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Article Open Access

FOXM1 inhibitor, RCM‑1, enhances venetoclax mediated apoptosis through downregulation of ATP2B4 in rhabdomyosarcoma

  • Authors:
    • Nawal Merjaneh
    • Ying-Wei Lan
    • Zicheng Deng
    • Johnny Donovan
    • Guolun Wang
    • Jonathan Do
    • Tiffany Juan
    • Xiaomei Xia
    • Vladimir V. Kalinichenko
    • Tanya V. Kalin
  • View Affiliations / Copyright

    Affiliations: Center for Cancer and Blood Disorders, Phoenix Children's Hospital, Phoenix, AZ 85016, USA, Phoenix Children's Research Institute, The University of Arizona College of Medicine‑Phoenix, Phoenix, AZ 85004, USA, Division of Neonatology and Pulmonary Biology, Perinatal Institute, Cincinnati Children's Research Foundation, Cincinnati, OH 45229, USA
    Copyright: © Merjaneh et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 52
    |
    Published online on: March 4, 2026
       https://doi.org/10.3892/ijo.2026.5865
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Abstract

Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children. Intensifying chemotherapy has failed to improve patient survival for metastatic or relapsed RMS and RMS survivors often suffer from significant long‑term toxicities. More efficient and less toxic new therapies are critically needed. RMS expresses high levels of anti‑apoptotic protein Bcl‑2 and an oncogenic transcription factor Forkhead box protein M1 (FOXM1), which is also known to inhibit tumor cell apoptosis. The present study used a combination therapy of a recently developed non‑toxic FOXM1 inhibitor, RCM‑1 and the FDA‑approved Bcl2 inhibitor, venetoclax, which is not effective as a monotherapy in solid tumors. Compared with venetoclax alone, the combination therapy efficiently inhibited RMS growth in the animal model by decreasing tumor cell proliferation and inducing tumor cell apoptosis. RNA‑sequencing analysis demonstrated that the combination therapy uniquely decreased expression of ATPase Plasma Membrane Ca2+ Transporting 4 (ATP2B4), a plasma membrane calcium channel that is highly expressed in RMS compared with normal muscle cells. RCM‑1, but not venetoclax treatment, inhibited ATP2B4 and enhanced the sensitivity of RMS cells to apoptosis. Knockdown of ATP2B4 decreased RMS tumor cell proliferation, migration and colony formation in vitro. Furthermore, knockdown of ATP2B4 increased tumor cell apoptosis, while overexpression of ATP2B4 decreased tumor cell apoptosis in vitro. In the animal model of RMS, depletion of ATP2B4 decreased tumor growth. In summary, combining RCM‑1 with venetoclax sensitized RMS cells to apoptosis by decreasing ATP2B4. This made ATP2B4 a promising therapeutic target for RMS and provides a rationale for exploring this combination in early‑stage clinical trials.
View Figures

Figure 1

RCM-1 and venetoclax synergistically
reduce RMS tumor cell viability and induce apoptosis in
vitro. Combination therapy of RCM-1 and venetoclax using the
IC50 dose significantly reduced RD and 76-9
rhabdomyosarcoma cell viability compared with a single agent. (A)
Light microscopy images of RD cells treated with vehicle, single
agent RCM-1, venetocalx, or combination therapy. (B) The same
experiment was performed using 76-9 tumor cells. (C) Combination
therapy significantly enhanced apoptosis in RD and 76-9 cells
compared with either agent alone. Values are shown as mean ± SD.
*P≤0.05, **P≤0.01, ***P≤0.001,
****P≤0.0001. Scale bar, 100 μm. RMS,
rhabdomyosarcoma; IC50, half-maximal inhibitory
concentrations.

Figure 2

The combination of
RCM1-NPFA and venetoclax decreases tumor growth and
enhances caspase-mediated apoptosis in a murine RMS model. (A)
Schematic diagram of tumor cells inoculation and treatment. The
figure is created in BioRender. Merjaneh, N. (2024) https://BioRender.com/z67t342. (B) Combination
therapy significantly reduced tumor burden compared with vehicle.
The mean vehicle tumor volume on day 21 was 685 mm3,
compared with the average tumor volume of the combination therapy
of 361 mm3. The maximum tumor diameter was 17.3×11.5 mm
and the corresponding maximum tumor volume was 1,144
mm3. Tumor volume was measured at different time points
during the experiment (P≤0.01; n=12). (C) Combination therapy
inhibited proliferation, as indicated by the decreased number of
Ki67-positive cells. Combination therapy increased apoptosis, shown
by the increased number of (D) BAX-positive cells and (E) the
number of caspase3-positive cells compared with single agents
and/or vehicle. A total of five random fields per sample were used
to quantify the number of Ki67, BAX and cleaved caspase 3 positive
cells per group. Values are shown as mean ± SD. Scale bar, 10
μm. *P≤0.05, **P≤0.01,
****P≤0.0001. RMS, rhabdomyosarcoma.

Figure 3

Combination treatment with RCM-1 and
venetoclax reveals a unique genetic profile in 76-9 cells. (A) Venn
diagrams illustrated the overlap of differentially expressed genes
between the combination therapy and venetoclax compared with the
control. (B) Gene set enrichment analysis of RNA-sequencing data
highlighted the upregulated pathways in the combination therapy
relative to venetoclax, such as ferroptosis and elevated cytosolic
Ca+2 level, as well as the downregulated pathways, such
as WNT signaling pathway, mitotic cell cycle and angiogenesis
pathway. (C) Volcano plot identified the differentially expressed
genes in the combination therapy vs. venetoclax. Atp2b4 was
notably downregulated in the combination therapy. (D) Reverse
transcription-quantitative PCR showed the downregulation of
Atp2b4 in 76-9 cells treated with RCM-1 and combination
therapy. Actb mRNA was used for normalization (E) Protein
levels of ATP2B4 were decreased after RCM-1 and the combination
treatments, as shown by immunofluorescence using ATP2B4 antibodies.
A total of five random fields per sample were used to quantify the
percentage of ATP2B4-positive cells per group. Scale bar, 10
μm. Data presented as mean ± SD. **P≤0.01,
***P≤0.001, ****P≤0.0001.

Figure 4

ATP2B4 is differentially
overexpressed in RMS cells vs. normal muscle cells and its
knockdown decreases tumor cell proliferation, migration and colony
formation. (A) Left panel, Human myoblast (muscle progenitor
cells), RMS, normal skeletal muscle and smooth muscle scRNA
sequencing datasets were visualized using UMAP. Data were extracted
from GSE143704 for normal muscle tissue, GSE 195709 for RMS (RMS1
and 3 are fusion-negative RMS, RMS2 is fusion-positive and RMS4 is
spindle (fusion-negative). Right panel, ATP2B4 mRNA
expression was higher in RMS cells compared with normal muscle
cells. (B) Reverse transcription-quantitative PCR showed the
upregulation of ATP2B4 mRNA level in RMS cells (RD and RH30)
compared with HSkMC. (C) Knockdown of ATP2B4 increased the
intracellular calcium level. Calcium level was assessed using the
calcium-sensitive fluorescent dye fura-2 AM. Scale bar, 10
μm. (D) Knockdown of ATP2B4 inhibited colony
formation in 76-9 RMS cells. Data presented as mean ± SD. (E)
Knockdown of ATP2B4 suppressed 76-9 cell proliferation in
culture. Data presented as mean ± SD. (F) ATP2B4 knockdown
decreased RMS cell migration. The percentage of wound closure
presented as mean ± SD (magnification, ×4). *P≤0.05,
**P≤0.01, ***P≤0.001. RMS, rhabdomyosarcoma;
UMAP, Uniform Manifold Approximation and Projection; HSkMC, human
skeletal muscle cells; SMC, smooth muscle cells.

Figure 5

ATP2B4 knockdown enhances
venetoclax-mediated apoptosis in 76-9 RMS cells. (A) Representative
immunofluorescence images from control and siATP2B4 76-9 cells
treated with DMSO, 5 and or 8 mM of venetoclax for 24 h.
Venetoclax-induced dose-dependent apoptosis was measured by caspase
3/7 (DEVD) activity (red) and annexin V (green). Scale bar, 100 mm.
The knockdown of ATP2B4 increased RMS apoptosis compared
with control cells. (B) The percentage of annexin and DEVD positive
cells were counted in five random fields using the EVOS imaging
software and presented as mean ± SD from triplicates of one
experiment. **P≤0.01, ***P≤0.001,
****P≤0.0001. ATP2B4; ATPase Plasma Membrane
Ca2+ Transporting 4; RMS, rhabdomyosarcoma.

Figure 6

Overexpression of ATP2B4 increases
the resistance of RMS cells to apoptosis. (A) Representative
immunofluorescence images depicting control, pATP2B4a and pATP2B4b
76-9 cells treated with DMSO, 5 mM, or 8 mM of venetoclax for 24 h.
The overexpression of both ATP2B4 isoforms reduced apoptosis
compared with control RMS cells following venetoclax treatment.
Scale bar, 100 mm. (B) The percentage of positive annexin and DEVD
cells were counted in five random fields using the EVOS imaging
software and presented as the mean ± SD from triplicates of one
experiment. ***P≤0.001, ****P≤0.0001. ATP2B4;
ATPase Plasma Membrane Ca2+ Transporting 4; RMS,
rhabdomyosarcoma.

Figure 7

FOXM1 binds the ATP2B4 promotor and
regulates the expression of ATP2B4 in RMS cells. (A) shRNA-mediated
knockdown of Foxm1 reduced Atp2b4 mRNA in 76-9 murine
RMS cells. RT-qPCR showed the efficient knockdown of Foxm1
in 76-9 cells. n=3. P≤0.0001. (B) shRNA-mediated knockdown of
FOXM1 reduced ATP2B4 mRNA in RH30 human RMS cells.
RT-qPCR confirmed the efficient knockdown of FOXM1 in RH30
cells. n=3. P≤0.01 (C) ChIP-seq showed the direct binding of FOXM1
protein to the ATP2B4 promoter region in K562 (human
lymphoblasts). (D) Upper panel, schematic drawing of the
pGL4.23-ATP2B4-Luc construct. Lower panel, 76-9 cells were
co-infected with the luciferase reporter, Renilla and either
a CMV-empty or CMV-Foxm1 overexpressed plasmid. The CMV-Foxm1
expression vector increased the transcriptional activity of the
Atp2b4 promoter region compared with the CMV-empty vector
(P<0.0001). *P≤0.05, **P≤0.01,
****P≤0.0001. FOXM1, Forkhead box protein M1; ATP2B4;
ATPase Plasma Membrane Ca2+ Transporting 4; RMS,
rhabdomyosarcoma; sh, short hairpin; RT-qPCR, reverse
transcription-quantitative PCR.

Figure 8

The knockdown of ATP2B4 slows RMS
tumor growth. (A) Control and shAtp2b4 76-9 cells were
inoculated subcutaneously into the flank of C57Bl/6J mice. Tumor
volume measured at different time points. Knockdown of ATP2B4
significantly impaired tumor growth compared with control mice (n=5
per group; presented as the mean ± SD). (B) Tumor volumes measured
after tumor harvest show smaller shAtp2b4 tumors. The
average vehicle tumor volume was 1,321 mm3 compared with
592 mm3 for shAtp2b4 C and 468 mm3 for
shAtp2b4 D. The maximum tumor diameter was 15.56×13.39 mm
and the corresponding maximum tumor volume was 1,395
mm3. (C) Gross images of control and shAtp2b4 RMS
tumors. (D) Reverse transcription-quantitative PCR analysis of mRNA
isolated from tumors confirmed ~50% knockdown efficiency in
shAtp2b4-expressing tumors. *P≤0.05,
**P≤0.01, ****P≤0.0001. ATP2B4; ATPase Plasma
Membrane Ca2+ Transporting 4; RMS, rhabdomyosarcoma; sh,
short hairpin.
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Copy and paste a formatted citation
Spandidos Publications style
Merjaneh N, Lan Y, Deng Z, Donovan J, Wang G, Do J, Juan T, Xia X, Kalinichenko VV, Kalin TV, Kalin TV, et al: FOXM1 inhibitor, RCM‑1, enhances venetoclax mediated apoptosis through downregulation of ATP2B4 in rhabdomyosarcoma. Int J Oncol 68: 52, 2026.
APA
Merjaneh, N., Lan, Y., Deng, Z., Donovan, J., Wang, G., Do, J. ... Kalin, T.V. (2026). FOXM1 inhibitor, RCM‑1, enhances venetoclax mediated apoptosis through downregulation of ATP2B4 in rhabdomyosarcoma. International Journal of Oncology, 68, 52. https://doi.org/10.3892/ijo.2026.5865
MLA
Merjaneh, N., Lan, Y., Deng, Z., Donovan, J., Wang, G., Do, J., Juan, T., Xia, X., Kalinichenko, V. V., Kalin, T. V."FOXM1 inhibitor, RCM‑1, enhances venetoclax mediated apoptosis through downregulation of ATP2B4 in rhabdomyosarcoma". International Journal of Oncology 68.5 (2026): 52.
Chicago
Merjaneh, N., Lan, Y., Deng, Z., Donovan, J., Wang, G., Do, J., Juan, T., Xia, X., Kalinichenko, V. V., Kalin, T. V."FOXM1 inhibitor, RCM‑1, enhances venetoclax mediated apoptosis through downregulation of ATP2B4 in rhabdomyosarcoma". International Journal of Oncology 68, no. 5 (2026): 52. https://doi.org/10.3892/ijo.2026.5865
Copy and paste a formatted citation
x
Spandidos Publications style
Merjaneh N, Lan Y, Deng Z, Donovan J, Wang G, Do J, Juan T, Xia X, Kalinichenko VV, Kalin TV, Kalin TV, et al: FOXM1 inhibitor, RCM‑1, enhances venetoclax mediated apoptosis through downregulation of ATP2B4 in rhabdomyosarcoma. Int J Oncol 68: 52, 2026.
APA
Merjaneh, N., Lan, Y., Deng, Z., Donovan, J., Wang, G., Do, J. ... Kalin, T.V. (2026). FOXM1 inhibitor, RCM‑1, enhances venetoclax mediated apoptosis through downregulation of ATP2B4 in rhabdomyosarcoma. International Journal of Oncology, 68, 52. https://doi.org/10.3892/ijo.2026.5865
MLA
Merjaneh, N., Lan, Y., Deng, Z., Donovan, J., Wang, G., Do, J., Juan, T., Xia, X., Kalinichenko, V. V., Kalin, T. V."FOXM1 inhibitor, RCM‑1, enhances venetoclax mediated apoptosis through downregulation of ATP2B4 in rhabdomyosarcoma". International Journal of Oncology 68.5 (2026): 52.
Chicago
Merjaneh, N., Lan, Y., Deng, Z., Donovan, J., Wang, G., Do, J., Juan, T., Xia, X., Kalinichenko, V. V., Kalin, T. V."FOXM1 inhibitor, RCM‑1, enhances venetoclax mediated apoptosis through downregulation of ATP2B4 in rhabdomyosarcoma". International Journal of Oncology 68, no. 5 (2026): 52. https://doi.org/10.3892/ijo.2026.5865
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