PTPσ inhibitors promote hematopoietic stem cell regeneration

Article abstract of DOI:10.1038/s41467-019-11490-5

Receptor type protein tyrosine phosphatase-sigma (PTPσ) is primarily expressed by adult neurons and regulates neural regeneration. We recently discovered that PTPσ is also expressed by hematopoietic stem cells (HSCs). Here, we describe small molecule inhi

Full text of DOI:10.1038/s41467-019-11490-5

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https://doi.org/10.1038/s41467-019-11490-5
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Yu r un Zh ang
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Mi c he ll e Li , Li m an Zh ao , J en ny Kan , T ian c hen g F ang , Xi ao Yan 2, 5 , Kat her i ne Po hl , Em el yn e D i ers ,
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Re c e p to r ty p e p ro te in t yro s ine ph os p hat as e -s ig m a (P T P σ) is p ri mar ily e xp re ss ed b y ad u lt
n e ur on s a nd r e g ul a te s ne u ra l re g e n e rat io n. We re cen t ly d isc ov er e d t h at PT P σ is als o
e xp re s s e d b y h e ma to po ie ti c s te m c e lls (H S Cs ). H e re , w e d es cr ibe sm all mo le cu le in h ibi to r s
o f P T P σ th a t p ro m o te H SC re g e n e rat io n in vi vo. S ys t em ic ad min is t rat io n o f t h e PT P σ in h i-
b ito r, DJ 001 , o r i ts an alo g , t o ir rad iat e d m ic e pr om o t es H SC r eg e ne r at ion , acc ele ra te s
h e mato l o gi c r e co ve r y , an d im p rov e s s ur viva l. S im ilar ly, D J00 1 ad min is t rat io n acc ele ra te s
h e mato l o gi c r e co ve r y in m ic e t re at e d wi th 5- fluo ro ura cil ch e mo th e ra py. D J00 1 d isp lays h ig h
sp e c ifici ty fo r P TP σ an d an ta g oni ze s PT P σ vi a un iq ue n on -c om pe t it ive , allo s te r ic b in din g .
M e ch an i s ti ca ll y , DJ 001 s up pre s s e s ra dia ti on- ind uc e d H SC ap o pt o sis v ia act iv at io n o f t h e
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HS C s w ith DJ 001 p ro m ot e s t he re g e n e rat io n of hu m an H SC s cap ab le o f mu lt ili ne ag e in v ivo
re p o pu l at io n . Th e s e s tu di e s de m o ns t rat e t he t he rap e ut ic p ot e nt ia l o f se le ct ive , sm all-
mo le cu l e P TP σ i nh i b it ors fo r hu m an he m a to po ie ti c re g e ne r at ion .
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Molecular Biology Institute, University of California, Los Angeles (UCLA), Los Angeles, CA 90095, USA. Division of Hematology/Oncology, Department of
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Medicine, UCLA, Los Angeles, CA 90095, USA. Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, UCLA, Los Angeles, CA
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0095, USA. 4 Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA 90095, USA. 5 Department of Molecular and Medical Pharmacology, UCLA,
Los Angeles, CA 90095, USA. Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA 90095, USA. 7 California Nanosystems Institute, UCLA,
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Los Angeles, CA 90095, USA. Department of Psychiatry and Behavioral Sciences, UCLA, Los Angeles, CA 90095, USA. Department of Radiation
Oncology, UCLA, Los Angeles, CA 90095, USA. These authors contributed equally: Yurun Zhang, Martina Roos. Correspondence and requests for
materials should be addressed to J.P.C. (email: jchute@mednet.ucla.edu)
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ematopoietic stem cells (HSCs) express several receptor compound, 6545075 (Chembridge), which was reported to have
tyrosine kinases (RTKs), including TIE2, fetal liver tyr- binding and inhibitory properties against the catalytic domain of
osine kinase 3 (FLT3), and vascular endothelial growth PTPσ . We searched a library of 80,000 small molecules in an
2
1
H
factor receptor 1, which regulate HSC maintenance, proliferation, attempt to find compounds with similar structural features,
1
–5
and differentiation . RTK signaling is carefully balanced by the namely 2-arylamino-1-arylpropenones, and identified two addi-
action of intracellular and receptor protein tyrosine phosphatases tional compounds, 6515205 and 5483071, which we hypothesized
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(
PTPs), which dephosphorylate tyrosine residues on RTKs
.
to have the potential to bind to PTPσ (Fig. 1a). We tested the
Small-molecule RTK inhibitors have been successfully developed inhibitory activity of these three molecules in a direct PTPσ
2
2
for clinical practice and are widely utilized in oncology, including enzymatic assay (Fig. 1b) . Compound 5483071 (3071) displayed
imatinib (BCR-ABL, PDGFR, c-KIT) and other BCR-ABL inhi- strong inhibitory activity against PTPσ, with a half-maximal
bitors, erlotinib, and gefitinib (epidermal growth factor receptor), inhibitory concentration (IC50) of 1.5 μM, whereas the other
8
–10
and lapatinib (HER-2)
. Therapeutic targeting of PTPs in compounds demonstrated lower PTPσ inhibitory activity. We
clinical practice has progressed more slowly, although the then prepared a synthetic sample of 3071, named DJ001, and
immunosuppressive agents, cyclosporine and FK506, which confirmed the inhibitory effect of DJ001 in the PTPσ enzymatic
indirectly inhibit a phosphatase, calcineurin, provide evidence assay (Fig. 1c). Importantly, DJ001 displayed no inhibitory
that phosphatase inhibitors can provide important clinical ben- activity against 20 other phosphatases, with only modest inhibi-
1
1
efits . Anti-sense oligonucleotides targeting PTP1B also have tory activity against Protein Phosphatase 5 (Supplementary
been demonstrated to lower blood glucose and increase insulin Fig. 1a). These data suggested that DJ001 was a highly specific
sensitivity in Type II diabetes mellitus, and have advanced in inhibitor of PTPσ.
12
clinical trials . Challenges to the successful development of PTP
We next determined that DJ001 can exist in two different
modulators include lack of selectivity, as the active enzymatic stereo-isomeric forms, the (Z)- and (E)-isomers (Supplementary
sites of PTPs may be conserved across the PTP family, and cell Fig. 2a). The (Z)-isomer displayed significantly increased
permeability, as highly charged anionic phosphate mimetics that binding affinity to both the PTPσ active (catalytic) site and the
1
1,13
could inhibit PTPs do not cross the cell membrane
.
allosteric site compared with the (E)-isomer (Supplementary
Genetic studies have revealed the function of two intracellular Fig. 2b, c). Importantly, the (Z)-isomer also demonstrated a
PTPs, Src-homology-2-domain containing tyrosine phosphatase 1.3 kcal/mol preference for binding to the allosteric site, located
2
(SHP2) and phosphatase of regenerating liver 2 (PRL2), in between domains 1 and 2 of PTPσ, over the catalytic site
14,15
regulating HSC fate
. A gain-of-function mutation in SHP2 (−9.0 kcal/mol vs. −7.7 kcal/mol) (Fig. 1d). Enzyme kinetic
promoted HSC cycling and increased HSC-repopulating capacity analysis via substrate titration studies demonstrated that DJ001
and the development of myeloproliferative disease in mice . functions as a non-competitive inhibitor of PTPσ, consistent with
1
4
Deletion of PRL2 was associated with a decline in HSC self- its allosteric binding properties (Fig. 1e). These studies revealed
1
5
renewal capacity in mice . However, the functions of receptor that DJ001 non-competitively inhibits PTPσ through a unique
PTPs in regulating HSC fate remain largely unknown. We mechanism.
recently discovered that pleiotrophin (PTN), a paracrine growth
factor secreted by bone marrow (BM) stromal cells and endo-
thelial cells (ECs), promoted HSC self-renewal and regeneration PTPσ inhibition promotes hematopoietic regeneration. In
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via binding and inhibition of receptor PTPζ
. PTPζ-deficient order to determine whether PTPσ inhibition could affect
mice display myeloid proliferation, increased numbers of BM HSC growth during homeostasis, we cultured non-irradiated BM
−
+
+
−
−
HSCs and progenitors, and increased HSC competitive repopu- CD34 ckit sca1 lin (CD34 KSL) HSCs with media (contain-
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lating capacity . We subsequently discovered that murine and ing 20 ng/mL Thrombopoietin, 100 ng/mL stem cell factor (SCF),
human HSCs differentially express another receptor PTP, 50 ng/mL Flt3 ligand, TSF) with or without 10–1,000 ng/mL
20
PTPσ . BM cells from PTPσ-deficient mice displayed sig- DJ001 for 7 days. Treatment with DJ001 increased the percentages
nificantly increased competitive repopulating capacity compared and numbers of BM KSL cells in culture compared with control
with PTPσ-expressing BM cells and human CB HSCs negatively cultures (Supplementary Fig. 3a). DJ001 treatment also sig-
selected for PTPσ surface expression demonstrated >10–fold nificantly increased the numbers of colony forming cells (CFCs) in
increased repopulating capacity in NOD/SCID IL2 receptor-γ 3 day culture of BM KSL cells (Supplementary Fig. 3b). Systemic
2
0
chain-deficient (NSG) mice . Based on these results, we hypo- treatment of adult C57BL/6 mice with 5 mg/kg DJ001, sub-
thesized that PTPσ negatively regulates HSC self-renewal, and cutaneously every other day for 30 days, caused no significant
that pharmacologic inhibition of PTPσ could therapeutically changes in complete blood counts, BM HSC or progenitor cell
promote HSC self-renewal. Here we report the development of a content, or BM CFCs compared with vehicle-treated controls
small molecule that selectively inhibits PTPσ via allosteric binding (Supplementary Fig. 3c–e).
in BM HSCs. Systemic administration of the PTPσ inhibitor, or
In order to determine whether PTPσ inhibition could promote
its analog, promoted HSC regeneration, accelerated hematologic HSC regeneration following myelosuppressive injury, we irra-
recovery, and improved survival in irradiated mice. Furthermore, diated BM KSL cells with 300 cGy and placed in TSF media with
treatment of irradiated, human HSCs with the PTPσ inhibitor and without 1 μg/mL DJ001 for 3 days. DJ001 treatment
promoted the regeneration of human HSCs capable of multi- increased recovery of BM CFCs and multipotent colony-
lineage repopulation in NSG mice. These studies reveal a class of forming unit–granulocyte erythroid monocyte megakaryocyte
selective PTPσ inhibitors with therapeutic potential to promote (CFU-GEMM) colonies compared with control cultures (Fig. 2a
human hematopoietic regeneration.
and Supplementary Fig. 4a). In a complementary study, we
irradiated mice bearing constitutive deletion of Ptprs, the gene
−
/−
that encodes PTPσ (Ptprs
mice), with 600 cGy total body
−
/−
Results
irradiation (TBI). Irradiated Ptprs
mice displayed increased
Development of a selective and allosteric PTPσ inhibitor. We recovery of BM CFCs at day + 10 compared with irradiated
+
/+
hypothesized that PTPσ inhibition could promote HSC self- Ptprs
mice (Fig. 2b). These results suggested that deletion
renewal and sought to develop a small molecule with specific of Ptprs or PTPσ inhibition comparably promoted hematopoietic
inhibitory activity against PTPσ. We identified an organic progenitor cell regeneration following irradiation.
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Fig. 1 DJ0 01 is a no n-c o mpe t it i ve , al l os te r i c i nhi bi t or of P T Pσ. a Che mic al st r uct ur e s of c ompounds 30 7 1, 5 2 0 5 , and 5 0 7 5 . b Conc e nt ra tion- i nh ib i t io n
c u rv es a nd IC50 v a lue s f o r 30 7 1, 5 2 0 5 , 5 0 7 5, an d co nt r ol (D MS O) f oll owing inc uba t ion wi t h P T P σ (n = 3). c Conc e ntra t ion- inhi bition c urv e s a nd I C5 0
va l u es f or DJ0 01 a nd c ont r ol fo ll ow i ng i ncu bat i on w it h P T Pσ ( n = 3). d I n si lic o t hre e - dime nsi onal doc k ing of D J0 0 1 (Z is ome r) (r e pre se nte d a s b al l a nd
s ti c k i n y e ll ow c ol or ) t o t he P T Pσ al l os te r i c bi ndi ng si t e l oca t e d be tw e e n doma in 1 (g re e n) and doma in 2 (bl ue ) of P T P σ. e At le f t , subs t rat e t it r at i o n reve al s
DJ0 0 1 a s a no n-c o mpe t it i ve i nhi bi t or t ha t i nhi bi t s su bst r a te ca t al ysis (Vma x), but not subs t rat e bin ding of P T Pσ (c onst a nt K
m a x a s a f un c t io n of c ompo und co nce nt r a tion de l iv e r e d fr o m nonl ine ar re g r e ssion in subs t rat e t it r at ion. E rr or bar s re pr e se nt SE M . So urc e da t a a re
p ro vi d e d a s S ou rc e Da t a F i le
m m
). At ri g ht , plo ts o f K a nd
V
In order to determine whether PTPσ inhibition could promote neutrophils, and lymphocytes compared with vehicle-treated
hematopoietic regeneration in vivo, we irradiated adult C57BL/6 controls (Fig. 2c). DJ001 treatment also accelerated the
mice with 750 cGy TBI and treated with 5 mg/kg DJ001 or recovery of BM KSL cells, which are enriched for hematopoietic
+
−
−
vehicle subcutaneously every day from day + 1 to day + 10. At stem/progenitor cells (HSPCs), ckit sca-1 lin myeloid pro-
day + 10, DJ001-treated mice displayed more than twofold genitors, and CFCs following TBI (Fig. 2d, e and Supplementary
increased peripheral blood (PB) white blood cells (WBCs), Fig. 4b, c). DJ001 treatment did not alter the percentages of BM
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megakaryocyte erythroid progenitors (MEPs), common myeloid post injection (Supplementary Fig. 4f), a concentration range
progenitors (CMPs), granulocyte monocyte progenitors (GMPs), that promoted BM hematopoietic progenitor cell recovery
or PB myeloid, B-cell or T-cell populations, but did cause a in vitro (Supplementary Fig. 3b).
modest increase in percentages of BM common lymphoid
As radiation exposure can cause acute mortality via hemato-
progenitors (CLPs)(Supplementary Fig. 4d, e). Of note, pharma- poietic toxicities, we evaluated whether daily administration of
cokinetic (PK) analysis of adult C57BL/6 mice treated sub- DJ001 from day + 1 to day + 10 could increase the survival of
cutaneously × 1 with 5 mg/kg DJ001 demonstrated DJ001 mice following lethal dose TBI. Following 750 cGy TBI, 12 of 29
concentrations of 10–100 ng/mL in the PB over several hours adult C57BL/6 mice (41%) survived to day + 40. Conversely, 27
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NA TUR E COMM UNICAT IO NS | ht t ps ://doi .or g / 10 .1 0 38/ s 41 46 7 -019- 114 90 -5
A R T I C L E
Fig. 2 P TPσ in hi bi ti on pr o mot e s he ma to poi e ti c r e ge ne r a t ion. a Me an numbe r s of CFCs f rom B M KS L c e lls f oll owing 30 0 c Gy ir ra diat i on and cu l t ure f or
d a y s in TS F me di a ± DJ0 0 1 ( n = 1 2) . b M ea n numb e rs of BM CFCs in Ptprs+/+ and Ptprs−/− mic e at day + 10 f oll owing 60 0 c Gy T BI (n = 1 2 ). c M ea n
n u mb er s of P B WB Cs , ne ut r ophi l s (N EU ) , an d l ymph ocy t es (L Y MP H ) in mic e at day + 10 f oll owing 7 5 0 c Gy and t re a tm en t wi t h D J0 0 1 or 1 0% D M SO
3
+
−
(
v eh i cl e ) ( n = 10 ). d R ep r es en tat i v e flow cy t ome t ri c an al ys i s of p e rc e nt ag e s of KS L c e lls and c -k it sc a-1 pr oge ni t or c e lls at day + 10 post 7 5 0 cGy i n t h e
+
−
t rea t me nt gr ou ps s ho wn. e M ea n pe r ce nt a ge s an d numb e rs of B M KS L c e lls and c -k it sc a-1 c e lls at day + 10 post 7 5 0 c Gy ( n = 9). f P e rc e nt su rvi va l o f
+
mi c e a f t er 7 5 0 c Gy a nd t r e at me nt w it h D J0 0 1 (2 7 / 2 9 al i v e ) or v e hic le (12 / 2 9 al ive ); L og -ra nk t e st . g M e an pe rc e nt ag e s of donor CD 45 . 2 c el l s i n t h e P B
+
5
+
o f CD4 5 .1 r e ci pi e nt mi ce ov e r t i me fo ll ow i ng t r ans pl an ta t ion o f 5 × 10 B M c e lls f rom CD 45 . 2 mic e at day + 10 f oll owing 7 5 0 c Gy T BI an d t re at me nt
5
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+
+
wi t h D J00 1 or v eh ic l e, a lon g w it h 1 × 1 0 CD 45 .1 BM co mpe t it or c e lls (n = 18–2 1/ gr oup). h M e an pe rc e nt ag e s of t ot al donor CD 45 . 2 c e lls and C D4 5 .2
+
+
+
+
+
+
Ma c 1 Gr 1 my e lo id c e lls , CD 45 .2 B2 2 0 B ce l l s, an d CD 45 .2 C D3 T c e lls in t he P B of re c ip ie nt mic e at 2 0 we e k s post t ra nspla nt at ion ( n = 1 8/g r ou p) .
i Mea n nu mbe r s of P B W BCs an d N EU co unt s i n mi ce at da y + 0 (pr e -5 FU ), + 8, + 11 , and + 14 f oll owing 5 FU t re a tm en t ( n = 4 –7 /g r oup). E rro r b ars
r ep re se nt S EM. S ou rc e da t a ar e pr ov i de d as S our c e D at a F i le . * P < 0 .0 5 , **P < 0 .0 1 , ***P < 0 .0 0 1, *** *P < 0 .0 0 0 1
of 29 irradiated C57BL/6 mice treated with DJ001 (93%) PTPσ inhibitor effects on cytokine levels. Although our in vitro
remained alive and healthy through day + 40 (P < 0.0001, Log- studies suggest that PTPσ inhibition directly promotes HSPC
rank test, Fig. 2f). Therefore, systemic administration of a PTPσ regeneration, it is also possible that treatment with DJ001 may
inhibitor also improved survival following myeloablative have caused indirect effects via alterations in the production of
irradiation.
cytokines by hematopoietic progenitor cells or their micro-
In order to determine whether DJ001 promoted the environment. Indeed, hematopoietic progenitor cells have been
regeneration of HSCs with in vivo repopulating capacity, we shown to secrete several inflammatory cytokines in response to
performed competitive repopulation assays using B6.SJL lipopolysaccharide and Pam3CSK4, a Toll-like receptor 2
+
26
(
1
CD45.1 ) recipient mice and donor BM collected at day + ligand . In order to address this, we measured 38 cytokines in
+
0 from C57BL/6 (CD45.2 ) mice irradiated with 750 cGy TBI cultures of non-irradiated and irradiated BM KSL cells treated
and treated daily with 5 mg/kg DJ001 or vehicle. Recipient mice with and without DJ001, and also measured the identical cyto-
5
transplanted with 5 × 10 BM cells from irradiated control kines in the BM of non-irradiated C57BL/6 mice and irradiated
5
+
mice, along with 1 × 10 competitor CD45.1 BM cells, C57BL/6 mice at day + 5 following treatment with DJ001 or
displayed declining donor-derived hematopoietic reconstitu- vehicle. At 12 h following 300 cGy irradiation of BM KSL cells
tion over time following transplant (Fig. 2g). This is consistent in vitro, we detected increased levels of lipopolysaccharide-
with our prior observations that high-dose TBI depletes HSCs induced CXC chemokine (LIX), macrophage inflammatory
2
3
with long-term repopulating capacity . In contrast, mice protein-1α (MIP-1α) and MIP-1β, and no other changes; DJ001
transplanted with the identical dose of BM cells from irradiated, treatment decreased LIX levels following irradiation but had no
DJ001-treated mice displayed significantly increased donor effects on any other cytokines in vitro (Supplementary Fig. 6a, b).
hematopoietic reconstitution over time compared with controls At day + 5 following 750 cGy TBI in C57BL/6 mice, we detected
(
Fig. 2g). Of note, mice transplanted with BM cells from decreased levels of LIX and CXCL12 in the BM and increased
irradiated, DJ001-treated donor mice displayed significantly levels of SCF and PTN. DJ001 treatment had no effects on
+
+
+
+
increased percentages of donor CD45.2 Mac1 /Gr1 myeloid cytokine levels in non-irradiated mice, but caused a modest
+
+
+
cells, CD45.2 B220 B cells, and CD45.2 CD3 T cells in the increase in CXCL12 levels and a decrease in PTN levels in the BM
PB at 20 weeks post transplant compared with mice of irradiated mice compared with irradiated, vehicle-treated mice
transplanted with control, irradiated BM cells (Fig. 2h).
In order to determine whether systemic treatment with DJ001
could also promote hematologic recovery following myelosuppres-
sive chemotherapy, we treated adult C57BL/6 mice intravenously
with 250 mg/kg 5-fluorouracil (5FU), an antimetabolite chemother-
apeutic that is widely utilized in clinical practice and causes
(Supplementary Fig. 6c, d).
PTPσ inhibition promotes HSC survival via induction of
RAC1. The mechanisms through which PTPσ regulates cellular
functions are not well understood . In a yeast-two-hybrid sys-
tem, PTPσ was shown to dephosphorylate p250GAP, a RhoGT-
Pase that attenuates RAC1 activity . In the presence of PTPσ,
27
2
4,25
depression of WBCs and neutrophils
. Control mice treated with
27
5FU displayed leukopenia and neutropenia from day + 8 through
27
p250GAP activity increased and RAC1 activity declined . Here
day + 14 (Fig. 2i). Mice treated with 5FU followed by 5 mg/kg
DJ001 SQ daily through day + 10 demonstrated significantly
increased PB WBCs and neutrophils at day + 14.
we tested whether PTPσ inhibition with DJ001 could alter
p250GAP phosphorylation and RAC1 activation in primary
hematopoietic progenitor cells. For this purpose, we developed an
enzyme-linked immunosorbent sandwich enzyme-linked immu-
nosorbent assay (ELISA) employing immunoglobulin A capture
of p250GAP (Supplementary Fig. 7a). Treatment with 1 µg/mL
DJ001 significantly increased phosphorylation of p250GAP in
In order to validate our hypothesis that small-molecule
inhibition of PTPσ could facilitate hematopoietic regeneration
following myelotoxicity, we designed and synthesized more than
50 structural analogs of DJ001. One of them, DJ009, with a 3,5-
difluorophenyl ring substituted for the 3-nitrophenyl unit of
DJ001 (Supplementary Fig. 5a), strongly inhibited PTPσ activity
in vitro (Supplementary Fig. 5b). When C57BL/6 mice were
irradiated with 750 cGy TBI and subsequently treated daily with
−
BM lin cells (Fig. 3a). Furthermore, DJ001 treatment sig-
nificantly decreased co-localization of PTPσ and p250GAP in BM
KSL cells, as measured by proximity ligation assay (PLA; Fig. 3b).
DJ001 treatment also caused a rapid increase in RAC1-GTP levels
in BM KSL cells (Fig. 3c). Importantly, DJ001 treatment did not
alter the activation of other RhoGTPases, CDC42, or RhoA, in
5
mg/kg DJ009 for 10 days, we observed a significant increase in
PB WBCs, neutrophils and lymphocytes, BM KSL cells, and CFCs
in response to DJ009 treatment (Supplementary Fig. 5c–e).
Consistent with these findings, DJ009 treatment also significantly
increased survival of irradiated C57BL/6 mice (Supplementary
Fig. 5f). These results provided further evidence that small-
molecule PTPσ inhibitors can promote hematopoietic regenera-
tion in vivo following myelosuppression.
−
BM lin cells, suggesting that CDC42 and RhoA did not con-
tribute to DJ001-mediated effects in hematopoietic progenitor
cells (Supplementary Fig. 7b). Therefore, PTPσ repressed RAC1
activation in HSPCs via regulation of p250GAP and DJ001 pro-
moted RAC1 activation via inhibition of PTPσ and p250GAP
function. Of note, DJ001 treatment of BM KSL cells from
N A TU RE C OMM U N IC AT I ON S |
(2 0 1 9) 1 0 : 3 66 7 | h t t p s :/ /d o i . o r g /1 0 . 1 0 3 8 /s 4 1 4 67 - 0 1 9- 1 1 4 90 - 5 | w ww . na t u r e . co m /n at u r ec o m mu n i ca t i o n s
5

A
R
T
I
C
L
E
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
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N
S
|
h
t
t
p
s
:
/
/
d
o
i
.
o
r
g
/
1
0
.
1
0
3
8
/
s
4
1
4
6
7
-
0
1
9
-
1
1
4
9
0
-
5
a
b
c
2
00
100
80
60
40
20
0
n.s.
Media
DJ001
80
60
40
20
0
150
100
50
Media
DJ001
DsRed: PTPσ-p250GAP
0
Ptprs+/+
Ptprs–/–
DAPI: Nucleus
d
e
Vehicle
DJ001
1
1
0
5
4
70
30
0
60
50
40
30
10
3
20
0
3
1
9
11
EHT1864
–
10
DJ001 + EHT1864
10
0
10
5
4
1
1
0
3
0
0
21
25
–
10
3
–10
3
0
10
3
10
4
10
5
–10
3
0
10
3
10
4
10
5
AnnexinV-FITC
f
g
Vehicle
DJ001
DJ001 + EHT1864
Media
8
6
4
2
0
DJ001
100
50
0
0
10
20
30
Days
h
i
CFU-GM
BFU-E
GEMM
2
0
0
0
Non-IRR
50
5
4
3
2
1
0
0
0
0
0
0
Media
40
30
20
10
0
DJ001
DJ001 + EHT1864
1
n.s.
n.s.
10
3
BCL-X -FITC
Non-IRR 300 cGy
L
−
/−
Ptprs
mice caused no change in RAC1-GTP levels, suggesting following 500 cGy TBI compared with vehicle-treated controls
that DJ001-mediated activation of RAC1 occurred specifically via (Fig. 3e). Administration of the RAC inhibitor, EHT1864,
PTPσ (Fig. 3c). DJ009 also did not induce RAC1 activation in BM abrogated DJ001-mediated anti-apoptotic effects on BM KSL
−
/−
cells from Ptprs
mice, suggesting similar selectivity of DJ009 cells in irradiated mice (Fig. 3e). Similarly, treatment with
for PTPσ (Supplementary Fig. 7c). Treatment of BM KSL cells EHT1864 inhibited DJ001-mediated CFC generation from BM
with DJ001 increased phosphorylation of p21-activated kinase 1 KSL cells irradiated with 300 cGy in vitro (Supplementary
(
PAK1), a substrate of RAC1, and concomitant treatment with Fig. 7d). Systemic administration of EHT1864 also completely
2
8
the RAC inhibitor, EHT1864 , abrogated DJ001-mediated blocked DJ001-mediated increase in survival of irradiated mice
phosphorylation of PAK1 (Fig. 3d). (Fig. 3f). These results suggested that DJ001-mediated anti-
Ionizing radiation causes HSC death via direct DNA damage, apoptotic and regenerative effects on BM HSPCs were dependent
generation of reactive oxygen species, and induction of pro- on RAC pathway activation.
apoptotic mediators2 . DJ001 treatment significantly decreased
5,29
RhoGTPases such as RAC1 can affect multiple signaling
3
0–34
the percentage of apoptotic BM KSL cells in C57BL/6 mice at 24 h pathways that regulate cell survival
, so we next measured the
6
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
(
2
0
1
9
)
1
0
:
3
6
6
7
|
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t
t
p
s
:
/
/
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.
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/
1
0
.
1
0
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/
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7
-
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1
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-
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1
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9
0
-
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|
w
w
w
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.
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n
s

NA TUR E COMM UNICAT IO NS | ht t ps ://doi .or g / 10 .1 0 38/ s 41 46 7 -019- 114 90 -5
A
R T I C L E
−
Fig. 3 DJ0 01 pr o mot e s H SC r e ge ne r at i on v i a RAC 1 ac t i va t i on and ind uct i on of B CL- X
±
L
. a % p2 5 0G AP phos pho-t yr osine (pT yr ) in B M li n c e ll s cu l t ure d
+
DJ0 0 1 ( med ia , n = 10 ; D J0 0 1, n = 7 ). b At l e ft , r e pr e se nt a ti v e ima ge s of P L A in B M KS L c e lls t re a te d ± D J0 0 1. Re d = D sRe d , P T P σ-p2 5 0 GAP co mpl e x;
b l ue = DAP I , or i gin a l ma gnifica t i on ×6 3, sc al e ba r s = 1 0 µm). At r ig ht , numbe r s of D sRe d KS L c e lls in e ac h c ondit i on (n = 4 8, c ont rol ; n = 36, DJ 00 1 ). c %
+
+
+/+
−/−
RA C1- GTP K SL c e lls f r om Ptprs
an d Ptprs
mi ce t r e at e d ± D J0 0 1 (n = 5 ). T wo- way AN O VA wi t h Si dak’s mult i ple c ompar ison t e st . d P e rc en ta g es o f
+
p -P A K 1 K SL c e lls f ol lo wi ng t r e at me nt w it h me di a al on e, 1 μg / mL D J0 0 1, or D J0 0 1 + 6 μg /mL E H T1 864 (n = 5 ). O ne -wa y AN O VA wi t h T uk ey ’s mu lt i pl e
c o mp a r is on t es t . e At le f t , Ann ex in V / 7 AAD st a in in g of BM KS L c e lls f rom mic e at 2 4 h post 5 0 0 c Gy and t re a tm en t wi t h ve hi c le , 5 mg /k g D J 001 ,
DJ0 0 1 + 4 0 mg / k g EHT1 8 64, or E HT 18 64 al on e. N umbe r s r e pre s e nt pe rc e nt ag es in e ac h g at e . At ri g ht , % Anne xin 7 AAD− B M KS L c e lls ( med ia a nd
DJ0 0 1, n = 9 ; DJ0 01 + EH T1 864 , n = 7 ; E HT 18 64, n = 4) . f P e r c en t sur viv al of mic e f oll owing 7 5 0 c Gy and 10 -d ay t re a tm en t wi t h D J0 0 1 (11/ 1 5 a li ve ),
+
−
ΔΔCt
ve hi c l e ( 4/ 15 a li ve ) , or DJ0 0 1 + E HT 18 64 (2 / 1 5 al i v e ; Lo g- r ank t e st ). g Fol d c hang e s (2
t rea t me nt wi th or wi th ou t D J0 0 1 (n = 3–6/ g r oup). T wo- w ay AN O VA wi t h Si dak’s mult i ple c ompar ison t e st . h At le f t , B CL- X
) of g e ne e xpr e ssi on in B M KS L c e lls at 12 h post 300 cGy a nd
pr ot e in leve l s i n n on -
L
i r ra d i a t e d B M K SL c e lls ( re d) , 30 0 cG y- ir r a di at ed KS L ce l l s i n me dia al one (bl ac k) or t re a te d wi t h D J0 0 1 (bl ue ) or D J0 0 1 + E H T1 864 (g ra y). At ri g ht , %
+
B
C
L
-
X
L
K
S
L
c
e
l
l
s
(
n
=
6
)
.
O
n
e
-
w
a
y
A
N
O
V
A
w
i
t
h
T
u
k
e
y
’
s
m
u
l
t
i
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l
e
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m
p
a
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i
s
o
n
t
e
s
t
.
i
C
F
C
s
f
r
o
m
B
M
K
S
L
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s
a
t
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8
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f
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l
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g
3
0
0
c
G
y
a
n
d
c
u
l
t
u
r
e
w
i
t
h
a n d wi th ou t DJ0 01, a nd w it h an d w it ho ut shRac1 or shBcl2l1 (n = 3–9/ gr oup). T wo- way AN O VA wi t h Si dak’s mult i ple c ompar ison t e st . E rr or bars re pre se nt
SE M. S ou rc e da t a a r e pr o v id ed as S our c e D at a F i le . * P < 0 .0 5, **P < 0 .0 1 , *** P < 0 .0 0 1, *** *P < 0 .0 0 0 1
hairpin RNAs (shRNAs) targeting Rac1 or Bcl2l1 and measured
hematopoietic progenitor cell recovery following 300 cGy irradia-
Table 1 Primers used for mouse gene detection
tion. Silencing of either Rac1 or Bcl2l1 blocked DJ001-mediated
Target mRNA
Sequence 5′–3′
recovery of hematopoietic progenitor cells from irradiated BM
KSL cells (Fig. 3i). Taken together, these results suggested that
DJ001-mediated HSPC recovery after irradiation was dependent
on RAC1 and BCL-XL.
G a pd h F w
G a pd h R v
B c l 2l 1 F w
B c l 2l 1 R v
B a x F w
B a x R v
B c l 2 F w
B c l 2 R v
B i m F w
T GGAT T TGGAC GCATT GG TC
T TGC ACTGGT ACGT GTT GAT
GACA AGGAGA TGC AGG TA TT GG
T CCCGTAGAG ATC CACA AAAG
T GAAGACAGGG GCCT TTT T G
AAT T CGCCGGAG ACACTCG
T GAGT ACCT GAACCGGCAT CT
GCAT CCCAGC CTCCG TT AT
CGGA TCGGAG ACGAGT TCA
T TCCA GCCT CGCGGT AAT CA
AGCA GCACT T AGAGT CGC C
CCT GGGT AAGGGG AGGA GT
AAAG GCGGCT GCA TAAGT C
T GGCGGTAT AGGT CGT CCT C
CCT GCTT ATC AATGCAG AGGG
T GCGGGTCACCA TT T CA GC
AT GGCT GCCACT CGATAT GAA
T CCTC CATT AGGAAC TCT CACAC
GGCG TAC CCACA GAAACCAT A
AGGT AAGGGCC ATCT GAAAACT
CCT GGT GAT GTC CGACCT G
CCAT GAGCGC ATCGCAAT C
T CAAACGT GAGAG TGT CT AACG
CCGG GCCGAAG AGATT TCT G
GCCA GCCT TGG GACAATA ATG
CT TG CACGT TGA GT TT GGGT
GAGA CGGAGC TGT TGGT AAAA
AT AGGCC CAGAT T CACTGG TT
GCGT ACCCT GACACCAAT CT C
CT CCT CTTCGCA CTT CTG CTC
GAGT GGGAACT GGT AGTG TT G
CGCA CAGAGCGA TGA AGGT
RAC1 has been shown to activate ERK1/2 signaling via PAK1-
and PAK2-mediated phosphorylation of MEK1 to facilitate the
3
5
formation of the Raf/MEK/ERK complex . Consistent with this,
DJ001 treatment induced ERK1/2 phosphorylation in irradiated
BM KSL cells, which was suppressed by RAC inhibition
B
i
m
R
v
(
Supplementary Fig. 7e). Furthermore, treatment with the
P uma F w
P uma R v
Mc l -1 F w
Mc l -1 R v
Cd k 2 F w
Cd k 2 R v
Cd k 4 F w
Cd k 4 R v
Cd k 6 F w
Cd k 6 R v
Cd k n 1a F w
Cd k n 1a R v
Cd k n 1b F w
Cd k n 1b R v
Cy c l i n E F w
Cy c l i n E R v
Ra c 1 F w
ERK1/2 inhibitor, BVD523, blocked DJ001-mediated increase in
BCL-XL protein levels in BM KSL cells and abrogated
hematopoietic progenitor cell recovery following irradiation
(
Supplementary Fig. 7f, g). These data suggested that DJ001-
mediated induction of BCL-XL in HSPCs was dependent on
ERK1/2, downstream of RAC pathway activation.
PTPσ inhibition promotes HSPC proliferation via Cdk2.
Hematologic recovery following myelosuppressive irradiation
requires not only survival of long-term HSCs but also the pro-
liferation of HSCs and progenitor cells to facilitate regeneration of
essential blood elements. DJ001 treatment did not alter the cell
cycle status of non-irradiated BM KSL cells in culture with
thrombopoietin, SCF, and Flt3 ligand (Supplementary Fig. 7h).
However, DJ001 treatment of BM KSL cells for 36 h following
Ra c 1 R v
300 cGy irradiation significantly increased the percentage of KSL
Cy c l i n D1 F w
Cy c l i n D1 R v
Cy c l i n D2 F w
Cy c l i n D2 R v
cells in G /S/M phase compared with control, irradiated KSL cells
2
(
Fig. 4a). Evaluation of the expression of cell cycle regulatory
genes revealed that DJ001 treatment did not alter the expression
of cyclin-dependent kinase 4 (Cdk4), Cdk6, Cdkn1a, Cdkn1b,
Cyclin D1, or Cyclin D2 in irradiated BM KSL cells (Fig. 4b and
Table 1). However, DJ001 treatment significantly increased the
effect of DJ001 treatment on the expression of pro- and anti- expression of Cdk2 and Cyclin E in irradiated KSL cells. Trans-
apoptotic genes in irradiated KSL cells (Fig. 3g). DJ001 treatment duction of BM KSL cells with Rac1-shRNA blocked DJ001-
had no effect on the expression of pro-apoptotic mediators, Bim, mediated transcription of Cdk2 and Cyclin E, suggesting that
Bax, p53 upregulated modulator of apoptosis (Puma), or the anti- DJ001-induced expression of Cdk2 and Cyclin E in KSL cells was
apoptotic gene, Bcl2, in BM KSL cells (Table 1). However, DJ001 RAC1 dependent (Fig. 4c). Furthermore, treatment of irradiated
treatment increased the expression of Mcl-1 and Bcl2 like 1 BM KSL cells with SU9516, a specific CDK2 inhibitor, or the RAC
(
Bcl2l1), which encodes the anti-apoptotic protein, BCL-X , as inhibitor, EHT1864, suppressed DJ001-mediated induction of cell
L
well as BCL-X protein levels in irradiated BM KSL cells (Fig. 3g, cycle progression in BM KSL cells following irradiation (Fig. 4d).
L
h). RAC inhibition blocked the increase in BCL-X protein levels These results suggested that DJ001-mediated HSC cell cycle
L
in irradiated BM KSL cells in response to DJ001, suggesting that progression following irradiation was dependent on RAC path-
DJ001-mediated induction of Bcl2l1 expression was RAC pathway way activation and CDK2.
dependent (Fig. 3h). In order to confirm that DJ001-mediated
In order to test whether systemic treatment with DJ001 altered
effects on irradiated HSPCs were dependent on RAC1 and BCL- HSC proliferation in vivo following irradiation, we measured
XL, we transduced BM KSL cells separately with lentiviral short BrdU incorporation in BM KSL cells of C57BL/6 mice at day + 10
N A TU RE C OMM U N IC AT I ON S |
(2 0 1 9) 1 0 : 3 66 7 | h t t p s :/ /d o i . o r g /1 0 . 1 0 3 8 /s 4 1 4 67 - 0 1 9- 1 1 4 90 - 5 | w ww . na t u r e . co m /n at u r ec o m mu n i ca t i o n s
7

A
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N AT URE CO M MU N ICAT IO N S | ht t ps: //doi .or g /1 0 .10 38 /s4 14 67 -01 9- 1 1490 - 5
a
Media
DJ001
1
00
Media
DJ001
1
1
05
6
7
24
58
36
8
6
4
2
0
0
0
0
0
04
1
03
0
9
6
–
103
0
50 K
100 K
150 K
200 K
250 K
0
50 K
100 K
150 K
200 K
250 K
G0
G1
G SM
2
7AAD
b
c 2.5
4
2.5
Media
DJ001
Media
DJ001
Media
DJ001
2
1
1
0
.0
.5
.0
.5
2.0
1.5
1.0
0.5
0.0
3
2
1
0
n.s.
0.0
shCtrl
shRac1
shCtrl
shRac1
d
Media
20
DJ001
DJ001 + EHT1864
28
DJ001 + SU9516
25
6
1
8
2
51
40
44
27
46
1
1
1
04
03
02
8
27
50 K
100 K
150 K
50 K
100 K
150 K
50 K
100 K
150 K
50 K
100 K
150 K
7AAD
50
80
50
40
30
20
10
0
40
30
20
10
0
60
40
20
0
Fig. 4 DJ0 01 pr o mot e s HSC pr ol i fe r a ti on v i a i nduc t io n of CD K2 . a At le f t , re pr e se nt at iv e c e ll c yc le ana lysi s of B M KS L c e lls at 36 h f oll owing 3 00 cGy a nd
−
−
+
−
+
+
0 1 2
c u l tu re wi th me di a ± 1 μg / mL D J0 0 1. At r i ght , me an pe r ce nt a ge s of KS L c e lls in G (Ki 67 7 AAD ), G (Ki 67 7 AAD ) and G /S /M phas e (Ki 67 7 AAD )
a r e s h own (n = 7 ). b F old ch ang e s (2
(
−ΔΔCt
) of ce l l cy cl e r e gul a t or y g e ne e xpr e ssion in KS L c e lls at 36 h f oll owing 30 0 c Gy and c ult ur e wi t h med i a ± D J 001
n = 3 ) . Ge ne t r an s cr i pt c h ang e s ar e nor ma l iz e d t o Gapdh an d med ia t re a tm en t. c Fol d c hang e s (2−ΔΔCt ) o f Cdk2 and Cyclin E e xpr e ssi on in B M l in ce ll s i n
−
r es p on se t o DJ0 01 a t 4 8 h af t e r 30 0 cG y, w it h an d w it ho ut Rac1-s hRNA t re a tm en t ( n = 3–5 ). d T op pane l show s re pr e se nt at i ve c e ll c yc le ana ly si s o f K SL
c el l s a t 36 h f ol lo win g 300 cG y an d cu lt ur e w it h me di a ± 1 µg / mL D J0 0 1, D J0 0 1 ± 6 µg /mL E H T1 864 , and D J0 0 1 ± 2 4 0 ng /mL CD K2 inh ibit o r S U95 1 6.
B o tt o m pa n el s ho ws t he pe r ce nt a ge s of BM ce l l s i n G , G , an d G /S /M phas e f or e ac h c ondit i on (me di a, n = 6; D J0 0 1 and D J0 0 1 + SU 95 16 g ro up s, n = 5 ;
0 1 2
DJ0 0 1 + EHT1 86 4 , n = 8 ) . T wo- w ay AN OV A w it h S i dak’s mul t i pl e c ompar ison t e st f or a–c. O ne -wa y AN O VA wi t h T uk ey ’s mult i ple c ompar iso n t es t f or
d. So u rc e da t a a r e pr o vi d ed as S our c e D at a F i le . * P < 0 .0 5, **P < 0 .0 1 , *** P < 0 .0 0 1, *** *P < 0 .0 0 0 1
8
N AT U R E CO M MU NI C A TI O N S |
( 2 01 9) 1 0 : 3 667 | h tt p s : //d o i . o r g /1 0 . 1 0 3 8 /s 4 1 4 67 - 0 1 9- 1 1 4 90 - 5 | w ww . na tu r e . c o m /n atu r e c o m m u n ic a tio n s

NA TUR E COMM UNICAT IO NS | ht t ps ://doi .or g / 10 .1 0 38/ s 41 46 7 -019- 114 90 -5
A R T I C L E
following 750 cGy TBI and treatment with or without DJ001. BM mimetics also have poor bioavailability, as they are typically cell
4
0
KSL cells from DJ001-treated mice displayed increased bromo- impermeable . These barriers have hindered the development of
deoxyuridine (BrdU) incorporation compared with irradiated, PTP active site inhibitors for clinical use. A recent analysis of
vehicle-treated controls (Supplementary Fig. 7i). In order to non-receptor and receptor PTPs for “druggability” suggested that
determine whether DJ001 treatment caused durable effects on the majority of PTPs were poor drug targets due to the hydro-
3
8
HSC cell cycle status, we measured BrdU incorporation in donor philic or shallow nature of their catalytic pockets . As an alter-
+
+
CD45.2 cells in recipient CD45.1 mice at day + 7 and day + 21 native strategy, allosteric inhibitors have been described for
following competitive transplantation. Of note, donor cells were specific PTPs, including selective inhibitors for dual specificity
collected from the BM of CD45.2 mice at day + 10 following phosphatase 6 and the SHP2
50 cGy TBI and daily treatment with DJ001 or vehicle, and then inhibitor of SHP2 was recently shown to inhibit RTK-driven
+
40,42
. A small molecule, allosteric
7
+
43
transplanted into lethally irradiated CD45.1 recipient mice, as human cancer growth in xenograft models . Here we describe a
described in Fig. 2. We detected no difference in BrdU small molecule that non-competitively inhibits PTPσ via allosteric
+
incorporation between donor CD45.2 cells from either the binding and is highly selective for PTPσ. Based on these unique
DJ001- or vehicle-treatment groups at these time points in properties, we propose that DJ001, and DJ001 structural homo-
recipient mice (Supplementary Fig. 7j).
Deletion of Rac1 has been shown to decrease the homing,
logs, are compelling candidates for therapeutic development.
A large number of patients with cancer require myelosup-
engraftment, and niche localization of hematopoietic stem and pressive chemotherapy and/or radiotherapy in the treatment of
progenitor cells3
0,31,36
. Therefore, it is possible that systemic their disease . Hematopoietic toxicities occur commonly in such
44
treatment of mice with DJ001 altered Rac1-mediated migration patients, resulting in hospitalizations, delays in curative therapy,
45
and localization of endogenous HSCs in supportive BM niches, and potentially life-threatening infectious complications .
thereby facilitating hematopoietic regeneration in vivo. We Depletion of BM HSCs and progenitor cells, which occurs fol-
observed that DJ001 treatment did not alter the in vitro migration lowing repeated cycles of cytotoxic chemotherapy, contributes to
4
6
of BM KSL cells through EC monolayers and had no effect on prolonged myelosuppression in such patients . Therapies that
+
−
homing of transplanted BM ckit lin cells at 18 h in irradiated promote HSC and progenitor cell regeneration could lessen the
C57BL/6 mice (Supplementary Fig. 8a, b).
complications of myelosuppression and facilitate the timely
completion of potentially curative chemotherapy. However, the
mechanisms that govern HSC regeneration are poorly understood
and this gap in knowledge has impeded the development of
targeted therapies to drive human hematopoietic regeneration.
Here we demonstrate that systemic administration of a small
molecule inhibitor of PTPσ accelerated hematopoietic regenera-
tion in mice following irradiation or chemotherapy. Furthermore,
PTPσ inhibition promoted the recovery of human hematopoiesis
and human HSCs with long-term repopulating capacity in NSG
mice following irradiation. Taken together, these results suggest
the therapeutic potential for a small-molecule PTPσ inhibitor to
accelerate hematologic recovery in cancer patients receiving
myelosuppressive chemotherapy or radiotherapy.
PTPσ inhibition promotes human HSC regeneration. As DJ001
promoted murine hematopoietic regeneration following myelo-
suppression, we sought to determine whether DJ001 could also
accelerate human HSC regeneration. Irradiation of human BM
+
CD34 cells with 300 cGy in vitro caused a significant loss of
+
−
37
CD34 CD38 cells, which are enriched for human HSCs , at 36
h after irradiation (Fig. 5a). Treatment with 5 µg/mL DJ001
+
−
increased the recovery of viable human BM CD34 CD38 cells
following irradiation (Fig. 5a). DJ001 also increased the recovery
of multipotent CFU-GEMMs at 72 h following irradiation
+
of human BM CD34 cells (Fig. 5b). Mechanistically, DJ001
treatment decreased radiation-induced apoptosis of human
−
Our in vitro studies of non-irradiated BM CD34 KSL cells
+
−
CD34 CD38 cells following irradiation (Fig. 5c). RAC inhibi-
suggested the potential for DJ001 or DJ001 analogs to promote
hematopoietic progenitor cell expansion; however, in vivo
administration of DJ001 3 days per week for 4 weeks caused no
expansion of HSCs, progenitors, or mature blood counts in non-
irradiated mice. The absence of effect in non-irradiated mice may
have occurred due to insufficient concentrations of DJ001
achieved in the BM of non-irradiated mice following the cho-
sen treatment schedule or possibly due to indirect effects of
systemically administered DJ001 on cytokines or chemokines in
the BM that counteracted direct effects on HSCs. Our analysis of
the BM of non-irradiated mice revealed no significant changes in
cytokine or chemokine levels in response to DJ001 treatment.
Following TBI, DJ001 treatment was associated with modest
changes in concentrations of the HSC growth factors, CXCL12
and PTN, in the BM compared with controls. These results
suggest that DJ001 promotes HSC regeneration primarily via
direct effects on HSCs; however, indirect effects of DJ001 via
undiscovered mechanisms cannot be not excluded.
tion blocked DJ001-mediated survival of irradiated human
CD34 CD38 cells, suggesting that DJ001-mediated anti-apop-
totic effects on human HSCs were RAC pathway dependent
+
−
(Fig. 5c). As we observed in our murine studies, DJ001 treatment
increased the expression of the pro-survival genes, BCL2L1 and
+
MCL-1, in irradiated human CD34 cells (Fig. 5d and Table 2).
In order to determine whether PTPσ inhibition could promote
the recovery of human HSCs with in vivo repopulating capacity,
+
we irradiated human BM CD34 cells with 300 cGy, cultured
with TSF media with or without DJ001 for 36 h, and transplanted
the progeny into NSG mice to assess human hematopoietic
engraftment over time (Fig. 5e). Mice transplanted with
+
irradiated, DJ001-treated BM CD34 cells displayed substantially
+
+
increased engraftment of human CD45 cells, CD19 B cells,
+
+
CD33 myeloid cells, and CD3 T cells at 12 weeks post
transplant compared with mice transplanted with equal doses of
+
irradiated, control BM CD34 cells (Fig. 5f, g). Therefore, PTPσ
inhibition promoted the recovery of human HSCs with multi-
lineage repopulating capacity following irradiation.
PTPσ and related receptors within the leukocyte common anti-
gen-related (LAR) subfamily of PTPs are expressed by neurons
and act as receptors for heparan sulfate proteoglycans4
7–50
. In
Discussion
the developing nervous system, PTPσ regulates axon guidance
4
7–52
PTPs have been recognized as important potential drug targets and synapse formation
. PTPσ also serves as a receptor for
due to their involvement in the pathogenesis of numerous dis- inhibitory glycosylated side chains of chondroitin sulfate pro-
3
8,39
47,53–55
eases
. However, active site-directed PTP inhibitors frequently teoglycans (CSPGs)
. CSPGs, which are enriched in the
lack target specificity due to the high degree of homology between extracellular matrix surrounding injured nerves, inhibit axon
PTP active sites4 . Negatively charged phospho-tyrosine regeneration following spinal cord injury . Genetic deletion of
0,41
6,7
N A TU RE C OMM U N IC AT I ON S |
(2 0 1 9) 1 0 : 3 66 7 | h t t p s :/ /d o i . o r g /1 0 . 1 0 3 8 /s 4 1 4 67 - 0 1 9- 1 1 4 90 - 5 | w ww . na t u r e . co m /n at u r ec o m mu n i ca t i o n s
9

A
R
T
I
C
L
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A
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|
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1
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1
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4
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7
-
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9
-
1
1
4
9
0
-
5
a
b
2
1
1
0
5
0
5
0
15
25
20
Media
DJ001
1
1
0
0
5
4
1
0
5
0
15
1
0
3
10
5
0
5
16
–
10
3
–10
3
0
10
3
10
4
10
5
–10
3
0
10
3
10
4
10
5
0
CD34-PE
300 cGy
300 cGy
c
d
Media
DJ001
9
5
25
10
5
Media
76
DJ001
88
DJ001 + EHT1864
2
5
1
0
0
5
4
90
85
20
1
4
15
10
5
80
1
0
3
79
0
75
19
8
19
–
10
3
70
0
0
–10
3
0
10
3
10
4
5
10
–10
3
0
10
3
10
4
10
5
–10
3
0
10
3
10
4
5
10
AnnexinV-FITC
e
300 cGy
f
Media
5
1
0
1
CD34+ human
BM cells
0.2
0
.3
.4
0.2
0.2
10
4
10
3
Treatment
with media or
DJ001
0
–10
3
DJ001
5
4
1
0
0
10
1.3
4
1.4
1.9
1
+
36 h
10
3
0
–
10
3
12 weeks
–10
3
0
10
3
10
4
1
5
0
–10
3
0
10
3
10
4
10
5
–10
3
0
10
3
10
4
10
5
–10
3
0
10
3
10
4
10
5
–10
3
0
10
3
10
4
10
5
NSG Mice
Mouse CD45-
BV605
CD34-AF647
CD19-APC-Cy7
CD33-FITC
CD3-APC
g
1
00
10
1
10
1
10
1
10
1
0
1
1
0.1
0.01
0.1
0.1
0.01
0.1
0
.1
0.01
0.01
Media DJ001
Media DJ001
Media DJ001
Media DJ001
Media DJ001
PTPσ promoted axon regeneration in a model of spinal cord demonstrate that systemic delivery of a small-molecule PTPσ
injury, suggesting that PTPσ may be a therapeutic target to inhibitor drives HSC regeneration and hematopoietic recon-
4
7
enhance nerve regeneration . Subsequently, systemic delivery of stitution in vivo following myelosuppression. The combined
a peptide-mimetic of the PTPσ wedge domain was reported to benefits of PTPσ inhibition on hematopoietic and neural regen-
relieve CSPG-mediated inhibition and restore serotonergic eration suggest an important role for PTPσ in regulating adult
5
3
innervation of the spinal cord in rats . In a model of myocardial somatic cell regeneration.
infarction, genetic deletion or injection of a PTPσ intracellular
Mechanistically, PTPσ inhibition suppressed radiation-induced
peptide beginning 3 days after injury promoted sympathetic apoptosis of murine and human HSCs. Downregulation of LAR
5
6
innervation in the area of myocardial scar . Here we protein, which, along with PTPσ and PTPδ, comprises the Type
1
0
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
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|
(
2
0
1
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)
1
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6
7
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.
1
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/
s
4
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4
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7
-
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9
-
1
1
4
9
0
-
5
|
w
w
w
.
n
a
t
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r
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.
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a
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n
s

NA TUR E COMM UNICAT IO NS | ht t ps ://doi .or g / 10 .1 0 38/ s 41 46 7 -019- 114 90 -5
A R T I C L E
+
-
Fig. 5 DJ0 01 pr o mot e s hu ma n H SC r e ge ne r at i on fo ll ow i ng i r r adi a t ion. a At le f t , re pr e se nt at i ve flow c yt ome t ri c ana lysi s of CD 34 CD 38 c e lls a t 3 6 h a ft e r
+
+
−
3
0 0 c G y ir r a d iat io n of B M CD 34 ce l l s an d cu lt ur e w it h me di a ± 1 µg /mL D J0 0 1. At ri g ht , me an pe rc e nt ag e s and numbe r s of CD 34 CD 38 ce ll s i n
ea c h gr ou p ( Da y 0, n = 6 ; me di a, D J0 0 1, n = 1 0) . O ne -w ay AN O VA wi t h T uk ey’s mult i ple c ompar ison t e st . b N umbe rs of CFU -GE MM s wi t hin cu l t ure s o f
+
CD3 4 c e lls a t 7 2 h a f t er 30 0 cG y an d cu lt ur e w it h me di a ± D J0 0 1 ( n = 6). c At le f t , re pr e se nt at i ve flow c yt ome t ri c ana lysi s of Anne x in V/ 7 AAD st a in i ng
+
+
−
o f CD3 4 c e lls a t 2 4 h a ft e r 30 0 cG y an d cu lt ur e w it h me di a ± DJ 00 1 ± E H T1 864 . At ri g ht , pe rc e nt ag e s of li ve and apop t ot ic CD 34 CD 38 c el l s i n ea ch
−
ΔΔC t
c o nd i ti on ( med ia a nd DJ00 1 , n = 1 0; D J0 0 1 + E HT 18 64, n = 5 ). O ne -wa y AN O VA wi t h T uk ey ’s mult i ple c ompar ison t e st . d Fol d c hang e s (2
BCL2L1 a nd MCL-1 ge ne e x pr e ssi on i n CD 34 CD 38 ce l l s at 1 2 h af t e r 30 0 c Gy in me dia ± D J0 0 1 ( n = 3). Ge ne t ra nsc rip t c hang e s we r e nor mal i z ed t o
GAPDH a nd me di a t r ea t m en t. T wo- w ay AN OV A w it h S i dak’s mult i ple c ompar ison t e st . e Sc he mat i c re pr e se nt ation of N SG mic e t ra nspla nt at i on a ssay
u s in g t he pr o gen y of hu ma n BM CD 34 ce l l s i r r adi a te d w it h 30 0 c Gy and t re a te d wi t h or wi t hout D J0 0 1 × 36 h. f Re pr e se ntat i ve flow c yt omet r ic a na ly si s
o f h u ma n CD4 5 c e lls , huma n CD 34 ce l l s, huma n CD 19 B ce lls , huma n CD 33 mye l oid c e lls , and huma n CD 3 T c e lls e ng ra ft me nt in t he B M o f N S G
) i n
+
−
+
+
+
+
+
+
mi c e a t 12 we ek s po s t t r ans pl an tat i on. g P e r ce nt e ng ra f tm ent of huma n he mat opo ie tic c e ll subs e t s in t he B M of N SG mic e at 12 we e k s post t ra nsp la nt a t io n
(n = 11–12 / gr oup) . S ou rc e da t a ar e pr ov i de d as S our c e D at a F i le . * P < 0 .0 5 , **P < 0 .0 1 , ***P < 0 .0 0 1, *** *P < 0 .0 0 0 1
favorably affected HSC transcriptional programs that regulate
myeloid and lymphoid cell differentiation and HSC repopulating
potential .
Table 2 Primers used for human gene detection
6
3
Target mRNA
Sequence 5′–3′
The paucity of therapeutics capable of accelerating HSC
regeneration and hematopoietic reconstitution in myelosuppressed
patients highlights an unmet medical need. Here we describe a
class of selective, allosteric PTPσ inhibitors that promote the
regeneration of murine and human HSCs capable of long-term
hematopoietic reconstitution. Our results provide the mechanistic
foundation for the development of selective PTPσ inhibitors to
promote hematopoietic regeneration in patients receiving myelo-
suppressive chemotherapy, radiotherapy, and those undergoing
myeloablative hematopoietic cell transplantation.
h G AP DH F w
h G AP DH R v
h B CL 2 L 1 F w
h B CL 2 L 1 R v
h MC L -1 F w
h MC L -1 R v
CCT GCACCACC AACT GCT T A
GGCC ATCCACA GTCTT CT GAG
GACT GAAT CGGAGA TGGAGACC
GCAG TT CAAACT CGT CGCCT
T GCT TCGGAAAC TGG AC ATC A
T AGCCACAAA GGCACCAAAAG
IIa receptor PTPs, has similarly been shown to have pro-survival
effects on neurons in the setting of serum deprivation5 . Our
7,58
results suggest that PTPσ inhibition promotes HSC survival fol- Methods
Animal models. All animal procedures were performed in accordance with animal
use protocols approved by the UCLA Animal Research Committee (Approval
lowing irradiation via RAC pathway activation and induction of
BCL-X in HSCs. Treatment with the PTPσ inhibitor, DJ001,
L
−/−
Number 2014-021-13 M). Ptprs
mice were provided by Dr Michel Tremblay
uncoupled PTPσ binding to p250GAP, causing RAC1 activation
McGill University). C57BL/6 mice, B6.SJL mice, and NOD.Cg-PrkdcscidIl2rgtm1Wjl/
(
in primary BM HSCs, without affecting other RhoGTPases, SzJ (NSG) mice between 8 and 12 weeks old were obtained from the Jackson
Laboratory.
CDC42 and RhoA. Deletion of Rac1 in the hematopoietic com-
partment of mice has been shown to cause decreased HSPC
3
0,31,36
engraftment, homing, and niche localization
expression of a dominant-negative Rac2 increased HSPC apop-
, whereas Synthesis of DJ001. A mixture of 1-phenylprop-2-yn-1-one* (0.303 g, 2.3 mmol),
-nitroaniline (0.427 g, 3.0 mmol), and copper (I) iodide (0.078 g, 0.4 mmol) in
dimethylformamide (6 mL) and water (60 μL) was stirred at 85 °C for 16 h. After
6 h, the reaction mixture was allowed to cool down to 21 °C and was diluted with
irradiated breast cancer cells, epithelial cells, and T cells via water (50 mL), and extracted with ethyl acetate (3 × 50 mL). The combined organic
induction of BCL-XL, MCL-1, and other BCL-2-like layer was washed with NH Cl/NH (1:1 v/v, 3 × 20 mL) and brine (30 mL), dried
, filtered, and concentrated under reduced pressure. The crude residue
3
3
6,59
tosis
. RAC1 has also been shown to inhibit apoptosis of
1
4
3
proteins32,60–62
over MgSO
4
. Here, PTPσ inhibition with DJ001 increased
was purified by column chromatography (n-hexane/ethyl acetate = 20:1) to
obtain (Z)-3-((3-nitrophenyl)amino)-1-phenylprop-2-en-1-one (DJ001,140 mg,
the expression and protein levels of BCL-X in HSCs and sup-
pressed HSC apoptosis following irradiation, dependent upon
L
1
3
0.52 mmol, 23%) as a yellow powder. H NMR (400 MHz, CDCl ) δ 12.26 (d, NH,
RAC pathway and ERK1/2 activation. These results suggest that J = 11.6 Hz), 7.96–7.91 (m, 3 H), 7.90 (ddd, 1 H, J = 8.0, 2.0, 0.8 Hz), 7.56–7.46
(
(
1
m, 5 H), 7.37 (ddd, 1 H, J = 8.0, 2.4, 0.8 Hz), 6.16 (d, 1 H, J = 8.0 Hz); 13C NMR
100 MHz, CDCl ) δ 191.8, 149.4, 143.4, 141.6, 138.6, 132.2, 130.6, 128.6, 127.5,
22.3, 117.8, 110.0, 95.7. High resolution mass spectrometry (HRMS) m/z calcu-
lated for C15 [M + H] + 269.0904, found 269.0921. *1-Phenylprop-2-yn-
RAC pathway activation and induction of BCL-X regulate HSC
survival in response to PTPσ inhibition.
In addition to regulation of HSC survival, our data suggest that
L
3
13 2 3
H N O
DJ001 promoted early HSPC proliferation compared with irra- 1-one was prepared by oxidation of 1-phenylprop-2-yn-1-ol in a two-step proce-
dure from benzaldehyde and trimethylsilylacetylene by a known method64
.
diated, control HSCs. Further, DJ001-mediated HSC cell cycle
progression was dependent on RAC pathway activation and
induction of CDK2. These findings are consistent with the
Synthesis of DJ009. (Z)-3-((3,5-difluorophenyl)amino)-1-phenylprop-2-en-1-
one (DJ009) was prepared by the same procedure using 3,5-difluoroaniline. 1
NMR (400 MHz, CDCl ) δ (p.p.m.): 12.0 (d, J = 11.7 Hz, 1 H), 7.94 (dt, J = 6.9, 1.6
Hz, 2 H), 7.55–7.50 (m, 1 H), 7.48–7.45 (m, 2 H), 7.38 (dd, J = 11.7, 8.2 Hz, 1 H),
As PTPσ regulates the phosphorylation of several kinases and the 6.64–6.57 (m, 2 H), 6.54–6.47 (m, 1 H), 6.09 (d, J = 8.2 Hz, 1 H); 13C NMR (100
RAC pathway regulates multiple pathways, PTPσ inhibition may MHz, CDCl ) δ (p.p.m.): 191.7, 164.0 (dd, J = 249, 15 Hz), 143.5, 142.8 (t, J = 13
3
0
−/−
H
observations by Gu et al. , who showed that Rac1
HSPCs
3
displayed decreased entry into G /S/M phase in response to SCF.
2
3
19
Hz), 138.7, 132.1, 128.6, 127.5, 99.3 (dd, J = 20, 8 Hz), 98.5 (t, J = 26 Hz), 95.3;
NMR (CDCl , 376 MHz) δ (p.p.m.): −107.9. HRMS m/z calcd. for C15 NO
M + H] + 260.0881, found 260.0866.
F
have altered the expression of other effectors that regulate HSC
proliferation beyond CDK2. However, we did not observe any
durable effect of DJ001 treatment on the proliferative potential of
HSCs or their progeny based on BrdU incorporation analysis of
3
12 2
H F
[
Phosphatase assay. The two intracellular catalytic domains (D1 and D2) of Ptprs
were cloned into a pET28a vector, overexpressed in Escherichia coli BL21 and
+
engrafted donor CD45.2 cells over time following transplanta-
+
65
tion into recipient CD45.1 mice. It is therefore perhaps more purified . Enzymatic activity of PTPσ was assayed using a modified version of the
6
6
Malachite Green Assay and the Tyrosine Phosphatase Assay Kit (Promega
Corporation). Unless stated otherwise, standard assays were carried out using
likely that the anti-apoptotic effects of systemic DJ001 treatment
on HSCs in irradiated mice contributed to the increase in
recovery of phenotypic HSPCs as well as HSCs with in vivo
5
0 nM PTPσ protein in 1× Buffer (10 mM Tris, 5 mM MgCl
2
, 10 mM NaCl, 0.02%
Tween) and Tyr Phosphopeptide as substrate (200 μM for Fig. 1b, c, 50–1200 μM
repopulating capacity. It is also possible that DJ001 treatment in Fig. 1c). Purified catalytic domain 1 and domain 2 of PTPσ were pre-incubated
N
A
T
U
R
E
C
O
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U
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(
2
0
1
9
)
1
0
:
3
6
6
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|
h
t
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s
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o
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o
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1
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.
1
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3
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-
0
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9
-
1
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-
5
|
w
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w
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c
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a
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11

A
R
T
I
C
L
E
N AT URE CO M MU N ICAT IO N S | ht t ps: //doi .or g /1 0 .10 38 /s4 14 67 -01 9- 1 1490 - 5
with test compounds 3071, 5205, 5075, or control for 15 min in a 96-well plate before
the addition of Tyr Phosphopeptide (DADE(pY)LIPQQG). For IC50 determination,
rates normalized relative to uninhibited controls (dimethyl sulfoxide (DMSO)) were
plotted against compound concentration and fitted using a four-parameter nonlinear
regression curve fit ((y = [(A − D) (1 + {xC − 1}B) − 1] + D) (Prism 6.0, Graphpad
Lentivirus-mediated shRNA silencing. Rac1 CDS-targeting- and Bcl2l1 CDS-
targeting shRNA in lentiviral plasmid (TRCN0000310888 and TRCN0000004685)
and control shRNA (SHC216V) were purchased from Sigma Aldrich. For viral
production, 293T cells were transfected with lentiviral gag/pol and VSV-G
(courtesy of Donald Kohn, UCLA) and the lentiviral plasmids, at a ratio of 2.2: 1.2:
Software). For mechanism studies and determination of the enyzme’s K
m
and Vmax
,
3.3 (in [μg], gag/pol:VSVG:Plasmid) using Lipofectamine 3000 and P300 Enhancer.
data were analyzed using a nonlinear regression fit according to the classical
Viral particles were collected after 24 h and 48 h. One milliliter of viral supernatant
6
−
Michaelis–Menten kinetics model, Y = Vmax*X/(K
m
+ X) (Prism 6.0, Graphpad
was used to infect 0.75 × 10 BM lin cells. Infected cells were collected the next
day and irradiated with 300 cGy followed by treatment with or without 1 μg/mL
DJ001, prior to CFC assay.
Software).
Phosphatase Profiler screen. Compound DJ001 was evaluated in a Phosphata-
seProfiler™ screen at 10 μM and 1 μM (2.7 μg/mL and 0.27 μg/mL) concentrations
at Eurofins Pharma Discovery Services UK (Study number UK022-0004033)
against a panel of 21 Phosphatases. In each experiment, the respective reference
antagonist/agonist was tested directly with DJ001 and the data were compared with
historical values determined at Eurofins. DJ001 compound inhibition was calcu-
lated as percentage inhibition of the enzymatic activity compared with control.
Immunofluorescence microscopy. Lab-Tek chamber slides were coated with
fibronectin (25 µg/mL, Millipore Sigma #341635). Sorted BM KSL cells (1 × 10 )
were resuspended in 200 µL TSF media and added to each of the pre-coated wells
2
and incubated overnight (37 °C/5% CO ). The next day, cells were serum starved
for 30 min and then stimulated with DJ001 (1 µg/mL), EHT1864 (6 µg/mL), or
equal volumes of DMSO for 5 min. Cells were washed once with PBS and fixed
with 4% PFA for 10 min. Cells were permeabilized with 0.5% Triton/PBS (PRM)
for 30 min and then blocked with 5% fetal bovine serum (FBS)/PRM for 1 h. The
slide was then incubated with a phospho-ERK1/2 primary antibody (Cell Signaling,
4
Pharmacokinetic study for DJ001. PK studies for DJ001 were performed at
Cyprotex (Study Number CYP1426-R1) under non-good laboratory practice
conditions. Mice were subcutaneously injected with a single dose of 5 mg/kg DJ001
#
4377, 1:200) for 1 h. The wells were washed three times and then incubated with a
donkey anti-rabbit Alexa Fluor 488 secondary antibody (Thermo Fischer, #A21206,
:200) at a 1:200 concentration for 1 h. The slide was washed three times and
(
in 10% DMSO, 40% phosphate-buffered saline (PBS), 50% polyethylene glycol
1
and plasma samples were collected at 0.08, 0.25, 0.5, 1, 2, 4, 8, and 24 h. Samples
were crashed with three volumes of methanol and analytical internal standard
mounted with ProLong Gold Antifade Reagent with DAPI. Cells were imaged
using a ×63 objective on a Zeiss Axio Imager M2 widefield fluorescence microscope
with all microscope settings derived from imaging a secondary only control. Data
were analyzed using Fiji (ImageJ). Briefly, the cell outline was identified by
threshold levels and the mean fluorescence intensity within the cell area was
quantified.
(
propranolol). Supernatant was subjected to liquid chromatography-mass spec-
trometry/mass spectrometry (MS/MS) using an acetonitrile-water gradient system
and electrospray ionization in multiple reaction monitoring mode for MS detec-
tion. All plasma samples were compared with an internal calibration curve pre-
pared in mouse blank plasma.
Flow cytometric analysis. Femurs and tibiae were collected from killed C57BL/6 or
Ptprs−/− mice and flushed with IMDM containing 10% FBS and 1%
penicillin–streptomycin for BM cells. PB was collected through sub-mandibular
puncture. Cells were filtered through a 40 μM strainer and then treated with ACK
Proximity ligation assay. A PLA assay was performed by using the Duolink In
Situ Red Starter Kit (Millipore Sigma) to detect the binding between PTPσ and
p250GAP proteins. BM KSL cells from C57BL/6 mice were sorted by fluorescence-
activated cell sorting and then plated onto fibronectin pre-coated chamber slides
overnight. The next day, cells were treated with 1 μg/mL DJ001 or vehicle (equal
volume of DMSO) for 1 h at 37 °C. Cells were then fixed with 4% paraformalde-
hyde (PFA) for 20 min. The slides were blocked with Duolink blocking solution
and incubated with rabbit anti-ARHGAP32 polyclonal antibody (Bioss Antibodies,
lysis buffer (Sigma Aldrich) before antibody staining for flow cytometry. For KSL and
CD150+CD48−KSL cell analysis, BM cells were stained with allophycocyanin (APC)-
and Cy7-conjugated anti-mouse Sca1 (BD Biosciences, #560654, 1:100), phycoery-
thrin (PE)-conjugated anti-mouse c-kit (BD Biosciences, #553355, 1:100), V450
lineage cocktail (BD Biosciences, 561301, 1:10), Alexa Fluor 488-conjugated anti-
mouse CD48 (BioLegend, #103414, 1:100), and Alexa Fluor 647-conjugated anti-
mouse CD150 (Biolegend, #115918, 1:100) antibodies. For MEP, CMP, GMP, and
CLP cell analysis, BM cells were stained with APC-Cy7-conjugated anti-mouse Sca1
(BD Biosciences, #560654, 1:100), PE-conjugated anti-mouse ckit (BD Biosciences,
#553355, 1:100), V450 lineage cocktail (BD Biosciences, #561301; 1:10), Alexa Fluor
488-conjugated anti-mouse CD127 (BD Biosciences, #561533, 1:100), Alexa Fluor
#bs-9296R, 1:50 dilution) and goat anti-PTPσ (K-19) polyclonal antibody (Santa
Cruz Biotechnology, #sc-10873, 1:50) overnight at 4 °C. Cells were imaged using a
Leica SP8 Confocal Microscope equipped with a ×63 objective lens. A white light
laser set to 580 nm was used to excite dsRed and a UV laser was used to excite 4′,6-
diamidino-2-phenylindole (DAPI). Analysis was performed using the spots
detection function in IMARIS 9.0.2 (Bitplane).
647-conjugated anti-mouse CD34 (BD Biosciences, #560230; 1:100), and BV605-
conjugated anti-mouse CD16/32 antibodies (BD Biosciences, #563006; 1:100). For
donor engraftment analysis in transplanted mice, PB or BM cells were stained with
BV605-conjugated anti-mouse CD45.2 (BioLegend, #109841, 1:100), fluorescein iso-
thiocyanate (FITC)-conjugated anti-mouse CD45.1 (BD Biosciences, #553775, 1:100),
PE-conjugated anti-mouse Mac-1 (BD Biosciences, #557397, 1:100) and anti-mouse
Gr1 (BD Biosciences, #553128, 1:100), V450-conjugated anti-mouse CD3 (BD Bios-
ciences, #561389, 1:100), and APC-Cy7-conjugated anti-mouse B220 (BD Biosciences,
p250GAP phospho-tyrosine sandwich ELISA. Clear, pre-blocked Protein A-
coated Microtiter wells (Fisher Scientific, #15132) were incubated with ARHGAP32
polyclonal antibody (Bioss, #bs-9296R, 1:500) in antibody dilution buffer (150 mM
NaCl, 25 mM HEPES pH 7.2, 0.5% bovine serum albumin, 0.05% Tween 20) for 3 h
at room temperature (RT). Subsequently, the plate was washed three times and
−
phosphorylated p250GAP from BM lin cell lysate was added to each well. BM
−
lin cells were collected in NP40 buffer (ThermoFisher Scientific; FNN0021)
552094, 1:100) antibodies.
supplemented with 1× complete, Mini, EDTA-free Protease Inhibitor Cocktail
Intracellular flow cytometric analysis was performed on irradiated (300 cGy) or
(
Sigma Aldrich; 4693159001) and 1× PhosSTOP (Sigma Aldrich; 4906845001).
non-irradiated, sorted KSL cells after treatment with 1 μg/mL DJ001 or control
equal volumes of DMSO) for 24 h. At 24 h after irradiation, cells were fixed with
% PFA for 10 min, followed by permeabilization using 0.25% saponin in PBS.
Following incubation for 2 h at 4 °C, the plate was completely emptied (without
washing) and exposed to 50 μL of a fixation solution (0.5% formaldehyde in 300
mM NaCl, 20 mM sodium phosphate buffer, pH 7) for 10 min. The plate-bound,
phosphorylated p250GAP was measured by a specific anti-phospho-tyrosine
peroxidase-coupled antibody (Sigma Aldrich, #A5964-1VL, 1:500) in antibody
dilution buffer. Incubation was for 1 h at RT. Peroxidase activity was measured by a
Tecan Infinity plate reader using BM Chemiluminescence ELISA Substrate (Sigma
Aldrich, #11582950001).
(
4
Cells were washed again and stained with antibody at the recommended
concentrations for 30 min at RT. Intracellular antibodies and phospho-flow
antibodies used were as follows: FITC-conjugated anti-BCL-X
:100), active RAC1-GTP antibody (NewEast Biosciences, #26903, 1:100) and anti-
PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) antibody
Abcam, #40795, 1:100), and FITC-conjugated goat anti-rabbit IgG H&L (Abcam,
97050, 1:200).
L
(Abcam, #26148,
1
(
#
G-LISA activation assays. The RAC1-GTP, RHOA-GTP, and CDC42-GTP
−
activation levels in BM lin cells were measured using a colorimetric-based RAC1-,
Gene expression analysis. For all studies, RNA was isolated using the Qiagen
RNeasy micro kit (Qiagen). RNA was reverse transcribed using the High-Capacity
cDNA Reverse Transcription Kit (ThermoFisher Scientific) and was then used for
quantitative PCR with SYBR Select Master Mix (Life Technologies). Values were
RHOA-, and CDC42 G-LISA Activation Assay Kit (Cytoskeleton, Inc.). BM cells
from femurs and tibias were isolated from 12-week-old Ptprs
mice. Cells were then depleted of lineage-committed cells with Direct Lineage Cell
Depletion Kit (Miltenyi Biotec, #130-110-470; 1:5). The BM lin cell fraction was
then serum starved in Iscove’s modified Dulbecco’s medium (IMDM) and treated
with either vehicle (equal amount of DMSO) or 1 μg/mL DJ001 for 10 min at 37 °C.
After treatment, cells were washed with ice-cold PBS and then placed in lysis buffer
supplemented with protease inhibitor. Lysate concentrations were measured by
+/+
−/−
and Ptprs
−
normalized to housekeeping gene Gapdh/GAPDH and given as ΔΔCt values nor-
67
malized to media-treated, non-irradiated BM KSL cells (2(−ΔΔC(T)) method)
.
Survival studies. For survival studies, 10-week-old female C57BL/6 mice were
TM
Pierce BCA Protein Assay Kit (ThermoFisher Scientific). G-LISA was performed
irradiated with 750 cGy TBI, which is lethal for ~50% of C57BL/6 mice by day + 30
(LD50/30), using a Shepherd Cesium-137 irradiator. Twenty four hours post
irradiation, mice were administered daily subcutaneous injections of 5 mg/kg
DJ001 or DJ009, or vehicle in a volume of 100 μL for 10 days. DJ001 or DJ009
injections were prepared in PBS, 0.5% Tween 80, and 10% DMSO. Corresponding
according to the manufacturer’s instructions. Briefly, 12.5 μg of lysates was added
to a GTP-binding protein pre-coated plate and active RAC1-GTP, RHOA-GTP, or
CDC42-GTP levels were measured at 490 nm using a PowerWave XS2 microplate
reader (BioTek).
1
2
N AT U R E CO M MU NI C A TI O N S |
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NA TUR E COMM UNICAT IO NS | ht t ps ://doi .or g / 10 .1 0 38/ s 41 46 7 -019- 114 90 -5
A R T I C L E
vehicle injections contained PBS, 10% DMSO, and 0.5% Tween 80. PB complete
blood counts were measured using a Hemavet 950 instrument (Drew Scientific) at
day + 10 post irradiation. For hematopoietic analysis, BM cells were collected at
day + 10 post irradiation. To study whether DJ001 increased survival rates through
activation of RAC signaling, the RAC inhibitor, EHT1864 (Selleckchem), was
dissolved in PBS and administered intraperitoneally, 40 mg/kg every other day, to
Biosciences, #553733, 1:100) antibodies. Sterile cell sorting was conducted on a
−
+
+
−
BD FACS-Aria cytometer. Purified KSL cells and CD34 c-kit sca-1 Lin
−
(CD34 KSL) cells were collected into IMDM (Life Technologies) + 10% FBS +
1% penicillin–streptomycin.
CFC assays and HSC competitive repopulation assays. CFC assays (colony-
750 cGy-irradiated mice until day + 10.
forming unit-granulocyte monocyte), burst-forming unit-erythroid, and CFU-
6
GEMM were performed using MethoCult GF M3434 (Stemcell Technologies) . For
−
−
Chemotherapy model. Ten-week-old female C57BL/6 mice received a single tail
vein injection of 250 mg/kg 5FU in 100 μL PBS. Two hours post 5FU injection,
mice were administered daily subcutaneous injections of 5 mg/kg (100 μg per
mouse) DJ001 or vehicle in a volume of 100 μL for 8 days. DJ001 injections were
prepared in PBS, 50% PEG-400, and 10% DMSO. Corresponding vehicle injections
contained PBS, 50% PEG-400, and 10% DMSO. Retro-orbital blood was collected
on day + 8, + 11, and + 14 after 5FU treatment with capillary pipettes (Fisher
Scientific). Complete blood counts were measured using a Hemavet 950 instrument
all in vitro assays, BM CD34 KSL cells, KSL cells, and Lin cells were cultured in
TSF media (IMDM, 10% FBS, 1% pen-strep, 20 ng/mL recombinant mouse TPO,
125 ng/mL recombinant mouse SCF, 50 ng/mL recombinant mouse Flt3 ligand).
Recombinant mouse SCF, Flt3 ligand, and TPO were purchased from R&D Sys-
tems. For competitive repopulation assays, BM cells were isolated from donor
+
+
10–12-week-old female CD45.2 mice. Recipient 10-week-old female CD45.1 B6.
SJL mice were irradiated with 950 cGy TBI using a Cs137 irradiator and donor BM
cells were administered via tail vein injection along with a competing dose of 1 ×
5
(
Drew Scientific).
10 non-irradiated host BM cells. Multilineage donor hematopoietic cell engraft-
ment was measured in the PB by flow cytometry
6
9
.
Cytokine analysis. For in vitro cytokine measurements, 3.5 × 104 BM KSL cells
were irradiated with 300 cGy and cultured in TSF media with and without 1 μg/mL
DJ001. Non-irradiated KSL cells were cultured in TSF media with and without 1
μg/mL DJ001. At 12 h of culture, culture supernatants were collected for analysis.
For in vivo cytokine measurements, adult C57BL/6 mice were irradiated with 750
cGy TBI and then treated with 5 mg/kg of DJ001 or vehicle, subcutaneously, from
day + 1 to day + 5. Non-irradiated mice received the identical regimen of 5 mg/kg
DJ001 or vehicle for 5 days. Mice were killed and BM cells were collected into
IMDM. Cells were pelleted and the BM supernatant was collected and sent to the
UCLA Immune Assessment Core for cytokine analysis. All samples were tested on
a mouse 32-plex Cytokine/Chemokine panel platform from EMD Milipore using
Luminex’s xMAP® immunoassay technology (Luminex, Inc.). In addition, the
following cytokine levels were assessed by solid-phase ELISA assay according to the
manufacturer’s description: CXCL12 (R&D Systems), SCF (R&D Systems),
Thrombopoietin (TPO) (R&D Systems), PTN (Biomatik), DKK1 (R&D Systems),
and EGF (R&D Systems).
Transendothelial migration assay. For the transendothelial migration assay,
TM
VeraVec
mouse spleen ECs (Angiocrine Biosciences) were cultured to con-
fluence in 8 µM pore transwells (Corning Incorporated). Transwells were then
seeded with 50,000 sorted BM KSL cells in IMDM with 10% FBS, 1% pen-strep, 20
ng/mL TPO, 125 ng/mL SCF, and 50 ng/mL Flt3 ligand with or without 1 µg/mL of
DJ001. SDF-1, 500 ng/mL (R&D Systems), was added to the bottom chamber of the
transwell. At 18 h post incubation, cells in the bottom chamber were collected and
cell counts were performed.
+
−
+
Homing study. Sorted BM sca-1 lin cells (100,000) from DsRed mice were
transplanted into lethally irradiated (950 cGy) recipient C57BL/6 mice followed by
subcutaneous treatment of 5 mg/kg DJ001 or vehicle × 1 dose. At 18 h post
transplantation, BM cells were collected from the recipient mice and were analyzed
+
17
for percentages of DsRed cells in the BM
.
Human BM cultures and human BM transplantation assays. Human BM
mononuclear cells were purchased from AllCells. Cryopreserved human BM cells
were recovered in IMDM + 10% FBS + 1% penicillin–streptomycin and then posi-
In vivo BrdU incorporation analysis. For BrdU incorporation analysis in BM KSL
cells in vivo, adult C57Bl/6 mice were irradiated with 750 cGy TBI and treated with
5
mg/kg DJ001 or vehicle daily from day + 1 to day + 10. On day + 10, mice were
+
tively selected for CD34 stem/progenitor cells by using CD34 MicroBead Kit
injected intraperitoneally with 2 mg BrdU (BD Biosciences). Sixteen hours later,
BM cells were collected and stained with anti-BrdU FITC (BD Biosciences,
+
(
(
Miltenyi Biotec, #130-046-702, 1:5). CD34 cells were cultured in human TSF media
IMDM, 10%FBS, 1% pen-strep, 20 ng/mL recombinant human TPO, 125 ng/mL
#557891, 1:50), APC-Cy7-conjugated anti-Sca1 (BD Biosciences, #560654, 1:100),
recombinant human SCF, 50 ng/mL recombinant human Flt3 ligand (R&D Systems).
PE-conjugated anti-c-kit (BD Biosciences, #553355, 1:100), and V450 lineage
cocktail (BD Biosciences, #561301, 1:10). We repeated this BrdU incorporation
5
+
The progeny of 2 × 10 irradiated, human BM CD34 cultured for 36 h and treated
with DJ001 at 5 μg/mL, were transplanted via tail vein injection into 10–12-week-old
NSG mice preconditioned with 275 cGy TBI. Multilineage donor hematopoietic cell
+
analysis on donor CD45.2 cells at day + 7 and day + 21 following competitive
+
transplantation into recipient CD45.1 mice.
20
engraftment was monitored in the PB and BM by flow cytometry . The PB or BM
cells were stained with BV605-conjugated anti-mouse CD45 (BioLegend, #103139,
1
:100), AF647-conjugated anti-human CD34 (BioLegend, #343618, 1:100), FITC-
Apoptosis assay and cell cycle analysis. Eight- to ten-week-old female C57BL/6
mice were irradiated with 500 cGy TBI followed by subcutaneous injections of 5
mg/kg DJ001 or vehicle, or 5 mg/kg DJ001 plus 40 mg/kg EHT1864. Twenty four
hours after irradiation, mice were killed to collect BM cells for Annexin V
apoptosis analysis. BM cells were stained with APC-Cy7-conjugated anti-mouse
Sca1 (BD Biosciences, #560654, 1:100), PE-conjugated anti-ckit (BD Biosciences,
conjugated anti-human CD33 (BD Biosciences, #555626, 1:100), V450-conjugated
anti-human CD45 (BD Biosciences, #560368, 1:100), APC-conjugated anti-human
CD3 (BD Biosciences, #555342, 1:100), and APC-Cy7-conjugated anti-human CD19
(
BD Biosciences, #557791, 1:100) antibodies.
#
553355, 1:100), and V450 lineage cocktail (BD Biosciences, #561301, 1:10)
In silico molecular docking studies. Molecular docking of DJ001, (Z)-isomer, to
antibodies for 30 min at 4 °C. Cells were then resuspended in 1× Annexin V
binding buffer and then stained with FITC-conjugated anti-Annexin V antibody
PTPσ (PDB ID: 2FH7) was carried out by AutoDock Vina, in which the Iterated
Local Search Globule Optimizer was applied as optimization algorithm70. Each
(
1
BD Biosciences, #556547,1:50) and 7AAD antibody (BD Biosciences, #559925,
structure of ligand was prepared in Maestro 10.5 (Schroedinger, LLC) and mini-
mized with the OPLS_2005 force field. All hydrogen atoms were added to each
protein and ligand to be docked and each coordinate file of protein and ligand was
generated as PDBQT file using AutoDockTools-1.5.6. A grid box for binding site
was set as 18 Å in the three dimensions (x, y, and z) that covered the catalytic site of
the protein or 40 Å in the three dimensions for allosteric binding site. The box had
1.0 Å grid spacing and centered at the geometric center of the protein. In each
docking experiment, the best binding mode was selected according to the binding
affinity calculated by the scoring function in AutoDock Vina. Docking results were
analyzed with PyMOL (Schroedinger LLC, NY) and visualized by VMD 1.9.2
(UIUC, IL).
6
8
:40) . For in vitro human BM cell apoptosis assays, isolated human BM
+
CD34 cells were plated into 96-well plates and then irradiated with 300 cGy.
Immediately after irradiation, cells were treated with 5 μg/mL DJ001 or vehicle
for 24 h and then collected for Annexin V cell apoptosis analysis. For cell cycle
analysis, BM KSL cells were irradiated at 300 cGy and then treated with 1 μg/mL
DJ001 or vehicle for 36 h. Subsequently, cells were resuspended in 200 μL of PBS,
fixed with 1 mL of ice-cold fixation solution (0.25% Saponin, 2.5% PFA, 2% fetal
calf serum (FCS) in PBS), and incubated for 30 min on ice. Cells were washed in
Saponin buffer (0.25% Saponin, 2% FCS in PBS) and resuspended in 200 μL of
Saponin buffer containing 20 μL of FITC-conjugated anti-Ki67 antibody (BD
Biosciences, #556026, 1:10) and 1 μL of RNase (Qiagen, #19101, 100 mg/mL).
After 30 min of incubation, cells were washed in Saponin buffer and resuspended
for flow analysis in 200 μL of Saponin buffer containing 5 μL 7AAD (BD Bios-
ciences, #559925, 1:40).
Statistical analysis. GraphPad Prism 6.0 was used for all statistical analyses. All
data were checked for normal distribution and similar variance between groups.
Data were derived from multiple independent experiments from distinct mice or
cell culture plates. Sample sizes for in vitro studies were chosen based on observed
effect sizes and SEs from prior studies. For all animal studies, a power test was used
to determine the sample size needed to observe a twofold difference in means
between groups with 0.8 power. A two-tailed Student’s t-test was utilized for all
comparison, except where otherwise noted in the figure legends. All animal studies
were performed using sex- and age-matched animals, with wild-type littermates as
controls. Animal studies were performed without blinding of the investigator.
Isolation of BM HSCs. BM cells were first treated with ACK lysis buffer (Sigma
Aldrich) and lineage-committed cells were removed using a Direct Lineage Cell
−
Depletion Kit (Miltenyi Biotec, #130-110-470, 1:5). Lin cells were stained with
APC-Cy7-conjugated anti-mouse Sca1 (BD Biosciences, #560654, 1:100), PE-
conjugated anti-ckit (BD Biosciences, #553355, 1:100), V450 lineage cocktail
(
BD Biosciences, #561301, 1:10), and FITC-conjugated anti-CD34 (BD
N
A
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C
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M
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N AT URE CO M MU N ICAT IO N S | ht t ps: //doi .or g /1 0 .10 38 /s4 14 67 -01 9- 1 1490 - 5
Values are reported as means ± SEM, unless stated otherwise. Results were con-
sidered significant when P < 0.05.
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hematopoietic stem and progenitor cells for regulation of stress – induced
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Data availability
The authors declare that all data supporting the findings of this study are available within
the article and its Supplementary Information files or from the corresponding author
upon reasonable request. The source data underlying Figs. 1b, c, e, 2, 3, 4, and 5 are
provided as a Source Data File.
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NA TUR E COMM UNICAT IO NS | ht t ps ://doi .or g / 10 .1 0 38/ s 41 46 7 -019- 114 90 -5
A R T I C L E
HL-086998-08 (J.P.C.), the California Institute for Regenerative Medicine Leadership
Awards LA1-08014 (J.P.C.) and DISC2-09624 (J.P.C.), NHLBI grant U54-HL-119893
J.P.C.), and the Tower Cancer Research Foundation Career Development Grant (M.R.).
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Author contributions
(
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J.P.C. and M.E.J. conceived of and designed the study. Y.Z. and M.R. performed the
majority of the experiments and analyzed the data. Y.Z. and M.R. designed experiments.
E.D., H.J.G. and M.E.J. designed and performed organic synthesis. M.Q., C.M.T. and
H.A.H. performed experiments and analyzed data. X.Y., J.K., L.Z., T.F., M.L., K.P. and
R.D. performed experiments. J.W. and W.M. contributed to the design and interpretation
of the studies, and contributed to writing the paper. Y.Z., M.R., M.E.J. and J.P.C. wrote
the paper.
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Competing interests: Authors M.R., M.E.J. and J.P.C. are inventors on patent
applications US 16/304,427 and EP 17803701.6, and PCT application US 2018/063,074,
which are relevant to the use of PTPσ inhibitors described here. All other authors declare
no competing interests.
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sigma in the sulfenic acid form. Mol. Cells 36, 55–61 (2013).
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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in
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Acknowledgements
We thank M. Tremblay (McGill University, Montreal, CA) for the Ptprs
−
/−
mice.
This work was supported, in part, by NIAID grant AI-067769-12 (J.P.C.), NHLBI grant
© The Author(s) 2019
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