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Rac guanosine triphosphatases represent a potential target in AML
Open accessAbstract
Acute myeloid leukemia (AML) is intrinsically prone to resistance to conventional chemotherapeutic agents. Here we explore the Rac family of small GTPases as novel biologic targets for AML treatment. The Rac subfamily of Rho guanosine triphosphatases (GTPases) plays an essential role in regulating normal hematopoiesis (1). Rac proteins cycle between active GTP-bound and inactive GDP-bound states. Of the three known Rac family members, Rac1 and Rac3 are expressed ubiquitously, while Rac2 is restricted to the hematopoietic system. In hematopoietic cells, Rac proteins integrate signals from growth factor, chemokine, and adhesion receptors to mediate a variety of cellular responses, including cell growth and survival, gene transcription, adhesion, motility, and formation of the actin cytoskeleton (1). We recently identified the Rac GTPases as molecular targets in BCR-ABL-induced myeloproliferative disease (2, 3). However, the role of Rac in AML has thus far not been clearly defined. Elevated levels of GTP-bound Rac have been described in CD34+ cells isolated from patients with AML. In these samples, Rac signaling was identified as a critical mediator of stem/progenitor cell and stroma interaction (4). Recently, Wei et al. observed a critical role of Rac signaling in a disease model of human CD34+ cells transduced with the Mixed Lineage Leukemia (MLL)-AF9 fusion oncogene (5). Interestingly, while MLL-AF9 transduced cells were sensitive to Rac inhibition, cells transduced with the AML-ETO oncogene did not depend on Rac signaling for survival and proliferation. These discrepancies prompted us to further investigate the role of Rac signaling in a panel of human AML cell lines, including the MLL gene-rearranged ML-2 cell line and cell lines not harboring MLL rearrangements such as the histiocytic lymphoma U937 and the acute promyelocytic HL-60 cell line. We demonstrate the presence of GTP-Rac in all cell lines via p21-activated kinase (PAK)-binding domain (PBD) pull-down and immunoblot (data not shown). Compared to purified normal human CD34+ cells, ML-2 cells, which contain a MLL-AF6 translocation (6), showed the most profound inhibition of cell proliferation upon pharmacologic inhibition of Rac using the small molecule Rac inhibitor NSC23766 (7) (Figure 1A). To determine whether a correlation exists between Rac activation and the observed decrease in proliferation of ML-2 cells, we analyzed the effect of NSC23766 on GTP-Rac via PBD pull-down assay and observed abrogation of Rac activation with drug treatment (Figure 1B). We next wanted to determine whether NSC23766 treatment would impact apoptosis (Figure 1C) and/or cell cycle progression of ML-2 cells (Figure 1D). ML-2 cells displayed an increase of early and late apoptosis as measured by Annexin V/7AAD 72 hours after drug exposure (Figure 1C). In addition, Rac inhibition led to increased cell cycle arrest in G0/G1 (Figure 1D). Importantly, these effects were specific to ML-2 cells, as normal CD34+ cells were not significantly affected by NSC23766 exposure. Analogous effects were observed in the MLL-AF9 containing THP-1 cell line (Supplemental Figure 1). In contrast to these MLL gene rearranged cell lines, no significant effect of NSC23677 on cell cycle or apoptosis was observed in U937 cells and only marginal effects were observed in HL- 60 cells (data not shown). To further analyze the potential therapeutic efficacy of NSC23766 in a murine xenograft model, 2 × 107 ML-2 cells were transplanted into irradiated (350 Gy) NOD/SCID mice. Alzet osmotic pumps containing NSC23766 (2 pumps, 75 mM NSC23766 per pump) or PBS were implanted on day 21 post transplant. The pumps were exchanged for new pumps on day 35 and removed on day 49 post transplant. Animals were monitored for survival and bone marrow chimerism of ML-2 cells (human CD45+) was assessed by flow cytometry post-mortem. Animals with less than 15% human CD45+ chimerism were censored from the study (3 animals in the NSC23766- and four animals in the PBS-cohort). We noted a significant difference in the survival of PBS vs. NSC23677-treated animals (Figure 2). Death due to disease progression in the treatment group occurred predominantly following pump removal.
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TL;DR
Rac GTPases are activated in a panel of human AML cell lines, and pharmacologic Rac inhibition with NSC23766 preferentially impairs MLL-rearranged AML cell lines while sparing normal CD34+ cells. In an ML-2 xenograft model, NSC23766 treatment was associated with a significant survival difference versus PBS, though deaths in the treatment group occurred predominantly after pump removal.
Key findings
- GTP-Rac was present in all tested human AML cell lines by PAK-binding domain (PBD) pull-down and immunoblot (data not shown).
- ML-2 cells, which contain an MLL-AF6 translocation, showed the most profound inhibition of proliferation after NSC23766; treatment abrogated Rac activation, increased early and late apoptosis by Annexin V/7AAD at 72 hours, and increased G0/G1 arrest. Normal CD34+ cells were not significantly affected.
- Analogous effects were observed in the MLL-AF9-containing THP-1 cell line. In contrast, U937 cells showed no significant effect on cell cycle or apoptosis, and HL-60 cells showed only marginal effects.
- In irradiated NOD/SCID mice transplanted with 2 × 10^7 ML-2 cells, NSC23766 delivered by Alzet osmotic pumps (2 pumps, 75 mM per pump) versus PBS produced a significant survival difference; deaths due to disease progression in the treatment group occurred predominantly after pump removal.
Methods
- In vitro studies used a panel of human AML cell lines, including MLL-rearranged ML-2 and MLL-AF9-positive THP-1, plus U937 and HL-60, compared with purified normal human CD34+ cells.
- Rac activation was assessed by PBD pull-down and immunoblot; NSC23766 effects were measured by proliferation, Annexin V/7AAD apoptosis, and cell cycle analysis.
- In vivo, ML-2 cells were transplanted into irradiated NOD/SCID mice; NSC23766 or PBS was delivered via osmotic pumps implanted on day 21, exchanged on day 35, and removed on day 49. Survival and bone marrow ML-2 chimerism (human CD45+) were assessed; animals with less than 15% human CD45+ chimerism were censored (3 NSC23766-treated and 4 PBS-treated animals).
Limitations
- The paper states that the mechanism by which other cell lines bypass Rac signaling will require further studies, and that more potent Rac GTPase inhibitors are needed for clinical use.
- Evident from the text: several key assays are reported as “data not shown,” including GTP-Rac presence across cell lines and the lack of significant effects in U937 and HL-60 cells.
- Evident from the text: the study relies on established cell lines and a xenograft model rather than primary AML patient samples, and the in vivo experiment uses a small number of censored animals and pump-limited drug exposure.
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“we analyzed the effect of NSC23766 on GTP-Rac via PBD pull-down assay and observed abrogation of Rac activation with drug treatment (Figure 1B).”
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In the paper
“Importantly, these effects were specific to ML-2 cells, as normal CD34+ cells were not significantly affected by NSC23766 exposure.”
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In the paper
“Compared to purified normal human CD34+ cells, ML-2 cells, which contain a MLL-AF6 translocation (6), showed the most profound inhibition of cell proliferation upon pharmacologic inhibition of Rac using the small molecule Rac inhibitor NSC23766 (7) (Figure 1A).”
- Also stated in: another passage · plain
“the MLL gene-rearranged cell line ML-2 displayed the most profound dependence on Rac signaling.”
What the sources say
- Not checked — no full text of the sourcecites [6] Drexler HG, MacLeod RA . Malignant hematopoietic cell lines: in vitro models for the study of anaplastic large-cell lymphoma. Leukemia 2004; 18: 1569–1571. source
- Not checked yetcites [7] Gao Y, Dickerson JB, Guo F, Zheng J, Zheng Y . Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc Natl Acad Sci USA 2004; 101: 7618–7623. source
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“We demonstrate the presence of GTP-Rac in all cell lines via p21-activated kinase (PAK)-binding domain (PBD) pull-down and immunoblot (data not shown).”
- Also stated in: another passage · plain
“While the drug target GTP-Rac was present in all cell lines (not shown)”
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“Analogous effects were observed in the MLL-AF9 containing THP-1 cell line (Supplemental Figure 1).”
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In the paper
“We noted a significant difference in the survival of PBS vs. NSC23677-treated animals (Figure 2).”
“Alzet osmotic pumps containing NSC23766 (2 pumps, 75 mM NSC23766 per pump) or PBS were implanted on day 21 post transplant.”
Citations
Cites
Rational design and characterization of a Rac GTPase-specific small molecule inhibitor
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Self-citationHematopoietic Cell Regulation by Rac1 and Rac2 Guanosine Triphosphatases
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Self-citationMicroenvironment Determines Lineage Fate in a Human Model of MLL-AF9 Leukemia
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Self-citationRac GTPases as key regulators of p210-BCR-ABL-dependent leukemogenesis
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Mack, N. A., Whalley, H. J., Castillo‐Lluva, S., & Malliri, A. (2011) · Cell Cycle · doi:10.4161/cc.10.10.15612 · Cited by 176
Rho GTPases in hematopoiesis and hemopathies
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Self-citationBosco, E. E., Kumar, S., Marchioni, F., Biesiada, J., Kordos, M., Szczur, K., Meller, J., Seibel, W. L., Mizrahi, A., Pick, E., et al. (2012) · Chemistry & Biology · doi:10.1016/j.chembiol.2011.12.017 · Cited by 62
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Favaro, P. M. B., Traina, F., Machado‐Neto, J. A., Lazarini, M., Lopes, M. R., Pereira, J. K. N., Costa, F. F., Infante, E., Ridley, A. J., & Saad, S. T. O. (2013) · Journal of Leukocyte Biology · doi:10.1189/jlb.0113057 · Cited by 59
Leukaemia cell of origin identified by chromatin landscape of bulk tumour cells
George, J., Uyar, A., Young, K. A., Kuffler, L., Waldron-Francis, K., Márquez, E. J., Ucar, D., & Trowbridge, J. J. (2016) · Nature Communications · doi:10.1038/ncomms12166 · Cited by 54
Overexpression of Rac1 in leukemia patients and its role in leukemia cell migration and growth
Wang, J., Rao, Q., Wang, M., Wei, H., Xing, H., Liu, H., Wang, Y., Tang, K., Peng, L., Tian, Z., et al. (2009) · Biochemical and Biophysical Research Communications · doi:10.1016/j.bbrc.2009.06.125 · Cited by 48
Targeted Elimination of Leukemia Stem Cells; a New Therapeutic Approach in Hemato-Oncology
ten Cate, B., de Bruyn, M., Wei, Y., Bremer, E., Helfrich, W., Mourabet, M. E., El‐Hachem, S., Harrison, J., & Binion, D. G. (2009) · Current Drug Targets · doi:10.2174/138945010790031063 · Cited by 45
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Rho GTPases in hematopoietic stem cell functions
Cancelas, J. A., & Williams, D. A. (2009) · Current Opinion in Hematology · doi:10.1097/moh.0b013e32832c4b80 · Cited by 40
Self-citationTransforming human blood stem and progenitor cells: A new way forward in leukemia modeling
Mulloy, J. C., Wunderlich, M., Zheng, Y., & Wei, J.-P. (2008) · Cell Cycle · doi:10.4161/cc.7.21.6951 · Cited by 30
Self-citationPharmacological Rac1 inhibitors with selective apoptotic activity in human acute leukemic cell lines
Cabrera, M., Echeverría, E., Lenicov, F. R., Cardama, G. A., González, N., Davio, C. A., Fernández, N., & Menna, P. L. (2017) · Oncotarget · doi:10.18632/oncotarget.21533 · Cited by 27
Chen, S., Li, H., Li, S., Yu, J., Wang, M., Xing, H., Tang, K., Tian, Z., Rao, Q., & Wang, J. (2016) · Stem Cells · doi:10.1002/stem.2348 · Cited by 26
Rac1 targeting suppresses p53 deficiency–mediated lymphomagenesis
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Self-citationFrat2 mediates the oncogenic activation of Rac by MLL fusions
Walf-Vorderwülbecke, V., de Boer, J., Horton, S. J., van Amerongen, R., Proost, N., Berns, A. J. M., & Williams, O. (2012) · Blood · doi:10.1182/blood-2012-05-432534 · Cited by 20
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Rho and Rap guanosine triphosphatase signaling in B cells and chronic lymphocytic leukemia
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Self-citation
Metadata
- Authors
- Müller, L. U. W., Schore, R. J., Zheng, Y., Thomas, E. K., Kim, M.-O., Cancelas, J. A., Gu, Y., & Williams, D. A.
- Affiliations
- Cincinnati Children's Hospital Medical Center (US)
- Corresponding author
- Williams, D A
- Venue
- Leukemia
- Published
- 2008-07-31
- Volume
- 22
- Issue
- 9
- Pages
- 1803-1806
- ISSN
- 0887-6924 (ISSN-L)
- 1476-5551
- PMC
- PMC4185433
- OpenAlex
- W2052574445
- Semantic Scholar
- 55b4a991266e57a18832e3abc684a4ec1074c8c6
- Cited by
- 30
- References
- 7
- Citations by year
- 2022: 2 · 2019: 1 · 2017: 1 · 2016: 2 · 2015: 2 · 2014: 1 · 2013: 7 · 2012: 2
- FWCI
- 2.55
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Leukemia (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- St. Baldrick's Foundation
- National Institutes of Health
- National Heart, Lung, and Blood Institute (P01HL069974)
- National Institute of Diabetes and Digestive and Kidney Diseases (R01DK062757)
- Topics
- Health Sciences › Medicine › Hematology › Acute Myeloid Leukemia Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Tyrosine Phosphatases
- Keywords
- acute myeloid leukemia, GTPase inhibitors, therapeutic target
- MeSH terms
- Aminoquinolines, Animals, Cell Cycle, Cell Survival, Drug Screening Assays, Antitumor, Enzyme Inhibitors, Humans, Pyrimidines, Transplantation, Heterologous, Leukemia, Myeloid, Acute, Survival Rate, Drug Delivery Systems, rac GTP-Binding Proteins, Cell Line, Tumor, Cell Proliferation, Mice
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