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Bimodal expression of RHOH during myelomonocytic differentiation: Implications for the expansion of AML differentiation therapy
Open accessAbstract
RhoH is an unusual member of the Rho family of small GTP‐binding proteins in that it lacks GTPase activity. Since the RhoH protein is constantly bound by GTP, it is constitutively active and controlled predominantly by changes in quantitative expression. Abnormal levels of RHOH gene transcripts have been linked to a range of malignancies including acute myeloid leukemia (AML). One of the hallmarks of AML is a block in the normal program of myeloid differentiation. Here we investigate how myeloid differentiation is controlled by the quantitative expression of RHOH. Our analysis demonstrates that increasingly mature myeloid cells express progressively lower levels of RHOH. However, as monocytic myeloid cells terminally differentiate into macrophages, RHOH expression is up‐regulated. This up‐regulation is not apparent in AML where myeloid differentiation is blocked at stages of low RHOH expression. Nevertheless, when the up‐regulation of RHOH is forced, then terminal macrophage differentiation is induced and the Cdc42 and Wnt intracellular signalling pathways are repressed. These results indicate that RHOH induction is a driver of terminal differentiation and might represent a means of effecting AML differentiation therapy. The potential of this therapeutic strategy is supported by forced up‐regulation of RHOH reducing the ability of AML cells to produce tumours in vivo.
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TL;DR
RHOH expression is bimodal during myelomonocytic differentiation: it declines through early/intermediate stages but is induced during terminal monocyte-to-macrophage differentiation. Forced RHOH expression in AML cells drives terminal differentiation, represses Cdc42/Wnt signalling, and reduces tumour growth in a xenograft model.
Key findings
- RHOH mRNA and protein progressively decline with myeloid maturation: M0/M1 vs M5 mRNA was 0.802 ± SD 0.603 vs 0.037 ± SD 0.033 (21.7-fold, P = 0.0067); patient protein was 2.12 ± SD 0.42 vs 1.29 ± SD 0.18 (P = 0.0012); cell-line protein was 2.06 ± SD 0.46 vs 0.94 ± SD 0.40 (P = 0.0008).
- Terminal differentiation induces RHOH: PMA or LPS treatment of HL60/OCI-AML3, and PMA treatment of primary monocytes and AML FAB M4 myeloblasts, increased RhoH protein while JunD was down-regulated.
- Forced RHOH in OCI-AML3 (OCI-RhoH vs OCI-Empty) reduced CD93 and CD11b and altered 72 transcripts; 42 were linked to mature myeloid functions, with 9 down-regulated Cdc42/Wnt-positive effectors and 4 up-regulated negative effectors plus RMRP.
- In xenografts, tumours developed in 12/13 mice injected with OCI-Empty vs 5/12 with OCI-RhoH; OCI-RhoH tumours had average volumes 3.6-fold smaller (P = 0.004).
Methods
- RHOH expression was analysed in FAB-subtyped de novo AML transcriptomes from five microarray datasets and one RNA-seq dataset (ALFA-0701, GEO GDS1064/GDS3057/GDS3312/GDS3329, TCGA), plus AML cell lines, primary monocytes, and primary AML blasts using Q-RT-PCR and Western blotting.
- OCI-AML3 daughter lines stably expressing empty vector (OCI-Empty) or RHOH (OCI-RhoH) were compared by Western blot and Agilent whole-human-genome microarray; 72 genes differed significantly (P ≤ 0.05; GEO GSE138479).
- OCI-Empty or OCI-RhoH cells (5 or 10 × 10^6) were injected subcutaneously into SHrNTM Hairless NOD.SCID mice; tumour volumes were measured 3 weeks later and compared by Mann-Whitney test.
Limitations
- The paper has no dedicated limitations section. It notes that cell lines may not mimic in vivo biology, so key findings were verified in primary monocytes and AML myeloblasts.
- Evident from the text: the in vivo therapeutic evidence comes from a single AML cell line (OCI-AML3) in immunocompromised mice, with small groups (13 OCI-Empty and 12 OCI-RhoH injected; tumour volume analysis n = 12 vs n = 11 after outlier removal) and a short 3-week endpoint.
- The conflict-of-interest statement notes that Carl S. Shelley is President and CEO of Leukemia Therapeutics, LLC, which is developing AML treatments based on targeting consequences of aberrant RHOH repression.
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![FIGURE 1 Repression of RHOH mRNA expression during the intermediate phases of myelomonocytic differentiation as assessed by the FAB subtype of AML patient samples and cell lines. (A– D) Regression curves of the means of relative RHOH mRNA levels as a function of FAB subtype established from the NCBI GEO Datasets GDS1064 [46] and GDS3057 [47] composed of AML patients with all karyotypes and GDS3312 and GDS3329 composed only of AML patients with a normal karyotype [48]. (E) Regression curves of the means of relative RHOH mRNA levels as a function of FAB subtype established from microarray analyses of AML patients recruited by the ALFA‐0701 study (ALFA) [43, 44, 45]. (F) Regression curve of the means of relative RHOH mRNA levels as a function of FAB subtype established from normalized RNA‐seq data generated by The Cancer Genome Atlas (TCGA) [45, 49]. The GDS DataSets as well as the ALFA‐0701 and TCGA data all reveal a linear decrease in RHOH mRNA expression as differentiation stage progresses from AML FAB subtype M0 to M5 (R2 values are presented at the bottom left of each panel in black text). In addition, all sets of data demonstrate a significant difference in RHOH mRNA expression between the early M0 and M1 stages of AML differentiation compared to the later M5 stage (P values presented at the bottom left of each panel in blue text). Mean RHOH mRNA levels for individual FAB subtypes are depicted as open blue lozenges with their standard deviations (SD) presented as vertical bars above and below. In addition, the number (n) of patients analysed in each FAB subtype is presented. (G) Total RNA was extracted from the cell lines Kasumi‐3 (K3), KG‐1 (KG) and KG‐1a (Ka) established from patients diagnosed with AML FAB subtype M0; from the cell lines GF‐D8 (GF) and YNH‐1 (YN) representing AML FAB subtype M1; and from THP‐1 (TH), Nomo‐1 (N1), Mono Mac‐1 (M1), MV4‐11 (MV), Molm‐14 (MO), SHI‐1 (SH) and SKM‐1 (SK) representing FAB subtype M5. The relative level of RHOH mRNA expression was evaluated for each cell line by Q‐RT‐PCR as previously described [37]. The mean relative expression of RHOH mRNA for three independent cultures of each cell line, each analysed twice in duplicate by Q‐RT‐PCR, are depicted as histograms with SD presented as vertical lines above. The mean relative RHOH mRNA expression in FAB subtypes M0 and M1 combined is 0.802 ± SD 0.603 while that of M5 is 0.037 ± SD 0.033. These two means are depicted as blue horizontal bars and their difference of 21.7 fold was determined to be significant (P = 0.0067). The P values presented in all panels were calculated using an unpaired Student's t‐test](/paper-figures/b3ec340bb23c429d96f707b85e885c17/bimodal-expression-of-i-rhoh-i-during-myelomonocytic-differentiation-figure-1.jpg)
![FIGURE 7 Nucleotide sequence of the P3 promoter of the human RHOH gene. The site of transcription initiated by the promoter is indicated as +1 with a black horizontal arrow depicting the 5′ end of exon 4 of the RHOH gene [14]. The location of potential binding sites for the transcription factor proteins c‐Myc, E2F1, HIF1, LEF1 and SRY that could activate the promoter are indicated with green horizontal bars and text [71, 72]. Each of these transcription factors is regulated by Wnt and/or Cdc42 signalling (Supplemental References 3). The location of binding sites for the JunD protein that represses the promoter are indicated with red horizontal bars and text [14]. The 7 CpG dinucleotides that could be methylated in AML are depicted in bold red text [70]](/paper-figures/6e14d829e7bd48a384901c29445cab9d/bimodal-expression-of-i-rhoh-i-during-myelomonocytic-differentiation-figure-7.jpg)
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In the paper
“These monocytes and myeloblasts were treated with phorbol ester for 48 h and terminal differentiation into macrophages confirmed by their acquisition of a strong adhesive phenotype, morphological spreading, and characteristic down‐regulation of CD11b and CD93 protein expression (Figure 4A, B, Supplemental Figure S1) [54, 55].”
§ Bimodal expression of RHOH during myelomonocytic differentiation: Implications for the expansion of AML differentiation therapy > RESULTS > RHOH expression is induced during the terminal stages of myeloid differentiation
What the sources say
- Not checked yetcites [54] Park H, Shelley CS, Arnaout MA. The zinc finger transcription factor ZBP‐89 is a repressor of the human β2‐integrin CD11b gene. Blood. 2003; 101: 894‐ 902. 12393719 source
- Not checked yetcites [55] Pilling D, Fan T, Huang D, Kaul B, Gomer RH. Identification of markers that distinguish monocyte‐derived fibrocytes from monocytes, macrophages, and fibroblasts. PLoS One. 2009; 4: e7475. 19834619 PMC2759556 source
plainstates a cause- Asserted: passes — The authors put it forward as true.
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In the paper
“Furthermore, when the AML cell line HL60 was induced to differentiate, with either phorbol ester for 1 hour or lipopolysaccharide for 7 days, RhoH protein levels were again repressed (Figure 3A).”
§ Bimodal expression of RHOH during myelomonocytic differentiation: Implications for the expansion of AML differentiation therapy > RESULTS > RHOH expression progressively declines during the early and intermediate stages of myeloid differentiation
plainstates a cause- Asserted: passes — The authors put it forward as true.
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In the paper
“Literature searches revealed that 42 of these 72 transcripts encode proteins or represent long non‐coding RNAs implicated in regulating functions characteristic of mature myeloid cells (Table 1).”
- Also stated in: § Induction of RHOH expression limits tumour growth in a xenograft model of AML · plain
“Transcriptome analysis implicates RHOH induction in regulating 42 genes controlling the function of mature myeloid cells.”
§ Bimodal expression of RHOH during myelomonocytic differentiation: Implications for the expansion of AML differentiation therapy > RESULTS > RHOH induction is associated with acquisition of a mature myeloid phenotype and alters expression of genes controlling Cdc42 and Wnt signalling
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- Also stated in: § Induction of RHOH expression limits tumour growth in a xenograft model of AML · plain
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In the paper
“In addition, RHOH induction is demonstrated to cause the down‐regulation of CD11b and CD93 protein expression characteristic of monocytes terminally differentiating into macrophages (Figure 4C) [54, 55].”
- Also stated in: § DISCUSSION · boosted
“We demonstrate here that as monocytes terminally differentiate into macrophages RHOH expression is induced and that this induction causes a down‐regulation in the levels of both CD93 and CD11b protein.”
§ Bimodal expression of RHOH during myelomonocytic differentiation: Implications for the expansion of AML differentiation therapy > RESULTS > Induction of RHOH expression limits tumour growth in a xenograft model of AML
What the sources say
- Not checked yetcites [54] Park H, Shelley CS, Arnaout MA. The zinc finger transcription factor ZBP‐89 is a repressor of the human β2‐integrin CD11b gene. Blood. 2003; 101: 894‐ 902. 12393719 source
- Not checked yetcites [55] Pilling D, Fan T, Huang D, Kaul B, Gomer RH. Identification of markers that distinguish monocyte‐derived fibrocytes from monocytes, macrophages, and fibroblasts. PLoS One. 2009; 4: e7475. 19834619 PMC2759556 source
boostedstates a cause- Asserted: passes — The authors put it forward as true.
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- Also stated in: § DISCUSSION · boosted
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In the paper
“In total, the tumours produced by RHOH differentiated AML cells had average volumes 3.6 fold smaller than those produced by their non‐differentiated counterparts (Figure 6).”
§ Bimodal expression of RHOH during myelomonocytic differentiation: Implications for the expansion of AML differentiation therapy > RESULTS > Induction of RHOH expression limits tumour growth in a xenograft model of AML
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Claims other papers make about this one
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In the citing paper
“Collectively, RhoH activity in the myeloid cell is critical in promoting macrophage terminal differentiation by the repression of the interlinked Cdc42 and Wnt intracellular signalling pathways and inflammatory effector proteins, CD11b and CD93.”
From The Role of RhoH in TCR Signalling and Its Involvement in Diseases (2021) · cites this paper as [63]
In the citing paper
“In the case of AML, lack of RhoH is presumed to contribute to AML pathogenesis by inhibiting terminal myeloid differentiation [63].”
From The Role of RhoH in TCR Signalling and Its Involvement in Diseases (2021) · cites this paper as [63]
In the citing paper
“Consistent with this finding, overexpression of JunD was found to repress RhoH gene in AML [61,63], indicating that regulation of RhoH mRNA expression by a transcriptional repressor may affect RhoH anti-tumour activity.”
From The Role of RhoH in TCR Signalling and Its Involvement in Diseases (2021) · cites this paper as [63]
In the citing paper
“Interestingly, RhoH-induced terminal myeloid differentiation may require Cdc42 and/or Wnt signalling inhibition because 13 affected genes were identified following RHOH induction.”
From The Role of RhoH in TCR Signalling and Its Involvement in Diseases (2021) · cites this paper as [63]
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A 17-gene stemness score for rapid determination of risk in acute leukaemia
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Self-citationDifferentiation therapy revisited
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Cited by
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Ahmad Mokhtar, A. M., Hashim, I. F., Mohd Zaini Makhtar, M., Salikin, N. H., & Amin-Nordin, S. (2021) · Cells · doi:10.3390/cells10040950 · Cited by 25
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Sanya, D. R. A., & Onésime, D. (2025) · Human Cell · doi:10.1007/s13577-025-01294-9 · Cited by 1
Full-length RNA-Seq of the RHOH gene in human B cells reveals new exons and splicing patterns
Leprêtre, F., Meneboo, J., Villenet, C., Delestré, L., Quesnel, B., Shelley, C. S., Figeac, M., & Galiègue‐Zouitina, S. (2024) · Scientific Reports · doi:10.1038/s41598-024-79307-0 · Cited by 1
Self-citationShelley, C. S., Galiègue‐Zouitina, S., Andritsos, L. A., Epperla, N., & Troussard, X. (2024) · Leukemia & lymphoma/Leukemia and lymphoma · doi:10.1080/10428194.2024.2438800
Self-citationВакилевна, Ш. Ю., Enikeeva, K., Korobeynikov, V., Shmelkova, P., Gainullina, D. K., Ринатовна, А. Э., & Павлов, В. Н. (2026) · Medical Immunology (Russia) · doi:10.15789/1563-0625-tho-3299
Metadata
- Authors
- Galiègue‐Zouitina, S., Fu, Q., Carton‐Latreche, C., Poret, N., Cheok, M., Leprêtre, F., Figeac, M., Quesnel, B., El Bouazzati, H., & Shelley, C. S.
- Affiliations
- Inserm (FR)
- California Institute for Biomedical Research (US)
- JPARC UMRS 1172 Inserm Lille University Lille France
- Centre National de la Recherche Scientifique (FR)
- Université de Lille (FR)
- LLC Leukaemia Therapeutics Hull Massachusetts USA
- Corresponding author
- Galiègue‐Zouitina, Sylvie
- Shelley, Carl S
- ORCID
- Venue
- eJHaem
- Publisher
- Wiley
- Published
- 2021-01-20
- Volume
- 2
- Issue
- 2
- Pages
- 196-210
- ISSN
- 2688-6146 (ISSN-L)
- PMC
- PMC9175762
- OpenAlex
- W3122045750
- Semantic Scholar
- 15fe3a033339383a91c14d85f9077b30e17446da
- Cited by
- 7
- References
- 73
- Citations by year
- 2026: 3 · 2025: 1 · 2024: 2 · 2021: 1
- FWCI
- 0.49
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- eJHaem (publishedVersion, cc-by)
- PubMed (publishedVersion)
- PubMed Central (publishedVersion, cc-by)
- HAL (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Funders
- Institut National de la Santé et de la Recherche Médicale
- Topics
- Health Sciences › Medicine › Hematology › Acute Myeloid Leukemia Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Retinoids in leukemia and cellular processes
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Keywords
- RhoH, acute myeloid leukemia, myelomonocytic differentiation, macrophage differentiation, terminal differentiation, Cdc42 signaling, Wnt signaling, differentiation therapy
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