MINI REVIEW ARTICLE

published: 01 May 2014

doi: 10.3389/fonc.2014.00093

Granulocyte colony-stimulating factor receptor mutations

in myeloid malignancy
Clifford Liongue 1,2 and Alister Curtis Ward 1,2 *
1
School of Medicine, Deakin University, Geelong, VIC, Australia
2
Strategic Research Centre in Molecular and Medical Research, Deakin University, Geelong, VIC, Australia

Edited by: Granulocyte colony-stimulating factor is a cytokine able to stimulate both myelopoiesis and
Mignon Lee-Cheun Loh, University of
hematopoietic stem cell mobilization, which has seen it used extensively in the clinic to
California San Francisco, USA
aid hematopoietic recovery. It acts specifically via the homodimeric granulocyte colony-
Reviewed by:
Ken Lieuw, Uniformed Services stimulating factor receptor (G-CSFR), which is principally expressed on the surface of
University of the Health Sciences, myeloid and hematopoietic progenitor cells. A number of pathogenic mutations have now
USA been identified in CSF3R, the gene encoding G-CSFR. These fall into distinct classes, each
Hélène Cavé, Hôpital Robert Debré,
of which is associated with a particular spectrum of myeloid disorders, including malig-
France
nancy. This review details the various CSF3R mutations, their mechanisms of action, and
*Correspondence:
Alister Curtis Ward , School of contribution to disease, as well as discussing the clinical implications of such mutations.
Medicine, Deakin University, 75 Keywords: G-CSF, G-CSFR, CSF3R, AML, SCN, CNL, MDS
Pigdons Road, Geelong, VIC 3216,
Australia
e-mail: [email protected]

G-CSF AND ITS RECEPTOR ROLE OF G-CSFR MUTATIONS IN MYELOID DISORDERS

Granulocyte colony-stimulating factor (G-CSF, also called CSF3) A large number of mutations in the gene encoding the G-CSFR,
augments the production and function of neutrophilic granu- designated CSF3R, have now been described. These mutations
locytes, which play an essential role combatting infection, espe- can be placed into a number of distinct classes that relate to the
cially those of a bacterial or fungal nature (1–5). G-CSF acts to type of mutation and their biological and clinical consequences
mobilize hematopoietic precursor cells and stimulate the pro- (Figure 1). Mostly these relate to perturbations of the myeloid
liferation and differentiation of myeloid cells, particularly along lineage, including SCN, Myelodysplastic syndrome (MDS), acute
the neutrophilic lineage, as well as activate various functions of myeloid leukemia (AML), and chronic neutrophilic leukemia
mature neutrophils (6). These properties have seen G-CSF widely (CNL). To avoid potential confusion over mutation nomencla-
used in the treatment of neutropenic conditions, including severe ture, this review provides residue numbers relative to those of the
congenital neutropenia (SCN) (7–9), and those associated with mature G-CSFR in the format suggested by the Human Genome
chemotherapy (10–12). G-CSF has also been extensively used for Variation Society, but with the alternate numbering that includes
harvesting of HSCs from the peripheral blood, thereby overcom- the cleaved signal sequence given in parenthesis in each case, since
ing the requirement for bone marrow transplantations in many these have also been used in the literature.
instances (13, 14).
The biological actions of G-CSF are mediated via docking to “CRIPPLING” EXTRACELLULAR MUTANTS
a homomeric receptor found on the surface of target cells, gran- One class of mutations has been identified affecting the extra-
ulocyte colony-stimulating factor receptor (G-CSFR) (also called cellular domain of the G-CSFR in patients with SCN (36–38) or
CSF3R) (15). The G-CSFR is a member of the hematopoietin chronic idiopathic neutropenia (CIN) (39). These mutations have
receptor superfamily, which has no intrinsic tyrosine kinase activ- in common the property of not only being defective themselves,
ity but upon ligand-binding undergoes conformational changes but also activating in a dominant-negative manner to cripple co-
to stimulate multiple tyrosine kinases associated with its cytoplas- expressed wild-type receptors (36–38). The first of these mutations
mic domain. These include Janus kinases (JAKs), especially JAK1 described was a germline p.Pro206His (p.Pro229His) change that
and JAK2 (16–19), members of the SRC kinase family, especially disrupted a conserved di-proline “hinge” motif located between
LYN and HCK (20–22), as well as SYK (20) and TNK (23). Impor- two halves of the ligand-binding cytokine receptor homology
tant pathways activated downstream include the signal transducer (CRH) domain. This disrupted the normal architecture of the
and activator of transcription (STAT) proteins, particularly STAT3 ligand/receptor complex, with severe consequences for G-CSF-
and STAT5 (17, 18, 24, 25), the phosphatidyl inositol 3-kinase mediated signal transduction and cellular responses (36). Two
(PI3-K)–AKT pathway (21, 26, 27), and the RAS–MAPK pathway other mutants represent deletions of the CSF3R gene and con-
(28–30). Signaling via the G-CSFR is tightly regulated, includ- comitant alterations in reading frame that yield G-CSFR proteins
ing by members of the SOCS family, especially SOCS3 and CISH consisting of extracellular regions truncated at the WSXWS motif
(31, 32), as well as the tyrosine phosphatases SHP-1 (26, 33) and followed by a novel sequence and a premature stop: the somatic
SHP-2 (34, 35). p.Ser296Gly,fs*29 (p.Ser319Gly,fs*29) mutation (38) and the

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Liongue and Ward G-CSFR mutations in myeloid malignancy

FIGURE 1 | Granulocyte colony-stimulating factor receptor rectangles) and important tyrosine residues within the cytoplasmic region.
perturbations in disease. Schematic representation of the mature The relative positions of various classes of mutation are indicated on the
G-CSFR (RefSeq NP_000751.3), showing important subdomains and right along with the respective clinical manifestations of these and other
residues conserved among members of the hematopoietin receptor G-CSFR perturbations. Abbreviations: Ig, immunoglobulin-like; CRH,
superfamily, including the N-terminal Ig domain, four conserved cysteines cytokine receptor homology; SCN, severe congenital neutropenia; CNL,
(thin line), and WSXWS motif (thick line) within the CRH domain, chronic neutrophilic leukemia; MDS, myelodysplastic syndrome; AML,
fibronectin, and transmembrane domains as well as Box 1–3 (gray acute myeloid leukemia.

germline p.Ser299Gly,fs*29 (p.Ser322Gly,fs*29) (37). Finally, the The p.Thr595Ile (p.Thr618Ile) mutation was initially described
CIN-associated p.Ser601Arg,fs*177 (p.Ser624Arg,fs*177) muta- as a late somatic mutation in the development of AML in an
tion involved a frameshift that truncates the receptor after the SCN patient already bearing an alternate G-CSFR mutation (46).
fibronectin domains (39). While not directly promoting malig- However, p.Thr595Ile has subsequently been identified as a com-
nancy, the neutropenic conditions that this class of mutation pro- mon mutation in CNL (23, 47), with the adjacent p.Thr592Ala
duces are likely to create susceptibility to other changes that can. (p.Thr615Ala) mutation alternatively found in other cases of CNL
Indeed, one SCN patient with this type of mutation subsequently (23). The p.Thr595Ile mutation is also less commonly observed
acquired additional truncating mutations in the G-CSFR (40), in atypical chronic myelogenous leukemia (aCML) (23), chronic
while the CIN patient went on to develop acute myeloid/natural myelomonocytic leukemia (CMML) (48), de novo AML (23, 48,
killer cell leukemia, although whether the CSF3R mutation played 49), as well as in cases of early T-cell precursor acute lymphoblas-
a role in the latter was not determined (39). tic leukemia (ETP-ALL) (23). G-CSFR forms containing either
the p.Thr595Ile or p.Thr592Ala mutation supported G-CSF-
“ACTIVATING” TRANSMEMBRANE MUTANTS independent growth of Ba/F3 cells, although growth was similar
Another class of CSF3R mutations affects the transmembrane to wild-type receptor at high G-CSF concentrations (48). Bone
domain and adjacent residues of the encoded receptor. This class marrow transduced with the p.Thr595Ile mutant also resulted
of mutations appears to act by stabilizing transmembrane helix– in G-CSF-independent growth (46), which could be replicated
helix interactions in the absence of ligand, creating an active by a p.Thr595Val mutant, suggesting the change to a hydropho-
dimeric configuration that leads to constitutive (and enhanced) bic amino acid was sufficient (49). Ba/F3 cells expressing the
activation (41). These are analogous to the activating mutations p.Thr595Ile mutant showed constitutive activation of JAK2, SRC,
found in the thrombopoietin receptor, c-MPL, which are associ- TNK, STAT3, and STAT5 (23, 48), but not AKT and ERK, as well
ated with hereditary or acquired thrombocythemia (42, 43), or as enhanced ROS production (48). Signaling from the mutant was
those in the βc chain of the heterodimeric IL-3R family identified found to be sensitive to various JAK kinase inhibitors, including
in vitro (44, 45). ruxolinitib and tofacitinib (23, 48), with some evidence of clinical

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Liongue and Ward G-CSFR mutations in myeloid malignancy

efficacy (23), but not to dasatinib that targets a number of tyrosine truncation displayed normal neutrophil numbers, although the
kinases, including SRC and TNK (23, 48). truncated form of the receptor was significantly overexpressed rel-
The p.Thr617Asn (p.Thr640Asn) mutation was first identified ative to the wild-type (59). However, all three studies revealed a
in a single case of AML (50). Further studies identified this – and hyper-responsiveness to G-CSF, with exogenous G-CSF produc-
the alternate p.Thr617Ile (p.Thr640Ile) – as rare, somatic muta- ing elevated numbers of neutrophils compared to wild-type mice
tions in de novo AML (49, 51). However, a germline p.Thr617Asn (57–59), due to increased myeloid progenitor proliferation (58,
mutation was also identified as the cause of autosomal dominant 65). Another study confirmed that G-CSFR truncations conferred
hereditary neutrophilia, where it showed complete penetrance a strong clonal HSC advantage that was also dependent on exoge-
(52). Interestingly, one of the affected individuals progressed to nous G-CSF (66), providing insight into how these mutants may
a myelodysplastic syndrome type disease (52), further implicat- contribute to their frequent progression to MDS/AML. Notably,
ing this mutation as predisposing toward myeloid malignancy. expression of the truncated receptor in mice was not by itself
In addition to neutrophilia, patients harboring p.Thr617Asn pos- leukemogenic, since no spontaneous leukemia has been reported
sessed increased numbers of CD34+ cells, which were able to in mice hetero- or homozygous for the mutation (57, 59). However,
proliferate and terminally differentiate in the absence of G-CSF, the truncated G-CSFR was found to co-operate with PML–RARa
and induce a myeloproliferative (MPD)-like disorder in mice. to induce AML in mice, where it decreased latency in a G-CSF-
Patient CD34+ cells showed constitutive phosphorylation of dependent manner, leading to higher blast counts and increased
JAK2, STAT3, STAT5, and ERK, which were hyperactivated by myelosuppression (67).
G-CSF compared to wild-type cells (52). Lineage-negative bone Investigation into the molecular mechanisms of G-CSFR signal
marrow cells retrovirally transduced with the p.Thr617Asn mutant transduction has helped to explain the dominant hyperprolif-
G-CSFR caused neutrophilia when transplanted into irradiated erative function of truncated G-CSFRs. These mutant receptors
mice (52). The p.Thr617Asn mutation also supported factor- exhibit higher and more sustained activation in comparison to
independent growth and survival in Ba/F3 cells, with weak con- wild-type receptors, with a heavily reduced “off-rate” (65, 68, 69).
stitutive phosphorylation of the receptor, JAK2, STAT3, and ERK, This is partly a result of impaired internalization due to the com-
and also enabled transduced CD34+ cells to undergo myeloid bined loss of a conserved di-leucine containing motif in Box 3 (69,
differentiation in the absence of G-CSF (51). 70), and a less well-defined motif spanning residues 756–769 (34).
Finally, an in-frame three nucleotide deletion has been iden- However, direct negative regulation is also blunted, due to the loss
tified in MDS that replaces two amino acids with an alter- of recruitment sites on the truncated receptors, including those for
nate residue, p.Asn630Lys,Arg631del (p.Asn653Lys,Arg654del). the receptor-associated tyrosine phosphatases SHP-1 (at an unde-
This mutation resulted in prolonged signaling following ligand fined site in the C-terminus) (71) and SHP-2 (at Y724) (34), and
stimulation (53). two members of the SOCS family, CISH (at Y729 and Y744) (32)
and SOCS3 (at Y729) (34), the latter exacerbated by decreased
“HYPERRESPONSIVE” INTRACELLULAR TRUNCATIONS SOCS3 transcription as a result of reduced STAT3 activation by
By far the most studied clinical abnormalities of the CSF3R gene truncated receptors (34).
are a series of acquired nonsense mutations identified in a sub- Cells expressing truncated G-CSFR receptors are also hyper-
set of SCN patients with a propensity to progress to leukemia. sensitive to ligand (54, 70). This is associated with an altered
These somatic mutations typically affect a single allele to trun- dose–response of STAT3:STAT5 activation, the ratio of which is
cate between 82 and 98 amino acids from the carboxyl-terminus drastically reduced at low concentrations of G-CSF (24). Since
of the receptor, such as p.Gln718* (p.Gln741*) and pGln731* STAT5 contributes to G-CSF proliferative responses (72), while
(p.Gln754*) (54, 55). These truncated receptors show normal STAT3 is inhibitory (73–75), the reduced STAT3:STAT5 ratio
affinity for G-CSF (56), but mediate heightened growth and may shift the balance toward proliferation, explaining the G-CSF
diminished maturation in response to ligand, acting dominantly hypersensitivity (54, 56).
over wild-type receptors (54). Truncated G-CSFRs are not the pri- Granulocyte colony-stimulating factor receptor truncation
mary cause of SCN, although they may exacerbate it to a modest impacts on the length and magnitude of receptor activation, and
extent (57–60). However, it is clear that SCN patients carrying particularly of STAT5 (69–71), pathways downstream of PI3-K,
truncating G-CSFR mutations show a particularly strong predis- such as AKT (27, 76), as well as SRC (23). Dominant-negative
position to both MDS and AML (61, 62). Indeed in SCN patients STAT5 has been shown to inhibit the hyperproliferative func-
progressing to AML, the most common mutations identified are tion of truncated G-CSFRs in vitro (77), while the absence of
in CSF3R (82%), followed by RAS (~50%) and monosomy 7 (63), STAT5 abrogated the clonal HSC advantage conferred by these
and when CSF3R mutations are present, 100% of blasts carry the receptors in vivo (66). Other pathways also contribute to prolifer-
mutation (54, 63). However, since mutations are not always seen ation and survival, including PI3-K, MAPK, and STAT3 (76–78).
in AML and can spontaneously disappear (64), progression to Interestingly, receptor truncations are sensitive to the multi-kinase
leukemia is not inevitable. inhibitor dasatinib, but not to JAK inhibitors (23), suggesting
A mouse line carrying a truncated G-CSFR “knock-in” allele an intrinsic difference in comparison to the activating trans-
(57) or one transgenically expressing a truncated human G-CSFR membrane mutants. Truncated receptors have also been shown
(58) exhibited mild neutropenia, with an increased percentage to increase ROS production (79), potentially creating genotoxic
of immature myeloid cells that were defective in maturation ex stress to facilitate addition mutations in cells expressing these
vivo (58, 65). An alternate mouse line with a targeted receptor receptors.

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Liongue and Ward G-CSFR mutations in myeloid malignancy

FIGURE 2 | Inhibitors of signaling pathways downstream of as well as the tyrosine residues that serve important docking sites for
G-CSFR. Schematic representation of the intracellular domain of the downstream signaling proteins indicated. Known inhibitors of these
G-CSFR, showing the important Box 1–3 sequences (gray rectangles), are shown.

DEFECTIVE SPLICE VARIANTS PATHOGENIC SNP

A presumably somatic single base change in CSF3R adjacent to a A CSF3R SNP that is present in ~6% of the population leads
cryptic splice-donor site has been identified in blasts of a de novo to a p.Glu785Lys (p.Glu808Lys) amino acid substitution in the
AML patient. This resulted in high expression of an alternate splice intracellular region of the G-CSF, which predisposes individuals
variant that generated a G-CSFR protein in which the C-terminal to high-risk MDS (82). Interestingly, blasts from an individual
130 amino acids are replaced with a different 34 amino acids from who developed AML following high-risk MDS were found to be
an alternate reading frame, p.Val684Ala,fs*34 (p.Val707Ala,fs*34) homozygous for this allele (83), providing further evidence of
(80). The primary AML blast cells of this patient failed to respond the potential pathogenicity of this SNP. Although the mechanism
to G-CSF in proliferation assays in vitro, despite responsiveness of action remains unknown, the variant receptor appears func-
to IL-3 or GM-CSF being maintained. This variant was unable to tional, but can act in a dominant-negative manner to reduce colony
transduce either proliferation or maturation signals in murine cell formation compared to the wild-type receptor (82, 83).
systems. By corollary, AML cells show a tendency for significantly
increased levels of a normally minor CSF3R transcript, class IV CONCLUSION
(81), which encodes a similar G-CSFR protein in which the C- Granulocyte colony-stimulating factor has proven to be an effec-
terminal 87 amino acids are replaced with the same alternate 34 tive therapy in a range of life-threatening conditions or to aid
amino acids, p.Val727Ala,fs*34 (p.Val750Ala,fs*34). The authors in the recovery of medical treatments, such as in the treatment
argue that the altered balance of class IV to normal (class I) recep- of neutropenia following chemotherapy. However, the evidence
tors might contribute to AML, due to the ability of the class IV suggests that G-CSFR mutations contribute to several disorders,
receptor to block maturation. including in settings where G-CSF may be used therapeutically. It

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Liongue and Ward G-CSFR mutations in myeloid malignancy

has been suggested that use of G-CSF in SCN may allow the selec- 10. Hartmann LC, Tschetter LK, Habermann TM, Ebbert LP, Johnson PS, Mail-
tive expansion of clones containing truncating CSF3R mutations. liard JA, et al. Granulocyte colony-stimulating factor in severe chemotherapy-
induced afebrile neutropenia. N Engl J Med (1997) 336:1776–80. doi:10.1056/
However, the available data are complicated, making conclusions
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age of MDS/AML onset and G-CSF dose or duration of therapy pediatric oncology. Curr Opin Hematol (2002) 9:222–7. doi:10.1097/00062752-
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in SCN patients increased with the degree of G-CSF therapy (84). 12. Bohlius J, Reiser M, Schwarzer G, Engert A. Impact of granulocyte colony-
stimulating factor (CSF) and granulocyte-macrophage CSF in patients with
However, higher doses may also reflect a more severe underlying
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disease with a higher propensity to MDS/AML. In addition, SCN doi:10.1046/j.1365-2141.2003.04450.x
patients developed AML prior to the advent of G-CSF therapy. 13. Bensinger WI, Weaver CH, Appelbaum FR, Rowley S, Demirer T, Sanders J,
In line with this, one SCN patient progressed to CMML in the et al. Transplantation of allogeneic peripheral blood stem cells mobilized by
absence of G-CSF treatment, but expressed a truncated G-CSFR recombinant human granulocyte colony-stimulating factor. Blood (1995) 85:
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(85). Thus it is possible that the mutant receptor form may have 14. Demirer T, Ayli M, Ozcan M, Gunel N, Haznedar R, Dagli M, et al. Mobi-
a selective advantage in the absence of treatment, perhaps due to lization of peripheral blood stem cells with chemotherapy and recombinant
the elevated G-CSF levels seen in SCN patients as a result of their human granulocyte colony-stimulating factor (rhG-CSF): a randomized eval-
neutropenia (63). However, G-CSF therapy is not a factor in other uation of different doses of rhG-CSF. Br J Haematol (2002) 116:468–74.
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these might specifically combat the aberrant signaling elicited by
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ACKNOWLEDGMENTS 86:3698–704.
The work was supported by funding from Deakin University via 18. Tian S-S, Tapley P, Sincich C, Stein RB, Rosen J, Lamb P. Multiple signaling path-
a Central Research Grant (to Alister Curtis Ward) and an Alfred ways induced by granulocyte colony-stimulating factor involving activation of
Deakin Postdoctoral Research Fellowship (to Clifford Liongue). JAKs, STAT5, and/or STAT3 are required for regulation of three distinct classes
of immediate early genes. Blood (1996) 88:4435–44.
19. Shimoda K, Feng J, Murakami H, Nagata S,Watling D, Rogers NC, et al. Jak1 plays
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granulocyte colony-stimulating factor-induced differentiation of myeloid cells. in the absence of any commercial or financial relationships that could be construed
J Biol Chem (1997) 272:25184–9. doi:10.1074/jbc.272.40.25184 as a potential conflict of interest.
74. de Koning JP, Soede-Bobok AA, Ward AC, Schelen AM, Antonissen C, van
Leeuwen D, et al. STAT3-mediated differentiation and survival of myeloid Received: 31 January 2014; accepted: 14 April 2014; published online: 01 May 2014.
cells in response to granulocyte colony-stimulating factor: role for the cyclin- Citation: Liongue C and Ward AC (2014) Granulocyte colony-stimulating factor recep-
dependent kinase inhibitor p27Kip1. Oncogene (2000) 19:3290–8. doi:10.1038/ tor mutations in myeloid malignancy. Front. Oncol. 4:93. doi: 10.3389/fonc.2014.00093
sj.onc.1203627 This article was submitted to Pediatric Oncology, a section of the journal Frontiers in
75. Lee CK, Raz R, Gimeno R, Gertner R, Wistinghausen B, Takeshita K, et al. Oncology.
STAT3 is a negative regulator of granulopoiesis but is not required for G- Copyright © 2014 Liongue and Ward. This is an open-access article distributed under
CSF-dependent differentiation. Immunity (2002) 17:63–72. doi:10.1016/S1074- the terms of the Creative Commons Attribution License (CC BY). The use, distribution
7613(02)00336-9 or reproduction in other forums is permitted, provided the original author(s) or licensor
76. Hunter MG, Avalos BR. Granulocyte colony-stimulating factor receptor muta- are credited and that the original publication in this journal is cited, in accordance with
tions in severe congenital neutropenia transforming to acute myeloid leukemia accepted academic practice. No use, distribution or reproduction is permitted which
confer resistance to apoptosis and enhance cell survival. Blood (2000) 95:2132–7. does not comply with these terms.

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