To the Editor
Colony stimulating factor 2 receptor beta (CSF2RB) is the shared beta-chain receptor
and essential signaling component for IL-3, IL-5 and GM-CSF receptor activation. In
the context of GM-CSF signaling, the ligand-specific alpha chain (CSF2RA) complexes
with CSF2RB and GM-CSF ligand, forming a dodecameric complex in which the proximity
of CSF2RB subunits allows associated JAK2 kinases to trans-phosphorylate
1
. CSF2RB signals through several pathways including JAK2/STAT5, PI3K/mTOR and MEK/ERK
to promote survival, proliferation and differentiation (reviewed in
2
). Both spontaneous transformation
3, 4
and random mutagenesis
4, 5
screens have shown that CSF2RB mutations can result in ligand-independent activation
in vitro. However, these predicted oncogenic mutations have never been observed clinically.
In fact, the only phenotypic CSF2RB mutations reported are recessive, loss-of-function,
germline mutations in pulmonary alveolar proteinosis
6
. As oncology moves toward an age of personalized and molecularly-targeted treatment
it will require the identification and in vitro characterization of targetable genetic
lesions. Here we describe the first case of a leukemia patient harboring a germline,
CSF2RB-activating point mutation (R461C) and identify small molecule inhibitors with
therapeutic potential against this mutation.
We obtained a pediatric T-cell acute lymphoblastic leukemia (T-ALL, Supplemental Figure
1A) with informed consent approved by the Institutional Review Boards of Oregon Health
& Science University and Erasmus University Medical Center - Sophia Children’s Hospital.
We isolated mononuclear cells and performed deep sequencing as previously described
7
, uncovering the CSF2RB R461C mutation at a 54% allele burden (Supplemental Figure
1B and C). The patient also presented with somatic NOTCH1-truncation, NOTCH1 missense,
and PTEN point mutations, all recurrent and leukemia-associated mutations
8
(other observed mutations in Supplemental Table 1). R461C was later confirmed by Sanger
sequencing as a heterozygous, germline variant using a minimal residual disease day
79 sample for normal DNA (Supplemental Figure 1D). CSF2RB R461C has never been previously
described in cancer patients (including public databases) but is listed as a SNP in
the 1000 Genomes database
9
(rs371045078) at a low allele frequency (MAF=0.000998, 5 observations in 1000 Genomes
9
; MAF=0.000272, 33 observations in the Exome Aggregation Consortium
10
) in exclusively heterozygous cases. Prior work has established that cancer-predisposing
mutations exist in the germline of healthy individuals at frequencies as high as 1.1%,
but those frequencies elevate to over 8% in children and adolescents with cancer,
most of which demonstrate no family history of cancer
11
. Congenital mutations in CSF3R have also been observed to precede the development
of acute myeloid leukemia
12
. In addition, there are numerous reports of families with gain-of-function germline
mutations in receptors with similar biology as CSF2RB (e.g., CSF3R
13
, EPOR
14
, MPL
15
) and in many of these pedigrees the affected family members do not develop overt
leukemia, though they sometimes exhibit elevated blood counts.
The location of this variant, within the putative membrane-spanning portion of the
receptor, is of particular interest as this region is a hot spot for activating mutations
in vitro
5
. Previously, mutations altering the number of membrane-spanning or membrane-adjacent
unpaired cysteines have been shown to activate the IL-7 receptor in T-ALL
16
and RET in medullary thyroid carcinoma
17
. CSF2RB has been shown to be partially activated in vitro by replacement of the extracellular
domain with a short, cysteine-containing sequence
4
. Lastly, multiple sequence alignment of CSF2RB homologues demonstrated that arginine
461 is conserved across most mammals (Supplemental Figure 2) increasing the potential
that R461C could have functional relevance and cooperate with canonical leukemogenic
mutations. Accordingly, we hypothesized that R461C could activate CSF2RB-signaling
and contribute to the patient’s leukemia.
We cloned CSF2RB R461C into the pMXs-IRES-Puro plasmid and transfected into IL-3-dependent
murine Ba/F3 cells by electroporation. Stably transfected cells were selected using
two weeks of continual 2µg/ml puromycin selection prior to performing IL-3 withdrawal
transformation assays as previously described
7
. R461C conferred factor-independent growth while wild-type (WT) CSF2RB did not (Figure
1A, Supplemental Figure 3). The full CSF2RB transgene was sequence confirmed in two
biologically replicate, IL-3 independent cell lines and used for further studies.
We identified that CSF2RB R461C expression resulted in an accumulation of surface
CSF2RB protein, relative to WT, prior to IL-3 withdrawal when assayed by flow cytometry
(Supplemental Figure 4). Furthermore, immunoblot analysis showed that the R461C protein
is significantly increased in IL-3-independent cells relative to WT cells grown in
IL-3 (Figure 1B, details and all antibodies included Supplemental methods and Supplemental
Table 2). As both exogenous versions of CSF2RB were expressed by identical promoters
we rationalized that the accumulation was due to changes in CSF2RB protein stability.
We performed cycloheximide time course experiments using 100ug/ml cycloheximide in
DMSO followed by staining with PE-conjugated anti-CSF2RB to determine alterations
in surface protein stability. The R461C mutant possessed a prolonged surface half-life
relative to WT CSF2RB (6.5 vs 3.6 hours; Figure 1C). Given the presence of a novel
cysteine and increased stability in the R461C mutant, we investigated disulfide-linked
receptor oligomerization by immunoblot under non-reducing conditions. Comparing the
WT- and R461C-expressing cells, we observed CSF2RB-containing complexes of ~500 kDa
that showed a reduced intensity when 2-mercaptoethanol was added to the lysate (normally
CSF2RB runs at 125 kDa, Figure 1D), indicating R461C enables novel disulfide interactions
with additional CSF2RB monomers or other endogenous receptors.
Previous studies have shown that mutations within receptor transmembrane domains can
result in constitutive receptor oligomerization and activation
15–17
. The role of the CSF2RB transmembrane domain in receptor stability and signal transduction
has not been characterized
1
. Even the boundaries of the membrane-spanning region have not been empirically studied,
though it is commonly assumed to span residues 444–460 based on early domain modeling
5
. Various modern models consistently predict a much larger domain (Supplemental Figure
5), including residue 461, suggesting R461C might be within the transmembrane domain
and alter receptor-receptor interactions. Another possibility is that the loss of
a charged residue near the membrane boundary could result in shifting or fluidity
of the transmembrane domain of the mutant receptor.
Using immunoblot and co-immunoprecipitation analysis, we showed that transformed Ba/F3
cells expressing CSF2RB R461C exhibited constitutive receptor phosphorylation and
signaled through several pathways, including STAT5, PI3K/mTOR1, and MEK/ERK (Figure
2A and B). To identify therapeutically targetable pathways we utilized a 104 small
molecule inhibitor library screen, as previously described
7, 18
, and compared IC50 values for R461C-expressing cells to WT-expressing cells. JAK
inhibitors (Tofacitinib, Ruxolitinib, and AZD1480) constituted the three top hits
in the screen (Figure 2C, Supplemental Figure 6A–C, full screen Supplemental Table
3). These treatments were specific and cytotoxic, inducing apoptosis in R461C-expressing
cells (Figure 2D and Supplemental Figure 6D). While these inhibitors have a differential
specificity for targeting JAK kinases (Tofacitinib: JAK2/3, Ruxolitinib: JAK1/2, AZD1480:
JAK2), the strongest target is JAK2. We also observed reduced levels of total JAK2
in R461C cells (Figure 2B), which is consistent with JAK2 activation and then degradation
through a STAT5/SOCS-1 negative feedback loop
19
. These results suggest a dependency on the JAK/STAT pathway that is consistent with
ligand-independent activation of CSF2RB in R461C-expressing cells. Targeted inhibition
of JAK2 using AZD1480 quickly reduced levels of phosphorylated STAT5, validating the
specific nature of the inhibition and highlighting JAK2 as the key mediator of CSF2RB
R461C signaling (Figure 2E).
In sequencing 449 primary hematopoietic malignancies, we found 7 additional CSF2RB
mutations (Supplemental Table 4) but none were membrane spanning and all 7 were incapable
of transforming Ba/F3 cells (data not shown). Several other CSF2RB-activating mutations
have been observed in vitro, but have not been observed in primary patient samples.
CSF2RB R461C is a rare germline variant and recent studies of germline cancer-predisposing
mutations have not included CSF2RB
11
, possibly because of the lack of previous evidence showing the oncogenic potential
of CSF2RB. Our data would support the inclusion of CSF2RB in future studies. CSF2RB
R461C is a transforming mutation in vitro, and further investigation is necessary
to determine its status as a predisposing or cooperative congenital mutation.
CSF2RB has previously been shown to become factor-independent through in vitro mutagenesis
screens. One such screen successfully predicted R461C to be an active variant
5
(though insufficient to transform Ba/F3 cells in a short-term withdrawal assay), demonstrating
the potential for predictive mutagenesis screens. In spite of this in vitro evidence,
there has never before been a reported case of cancer with a CSF2RB-activating mutation.
We have demonstrated for the first time that a targetable CSF2RB variant found in
a human leukemia confers factor-independent growth, receptor phosphorylation and accumulation,
and constitutive JAK/STAT pathway activation. Our findings contribute to ongoing efforts
to identify potential germline predisposition mutations in pediatric cancers, and
are consistent with observations that genetic alterations activating cytokine receptor
pathways are common in leukemia. Our research highlights the need for basic research
to investigate and characterize the functional role of the CSF2RB transmembrane domain
in signaling and recycling of the receptor. Finally, JAK inhibitors blocked the growth
of R461C-transformed cells, thereby providing a therapeutic rationale to consider
JAK inhibitors in any future cases of CSF2RB-activated leukemias.
Supplementary Material
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