouse model of AMKL via luciferase-tagged CMK cells
(DCAF15^WT or DCAF15^KO) and randomly divided them into three groups
(DCAF15^WT + vehicle, DCAF15^WT + indisulam, and
DCAF15^KO + indisulam). AMKL mice were then administered indisulam
(12.5 mg/kg/d) or vehicle for one week via intraperitoneal injection
(Fig. [163]8A). Bioimaging revealed that indisulam effectively reduced
the tumor burden in the DCAF15^WT group, whereas its efficacy was not
observed in the DCAF15^KO group (Fig. [164]8B, C, S8A). By examining
the expression of Ki67 (a proliferation marker) (Fig. [165]8D) and the
proportion of human CD45 + (leukemia cell surface antigen) cells in the
bone marrow, liver, and spleen (Fig. S8B-E), we found that the number
of Ki67- and human CD45- positive cells in the DCAF15^WT + indisulam
group was significantly lower than that in the other groups.
Additionally, we found that AMKL mice in the DCAF15^WT + indisulam
group also had better overall survival than did those in the other
groups (Fig. [166]8E). Furthermore, we found no significant difference
in body weight (for early identification of toxic reactions)
(Fig. [167]8F) or in the appearance of the liver or spleen (Fig. S8F)
among the three groups. During the treatment, no other adverse effects,
such as diarrhea, hair deterioration, skin lesions, loss of appetite,
or changes in movement were observed. Taken together, the in vivo
results demonstrated that indisulam could effectively treat AMKL in a
DCAF15-dependent manner without significant drug toxicity.
Fig. 8.
[168]Fig. 8
[169]Open in a new tab
Efficacy of indisulam in a xenograft mouse model. A Schematics of drug
treatment in AMKL mice. B Representative bioluminescence images of NSG
mice treated with indisulam at the indicated posttransplant times. C
The tumor fluorescence signal intensity in the spleen (upper), liver
(middle), femur and tibia (lower). D Representative Ki67-IHC staining
images from bone marrow (upper), spleen (middle), and liver (lower)
tissue sections (the scale bar represents 50 μm). Ki67-positive cells
are indicated by red arrows. E Kaplan–Meier survival curves of
DCAF15^WT + vehicle, DCAF15^KO + indisulam, and DCAF15^WT + indisulam
AMKL mice. F Mean body weights of the DCAF15^WT + vehicle group,
DCAF15^KO + indisulam group, and DCAF15^WT + indisulam group. The
Log-Rank test was used for survival analysis. P < 0.05 is considered
statistically significant. "n" refers to biological replicates
Discussion
Spliceosome-mediated pre-mRNA editing is a critical biological process
that generates mature RNAs as templates for protein synthesis.
Mutations in splicing factors such as SF3B1, SRSF2, U2AF1 et al. are
frequently found in AML patients [[170]37]. Additionally, splicing
factors are found to be upregulated in AML samples, confer
chemo-resistance, and are associated with poor prognosis, highlighting
the role of splicing factors in the pathogenesis of AML [[171]38,
[172]39]. Eric Wang et.al. identified a network of RBPs that are
crucial for maintaining RNA splicing and the survival of AML cells. In
particular, the loss of RBM39 affects RNA splicing and selectively
induces lethality in AML cells harboring spliceosome mutations
[[173]20]. In our study, we demonstrated that RBM39 inhibitor indisulam
exerted a profound anti-leukemic effect in AMKL-the M7 subtype of AML
by inducing substantial aberrant splicing events. These findings not
only underscore the importance of splicing in AMKL cell survival, but
also support the oncogenic role of RBM39 in AMKL, which is consistent
with research across various tumor types including AML [[174]25], liver
cancer [[175]26], and high-risk neuroblastoma [[176]40].
As an essential splicing factor, RBM39 regulates ~ 20% of the
alternative splicing events in multiple biological processes, including
cancers [[177]41]. Disturbing the splicing of essential oncogenes by
targeting RBM39 can be an effective strategy in cancers. The cancer
stem cell enriched KRAS4A isoform was regulated by RBM39, thus
providing treatment targeting cancer stem cells [[178]42]. RBM39
inhibitors induced the intron retention of mRNA of homologous
recombination repair (HRR) genes in cancers with homologous
recombination deficiency (HRD), causing a synthetic lethality of
HRD-positive tumor cells [[179]43]. A recent study has shown that RBM39
regulated cell proliferation by affecting the splicing of MRPL33 in
gastric cancer cells [[180]44]. Our study revealed that RBM39 is
responsible for maintaining the splicing of important oncogenes in AMKL
cells. Many cancer-related genes [[181]45–[182]47], such as ZMYND8,
ASAH1, and SELENBP1, were found mis-spliced following the degradation
of RBM39. However, in addition to splicing factors, RBM39 can also work
as a transcription factor [[183]12, [184]13, [185]48] and a metabolic
sensor [[186]26]. In our study, several genes involved in biosynthesis
of amino acids, cysteine, and methionine metabolism pathways were
identified as mis-spliced following RBM39 depletion, suggestive of a
possible role of RBM39 in regulating AMKL metabolism programs. The
mechanism of RBM39-regulated metabolism and its association with
RBM39-directed splicing events needs further investigation.
We focused on the ZMYND8, which underwent multiple forms of
mis-splicing after indisulam treatment or RBM39 depletion in AMKL
cells. ZMYND8 is a well-known chromatin reader which recognizes
acetylated or methylated histones, and involved in transcription
regulation, DNA damage and cancer [[187]26]. ZYMND8 promotes DNA repair
response by interacting with the NuRD chromatin remodeling complex
[[188]49]. Mutations or dysregulation of ZMYND8 expression are
associated with cancer development and progression [[189]50]. However,
the role of ZMYND8 in cancer is still controversial. Several studies
have demonstrated a tumor suppressive role of ZMYND8 by suppression of
superenhancer-regulated gene expression [[190]51, [191]52]. On the
other hand, ZYMND8 also plays an oncogenic role in intestinal
tumorigenesis via driving the enhancer-promoter interaction to
upregulate the cholesterol biogenesis mevalonate (MVA) pathway
[[192]53]. In aldehyde dehydrogenase-high (ALDHhi) breast cancer stem
cells (BCSC), ZYMND8 forms a positive feedback loop with NRF2 that
amplifies the antioxidant defense mechanism sustaining BCSC survival
and stemness [[193]47]. Previous findings also suggested that ZMYND8 is
essential for AML survival by binding to the ET domain of BRD4 through
its chromatin reader domain, thereby sustaining leukemia growth
[[194]36]. In AMKL cells, we revealed that ZMYND8 was overexpressed.
Knockdown of ZYMND8 suppressed AMKL cell growth and induced cell
apoptosis, indicating ZMYND8 is an oncogene in AMKL.
The activity of ZMYND8 was regulated by different types of
modification, such as phosphorylation [[195]54], and acetylation
[[196]55], while the splicing of ZMYND8 was not reported. The reduced
protein expression of ZMYND8 was observed after multiple splicing upon
indisulam treatment. The possible mechanism could be the translation of
the mis-spliced transcript was prevented by intrinsic RNA quality
control steps, such as nonsense-mediated decay, non-stop decay et al.
[[197]56] or the resultant product is unstable since it is reported
that the PBP (PHD-BRD-PWWP) domain of ZMYND8 was essential in binding
with USP7, thereby stabilizing ZMYND8 [[198]57]. The mechanism would be
interesting to explore.
There are some limitations of this study. First, ZMYND8 underwent
multiple splicing pattern changes following RBM39 deletion but the
function of specific spliced products of ZMYND8 in AMKL cells was not
tested in this study. Considering the eventual outcomes of multiple
splicing events was the significant reduction of protein expression of
ZMYND8, we demonstrated the role of ZMYND8 in AMKL cells by knockdown
of the protein expression. Second, we only focused on the splicing
changes of ZMYND8, while the splicing events of other genes (ASAH1 and
SELENBP1) which were shown to mis-spliced following RBM39 depletion and
their roles in AMKL survival were not fully demonstrated in this study.
Another limitation of our study is the lack of AMKL clinical samples
due to the rarity of AMKL patients. Also, the limited types of AMKL
cell lines may not fully capture the complexity of AMKL in real-world
clinical settings. Further validation using clinical samples and a
wider range of cell lines would enhance the generalizability and
robustness of our study.
Conclusion
Together, this study demonstrated that indisulam, an RBM39 degrader,
shows significant anti-AMKL activity both in vitro and in vivo.
Degradation of RBM39 by indisulam induced aberrant splicing of ZMYND8
resulting in cell cycle arrest and apoptosis in AMKL cells
(Fig. [199]9). Considering that indisulam was well tolerated by
patients in Phase II clinical trials of other tumor types, our study
provides potential treatment options for AMKL patients, and it could
warrant further investigation in clinical trials. Additionally, the
expression level of DCAF15 may serve as a biomarker for predicting
sensitivity to indisulam treatment which precisely allows inclusion of
AMKL patients.
Fig. 9.
[200]Fig. 9
[201]Open in a new tab
Model of potential targets of RBM39 in AMKL. Indisulam-induced
degradation of RBM39 leads to alternative splicing disruption of
ZMYND8 in AMKL cells, which inhibits AMKL cell growth
Supplementary Information
[202]13578_2025_1380_MOESM1_ESM.pdf^ (828.9KB, pdf)
Supplementary Material 1: Fig. S1. Indisulam induces the death of AMKL
cells via RBM39 protein degradation. Indisulam acts as a molecular glue
that links the DCAF15 E3 ubiquitin ligase and RBM39 together, leading
to polyubiquitination and degradation of RBM39 protein, and splicing
defects. Representative images of AMKL cells treated for 72 h with
indisulam or VC. The scale bar indicates 50 µm.
[203]13578_2025_1380_MOESM2_ESM.pdf^ (2.6MB, pdf)
Supplementary Material 2: Fig. S2. High epression of RBM39 is
associated with poor prognosis and reduced RBM39 expression by shRNA
inhibited AMKL cell survival. RBM39 expression in normal tissue and
tumor samples was explored via the TCGA and GTEx databases. The TCGA
and GTEx databases revealed that RBM39 was upregulated in AML samples
compared with normal samples. TPM, transcripts per million. T,
tumor/cancer. N, normal. Kaplan–Meier survival analysis of AML patients
with high or low RBM39 expression using the TCGA and GTEx databases.
The difference in prognosis was significant according to the log-rank
test. RT-qPCR analysis of RBM39 mRNA expression in different AML cell
lines. RT-qPCR was used to detect the knockdown efficiency of RBM39 in
AMKL cell lines. Flow cytometry analysis of Annexin V + cells in the
shNC and shRBM39 groups. Flow cytometry analysis of apoptotic cells in
the shNC and shRBM39 groups. The error bars denote the SD. P values
were determined via Mann–Whitney U test and are indicated as *P < 0.05,
***P < 0.001, and ****P < 0.0001. "ns" signifies not significant. "N"
refers to biological replicates. Each experiment was performed with
three technical replicates.
[204]13578_2025_1380_MOESM3_ESM.pdf^ (3.2MB, pdf)
Supplementary Material 3: Fig. S3. Knockdown of RBM39 led to a
decreased leukemic burden in the AMKL mouse model. The leukemia
burdenin the liver, spleen, and bone marrow was detected by flow
cytometry. Percentages of human CD45 + cells in the liver, spleen, and
bone marrow. The mean body weights of shNC- and shRBM39-treated AMKL
mice. The appearance of the liver and spleen in shNC and shRBM39 AMKL
mice. HE staining of bone marrow, spleen, and liver tissue sections.
The error bars denote the SD. P values were determined via Mann–Whitney
U test and are indicated as *P < 0.05, ***P < 0.001, and ****P <
0.0001. "ns" signifies not significant. Each experiment was performed
with three technical replicates.
[205]13578_2025_1380_MOESM4_ESM.pdf^ (382.1KB, pdf)
Supplementary Material 4: Fig. S4. RBM39 deletion results in altered
RNA splicing. The number of AS events in CMK cells treated with 5 µM
indisulam or VC for 24 h. Violin plot of the difference in the
inclusion level of AS in CMK cells treated with 5 µM indisulam compared
with those treated with VC.
[206]13578_2025_1380_MOESM5_ESM.pdf^ (1.8MB, pdf)
Supplementary Material 5: Fig. S5. Indisulam treatment resulted in
aberrant RNA splicing and protein changes. Violin plot of differences
in protein level changes for genes with different numbers of AS
types. Sashimi plots showing the aberrant RNA splicing of ASAH1, ECI2,
SELENBP1, and ZMYND8.mRNA expression levels of ASAH1, ECI2, SELENBP1,
and ZMYND8 in AML FAB subtypes from the TCGA database.
[207]13578_2025_1380_MOESM6_ESM.pdf^ (1.5MB, pdf)
Supplementary Material 6: Fig. S6. ZMYND8 is highly expressed in AMKL
and is associated with poor outcomes. ZMYND8 expression is higher in
AML samples than in normal samples. Kaplan–Meier survival analysis of
AML patients with high or low ZMYND8 expression. The difference was
significant according to the Log-Rank test. Pearson correlation
analysis between RBM39 and ZMYND8 mRNA expression in AML patients in
the TCGA database. TPM, transcripts per million. PCR analysis of the
MXE of ZMYND8 in shNC and shRBM39 CMK cells. RT-qPCR analysis of the
relative expression level of ZMYND8 in different AML cell
lines. RT-qPCR analysis of the knockdown efficiency of ZMYND8 in AMKL
cell lines. The error bars denote the SD. P values were determined via
Mann–Whitney U test and are indicated as *P < 0.05, **P < 0.01, and
****P < 0.0001. "ns" signifies not significant. Each experiment was
performed with three technical replicates.
[208]13578_2025_1380_MOESM7_ESM.pdf^ (1.7MB, pdf)
Supplementary Material 7: Fig. S7. DCAF15 is highly expressed in AMKL
and is required for the anti-AMKL effect of indisulam. The TCGA
database shows the expression of DCAF15 in AML patients based on FAB
classification. Assessment of apoptosis via flow cytometry. PCR
analysis of the SE of EZH2 in CMK and MEG01cells. Western blot analysis
of the EZH2 protein in CMK and MEG01 cells following indisulam
treatment. The flow cytometry experiments were performed with three
technical replicates. Each experiment was performed with three
technical replicates.
[209]13578_2025_1380_MOESM8_ESM.pdf^ (1.3MB, pdf)
Supplementary Material 8: Fig. S8. The efficacy of indisulam was
dependent on DCAF15 in a xenograft mouse model. The tumor fluorescence
signal strength of the DCAF15^WT + Vehicle, DCAF15^WT + indisulam, and
DCAF15^KO + indisulam groups. Flow cytometry was used to detect human
CD45 + cells in the liver, spleen, and bone marrow of the
DCAF15^WT + Vehicle, DCAF15^WT + indisulam, and DCAF15^KO + indisulam
groups. The percentages of human CD45 + cells in the liver, spleen, and
bone marrow in the three groups. The appearance of the spleen and liver
in the three groups. The error bars denote the SD. P values were
determined via Mann–Whitney U test and are indicated as *P < 0.05, ***P
< 0.001, and ****P < 0.0001. "ns" signifies not significant. Each
experiment was performed with three technical replicates.
[210]Supplementary Material 9^ (6.6MB, pdf)
[211]Supplementary Material 10^ (37.8MB, xlsx)
Acknowledgements