A Potential Global Role for Dipeptidylpeptidase 4 (DPP4/CD26) and Its Inhibition In the Regulation...

A Potential Global Role for Dipeptidylpeptidase 4 (DPP4/CD26) and Its Inhibition In The Regulation of

Hematopoiesis and Other Cell Systems

Hal E. Broxmeyer, PhD

Department of Microbiology/Immunology

Indiana University School of Medicine Indianapolis, Indiana, USA

Disclosure: Hal E. Broxmeyer, Ph.D.

Financial interests to disclose

Consulting:

Cord Use - a public cord blood banking company - MSAB and a founder of Cord Use Family Cord Blood Bank

Fate Therapeutics - Consultant

Since our initial scientific and clinical studies, Broxmeyer et al Proc. Natl. Acad. Science USA 86:3828-3832, 1989 Gluckman, Broxmeyer et al N. Engl. J. Med. 321: 1174-1178, 1989

there have now been over 30,000 cord blood transplants done to treat a wide variety of malignant and non-malignant disorders with hematopoietic stem cells

Broxmeyer, Farag, Rocha. 2013. Cord Blood Hematopoietic Cell Transplantation. In: Thomas’ Hematopoietic Cell Transplantation 5th Edition (Applebaum, Forman, Negrin, Antin, Eds) Wiley-Blackwell, England

Ballen, K.K., Gluckman, E., and Broxmeyer, H.E. 2013. Umbilical Cord Blood Transplantation – the first 25 years and beyond. Blood. In press

Goal

Understanding biology of HSC to enhance transplantation engraftment:

• CD26/Dipeptidylpeptidase (DPP) 4 influence on cytokine action, hematopoiesis, recovery from stress, and engrafting capability

A more global role for DPP4

CD26/DPPIV (dipeptidylpeptidase IV) cleaves dipeptides from the N-terminus

after a proline or an alanine

CD26/DPPIV

N- X1 - A/P2 - X3 - X4 … -C Cleavage of substrates after

the N-terminal pen-ultimate alanine or

proline

cleavage

Modulation of Hematopoietic Stem Cell Homing and Engraftment by CD26

K.W. Christophersen, Giao Hangoc, Charlie Mantel and Hal E. Broxmeyer

Science 30:1000-1003, 2004

CD26/Dipeptidylpeptidase IV Negatively Regulates Colony Stimulating Factor Activity and Stress Hematopoiesis

Hal E. Broxmeyer, Jonathan Hoggatt, Heather O’Leary, Charlie Mantel, Brahmananda R. Chitteti, Scott H. Cooper,

Steven Messina-Graham, Giao Hangoc, Sherif Farag, Sara L. Rohrabaugh, Xuan Ou, Jennifer Speth, Louis M. Pelus,

Edward F. Srour and Timothy B. Campbell

Nature Medicine 18: 1786-1796, 2012

In addition to SDF-1/CXCL12 and a number of other chemokines, there are other cytokines

that have putative CD26 truncation sites

Growth Factor Species Full length N-terminus Predicted

Mass Truncated N- Terminus Predicted Mass

GM-CSF Human APARSPSPSTQPWEH… 14,469 ARSPSPSTQPWEH… 14,301

Mouse APTRSPITVTRPWKH… 14,112 TRSPITVTRPWKH… 13,944

G-CSF Human TPLGPASSLPQSFLL… 18,661 LGPASSLPQSFLL… 18,463

Mouse VPLVTVSALPPSLPL… 18,940 LVTVSALPPSLPL… 18,744

IL-3 Human APMTQTTSLKTSWVN… 15,072 MTQTTSLKTSWVN… 14,904

Mouse ASISGRDTHRLTRTL… 15,673 N/A N/A

EPO Human APPRLICDSRVLERY… ~37 kDa † PRLICDSRVLERY… ~37 kDa †

Mouse APPRLICDSRVLERY… ~36 kDa † PRLICDSRVLERY… ~36 kDa †

M-CSF Human EEVSEYCSHMIGSGH… 18,403 * N/A N/A

Mouse KEVSEHCSHMIGNGH… 25,987 * N/A N/A

SCF Human EGICRNRVTNNVKDV… 18,458 N/A N/A

Mouse KEICGNPVTDNVKDI… 18,298 N/A N/A

FL Human TQDCSFQHSPISSDF… 18,050 N/A N/A

Mouse GTPDCYFSHSPISSN… 18,385 N/A N/A

* This may be in dimer form as well. † Glycosylated protein.

Cytokine

Cytokine + DPPIV(18 hrs)

Cytokine + Diprotin A + DPPIV (18 hrs)

GM-CSF IL-3

m/z14100 14600

Intensity

160

0

<14471>

m/z14100 14600Intensity

50

0

<14473>

m/z14100 14600

Intensity

18

0

14301

14470

m/z2000 20000

Intensity

160

0

14471

m/z2000 20000

Intensity

120

0

15080

m/z14700 15200Intensity

120

0

15080

m/z14700 15200

Intensity

40

0

14914

m/z14700 15200

Intensity

20

0

15085

Full Spectrum

14,471

14,471

14,301

14,473

15,080

15,080

15,085

14,914

Mass Spec Analysis of Human GM-CSF and IL-3

0 10 20 30 40 50 60 70 80 90 Colony Formation by Human Cord Blood Cells

Control DA w/ WASH

DA w/ NO WASH

Influence of Diprotin A Pretreatment of Human Cord Blood on Human Cytokine Stimulation of CFU-GM Colony Formation

rhuGM-CSF (20)

rhuGM-CSF (10)

rhuGM-CSF (1)

rhuG-CSFmet (20)

rhuG-CSFmet (10)

rhuG-CSFmet (1)

rhuIL-3 (20)

rhuIL-3 (10)

rhuIL-3 (1)

rhuM-CSF (1000)

rhuM-CSF (100)

rhuM-CSF (10)

rhuFlt3L

rhuSCF

rhuG-CSF (20)

rhuG-CSFmet (20)

Influence of Diprotin A (DPA) Pretreatment of Mouse Bone Marrow or Human Cord Blood on EPO Stimulation of BFU-E colony

Formation

Mouse Bone Marrow Stimulated by Recombinant Mouse (N=3) or Human (N=3) EPO

Human Cord Blood Stimulated by Recombinant Human (N=5) EPO

[Fold Increase of +DiProtinA / -DiprotinA]

a p<0.01 for +DPA compared to -DPA

0 0.5 1 1.5 2 2.5

a

a

• DPP4-truncated CSFs were much less active than full length CSFs

• Truncated CSFs blocked the activity of their own full length CSF form, acting as a dominant negative form of CSF

additional information (in vitro)

Effects of CD26 -/- on Cytokine

Actions In Vivo.

Control EPO: Full Length EPO: Truncated EPO: FL+T

+/+ CD26 -/- 0

2

4

6

8

10

12

14

16

(3.8X)

(1.4X) (1.3X)

(6.3X)

(1.6X) (1.8X)

* *

* *

*

* *

* P<0.01, N=4/group; 10U EPO (FL, T) or 10U each FL or T s.c. Blood assessed 24 hrs later

Influence of EPO On % PB Reticulocytes

Effects of Full Length and Truncated rmuGM-CSF, Alone and in Combination, on Absolute Numbers of Hematopoietic Progenitor Cells (HPC)

0 10 20 30 40 50 60 70 80

WT CD26

0 10 20 30 40 50 60 70 80

WT CD26

CFU-GM

HPC

per

Fem

ur (x

103 )

%

in S

-Pha

se

Control

GMCSF:Full Length (FL)

GMCSF:Truncated (T)

GMCSF:FL+T

Equilibrium receptor binding kinetic analysis using the factor dependent human

cell line, TF-1, as well as primary purified CD34+ cord blood cells demonstrated that:

• Truncated GM-CSF binds with higher affinity to GM-CSF receptor than does full length GM-CSF

• Truncated GM-CSF can block binding of full length GM-CSF

• This supports the functional progenitor cell GM-CSF stimulation assays that suggest that truncated GM-CSF can act as a negative regulator of full length GM-CSF function.

pJAK2

15 minutes

5 minutes

15 minute ratios

30 minutes

Unstimulated Full Length Truncated 1:1 5/10 2.5/10 1.25/10

N = 3 expts

% Ch

ange

in m

ean f

luores

cenc

e (m

ean +

SE)

0

10

20

30

40

50

60

70

80

90

100

N = 3 expts

% Ch

ange

in m

ean f

luores

cenc

e (m

ean +

SE)

0

10

20

30

40

50

60

70

80

90

100

N = 3 expts

% Ch

ange

in m

ean f

luores

cenc

e (m

ean +

SE)

0

10

20

30

40

50

60

70

80

90

100

% C

hang

e in m

ean f

luore

scen

ce

(mea

n + S

E)

0

10

20

30

40

50

60

70

80

90

100

FL GM-CSF T GM-CSF 1:1 2.5:10 1.25:10

p<0.001

p<0.001 p<0.001

N=1 exp

(TF1 Cells)

pSTAT5 Unstimulated Full Length Truncated 1:1 5/10 2.5/10 1.25/10

FL GM-CSF T GM-CSF 1:1 2.5:10 1.25:10

N = 3 expts

% C

hang

e in m

ean f

luore

scen

ce

(mea

n + S

E)

0

10

20

30

40

50

60

70

80

90

100

15 minutes

5 minutes

30 minutes

30 minute ratios

% C

hang

e in m

ean f

luore

scen

ce

(mea

n + S

E)

0

10

20

30

40

50

60

70

80

90

100

N = 3 expts

% C

hang

e in m

ean f

luore

scen

ce

(mea

n + S

E)

0

10

20

30

40

50

60

70

80

90

100

N = 3 expts

% C

hang

e in m

ean f

luore

scen

ce

(mea

n + S

E)

0

10

20

30

40

50

60

70

80

90

100

p<0.002

p<0.004 p<0.004

N=1 exp

(TF1 Cells)

% In

crea

se in

mea

n flo

ures

cenc

e (m

ean±

SE)

0

20

40

60

80

100

120

0

20

40

60

80

100

120

Truncated

Full Length

pJAK2

pSTAT5

GRAY = Untreated

BLUE = Truncated GM-CSF

RED = Full Length GM-CSF

N = 6 expts

N = 3 expts

Influence of Full Length and Truncated Human GM-CSF on Phosphorylation of JAK2 and STAT5 in Human CD34+ Cord Blood Cells

Effects of CD26/DPPIV -/- In Vivo on

Recovery from Stress

(radiation and drugs)

a

NS

a

a

NS

NS

a NS

NS

NS

NS NS

0

1000

2000

3000

4000

5000

6000

7000

0 12 24 36 48

Cell Lysate: 400 rads

PLASMA: 400 rads

PLASMA: 650 rads

Cell Lysate: 650 rads

Hours post irradiation

Rel

ativ

e Li

ght U

nits

DPPIV Activity in Plasma and Cell Lysates of Mice Pre- and Post- Radiation

CFU-GM Pr

ogen

itors

/fem

ur (t

hous

ands

)

0

5

10

15

20

25

30

35

40

45

Days Post 400cGy Radiation

D0 D7 D10 D21

1.7X

(<0.

04)

3.6X

(<0.

006)

3.9X

(<0.

03)

9.4X

(<0.

001)

6.9X

(<0.

002)

1.9X

a

WT

WT+Sitagliptin

CD26-/-

0

20

40

60

80

100

120

140 CFU-GM

Days Post 5FU

Prog

enito

rs/fe

mur

(tho

usan

ds)

D0 D7 D10 D21

3.0X

(<0.

001)

1.8X

(<0.

004)

9.5X

(<0.

001)

7.1X

(<0.

002)

ND

WT

WT+Sitagliptin

CD26-/-

Control

Sitagliptin (oral)

0

20

40

60

80

1 2 4 6

p=<0

.001 p=

<0.00

1 p=<0

.001

p=<0

.001

Primary Transplant (Competitive)

Months (post transplant)

% D

onor

(CD

45.2

) Chi

mer

ism

9

p=<0

.001

Effect of Oral Administration of DPP4 Inhibitor (Sitagliptin) to Lethally Irradiated Recipient Mice on Engraftment of Mouse Bone Marrow Cells

In-vivo inhibition to enhance engraftment of single-unit cord blood transplants in adults with

high-risk hematological malignancies

Farag, S.S., Srivastava, S., Messina-Graham, S., Schwartz, J., Nelson, R., Robertson, M., Abonour, R., Cornetta, K., Wood,

L., Secrest, A., Rojas, L., Strother, M.R., Jones, D., and Broxmeyer, H.E.

Stem Cells & Development 22:1007-1015, 2013

Sherif Farag M.D.

Sitagliptin: A clinical DPP-4 inhibitor

• Specific DPP-4 inhibitor • Only available PO • FDA approved for treatment of type 2 diabetes

mellitus (DM) • Extensively tested in healthy subjects

• Incretin stimulation of insulin release is glucose dependent; hypoglycemia not observed even at high doses

• PK and PD well characterized in healthy volunteers and patients with DM

Treatment Plan

Day of Transplantation -7 -6 -5 -4 -3 -2 -1 0 +1 +2

TBI (165 cGy bid) X X X X Cyclophosphamide (60 mg/kg/day)

X X

ATG (12.5 mg/kg/day)* X X X Sitagliptin (600 mg) X X X X UCB transplantation X

Filgrastim 5 μg/kg/day SC starting day +5 until ANC ≥2.0x109/l for 2 consecutive days

*ATG replaced by fludarabine 30 mg/m2 days -6 to -2 after first 11 patients to reduce risk of infections

Comparison to NYBC Data on engraftment of single UCB Units

Expected Outcome: < 80% engraftment by day 77 50% engraft by day ~28-30

Rubenstein et al. NEJM 1998, 351:2276

CD26 Inhibition

Days after transplantation

Cum

ulat

ive

inci

denc

e

of n

eutr

ophi

l eng

raftm

ent

Median days to engraftment 21 days

Activity of Plasma DPPIV (RCD CB Unit Transplant)*

* Maximum inhibition occurred 2-4 hrs after dosing, with DPPIV activity ≥80% of baseline by 16 hrs.

Pre-dose 0.5 1 2 4 8 12 16 24 2 4 8 16 24 2 4 8 16 24 2 4 8 16 24

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

Day -1 Day 0 Day +1 Day +2

Time after sitagliptin dose

Plas

ma

CD

26 A

ctiv

ity (%

of p

re-d

ose

leve

l)

Conclusions • DPP4 enzyme activity in patients receiving

sitagliptin was suboptimal • Opportunities to further improve the efficacy

of DPP-4 inhibition need to be explored: • Pharmacodynamic studies in transplant patients suggest

that more frequent doses of sitagliptin are required for sustained DPP-4 inhibition

• Longer duration of DPP-4 inhibition may be needed to take harness positive effects on hematopoeitic cytokines

• Modification in conditioning regimen

• Multicenter phase II trial sponsored by NHLBI is under development to confirm results

Influence of DPP4 Truncation on Negative Regulators of

Hematopoiesis

DEK • Mammalian nuclear phosphoprotein originally

identified as a fusion protein resulting from a t (6;9) translocation of a rare subtype of AML

Von Linderm et al Mol Cell Biol 12:1687, 1992

• Bears little resemblance to other known proteins, but is well conserved among higher eukaryotes

• In addition to its DNA-binding properties, it is found in association with mRNA splicing and export factors, as well as with spliced transcripts

• Plays an active role in maintaining higher order chromatin architecture

DEK • DEK can leave the cell, and in paracrine fashion, be

taken up by another cell, traffic to the nucleus of that cell and in bioactive form modulate global chromatin structure

David Markovitz and colleagues University of Michigan, Ann Arbor, MI

PNAS in press

DEK Regulates Hematopoietic Stem Engraftment and Progenitor Cell Proliferation

Broxmeyer, H.E., Kappes, F., Mor-Vakin, N., Legendre, M., Kinzfogl, J., Cooper, S., Hangoc, G.,

and Markovitz, D.M.

Stem Cells and Development 21: 1449-1454, 2012

0

10000

20000

30000

40000

Absolute Number of Progenitors / Femur

p=0.003

p=0.108 p=0.044

CFU-GM BFU-E CFU-GEMM

WT DEK (-/-)

0

5000

10000

15000

20000

Absolute Number of Progenitors /Spleen

p=0.006

p=0.001

p=0.129

CFU-GM BFU-E CFU-GEMM

0

20

40

60

80

100

Cycling: BM Progenitors (% S-Phase)

p=0.001 p=0.003 p=0.001

CFU-GM BFU-E CFU-GEMM 0

20

40

60

80

100

Cycling: Spleen Progenitors (% S-Phase)

p=0.001 p=0.001 p=0.001

CFU-GM BFU-E CFU-GEMM

DEK is myelosuppressive in vitro for colony formation by mouse CFU-GM,

BFU-E, and CFU-GEMM

0 10 20 30 40 50

CFU-GEMM

BFU-E

CFU-GM

Control

DEK (100nM)

Percent wells with colony

p<0.0001

p= 0.020

p= 0.059

0

20

40

60

80

100

1 2 4 6 1 3 5

Primary Mouse Recipients

Peripheral Blood Bone Marrow Peripheral Blood

Secondary Mouse Recipients WT

DEK (-/-)

Months: post transplant

NS NS p<0.002 p<0.04 p<0.002 p<0.002 p<0.002

% D

onor

Cel

l Chi

mer

ism

in

Com

petit

ive

Rep

opul

atin

g As

say

a) b)

A Role For DEK in Stem/Progenitor Cell Biology

Broxmeyer, H.E., Mor-Vaknin, N., Kappes, F., Legendre, M., Saha, A.K., Ou, X., O’Leary, H.A., Capitano, M., Cooper, S., and Markovitz, D.M.

Stem Cells in press

0 20 40 60 80

Control

FL DEK (50nM)

“ (10nM)

“ (1nM)

TR DEK (50nM)

“ (25nM)

FL DEK (50nM)+TR DEK (25nM)

“ + " (12.5nM)

“ + " (6.25nM)

0 25 50 75 100 125

Colony Formation

Exp #1 Exp #2

*, significantly different from control medium (p<0.05)

* *

* *

* ND

ND

ND

Influence of full length (FL) and DPP4-treated (=truncated, TR) DEK alone and in combination on 5x104 C57Bl/6 mouse BM cells/ml treated

with GM-CSF (Exp#1) or GM-CSF+SCF (Exp#2)

Potential Role for Dipeptidylpeptidase (DPP) 4 in Regulation of Many Different Cell and

Tissue Systems

Implications of DPP4 Modification of Proteins That Regulate Stem/Progenitor and More Mature Cell Types

Ou, X., O’Leary, H.A., and Broxmeyer, H.E. 2013 Blood in press

The Role of DPP4 in Hematopoiesis and Transplantation

O’Leary, H.A., Ou, X., and Broxmeyer, H.E. 2013 Current Opinions in Hematopoiesis (Hematology) in press

Chemokines Cytokines Peptide Species N-terminus Peptide Species N-terminus CXCL1 /GRO-α M APIAN… a,b Epo H APPRL… a,b CXCL2 /GRO-β/MIP-2α H APLAT… a,b Epo M APPRL… a,b CXCL4 /PF4 H EAEED… a,b GM-CSF H APARS… a,b CXCL5 /ENA-78 M APSSV… a,b GM-CSF M APTRS... a,b CXCL6/GCP2 H GPVSA… a,b G-CSF H ATPLG… a,b CXCL8/IL-8 H SAKEL… a,b G-CSF M VPLVT… a,b CXCL9/MIG H TPVVR… a,b IL-3 H APMTQ... a,b CXCL10/INP-10 H VPLSR… a,b IL-1 α H SAPFS… a,b CXCL10/INP-10 M IPLAR… a,b IL-1 β H APVRS… a,b CXCL11/I-TAC H IPLAR… a,b IL-1 α M SAPYT… a,b CXCL12/SDF-1 H KPVSL… a,b IL-1 β M VPIRQ… a,b CXCL12 (isoform α) M KPVSL… a,b IL-2 H APTSS… a,b CXCL12 (isoform β) M KPVSL… a,b IL-2 M APTSS… a,b CXCL12( isoform γ) M QPDAI… a,b IL-5 H IPTEI… a,b CCL2/MCP-1 H QPDAV… a,b IL-6 H VPPGE… a,b CCL2/MCP-1 M APYGA… a,b IL-6 M FPTSQ… a,b CCL3 /MIP-1α/LD78α M APLAA… a,b IL-8 (6-77) H SAEKL… a,b CCL3-L1/LD78β H APMGS… a,b IL-10 H SPGQG… a,b CCL4 /MIP-1β H APMGS… a,b IL-13 H GPVPP… a,b CCL4 /MIP-1β M APMGS… a,b IL-17B H QPRSP… a,b CCL5/RANTES H SPYSS… a,b IL-17A M AAIIP… a,b CCL5/RANTES M SPYGS… a,b IL-17C M DPPSW… a,b CCL7/MCP-3 H QPVGI… a,b IL-22 H APISS… a,b CCL7/MCP-3 M QPDGP… a,b IL-22 M LPVNT… a,b CCL8/MCP-2 H QPDSV… a,b IL-23 α H RAVPG… a,b CCL11/Eotaxin H GPASV… a,b IL-23 α M VPRSS… a,b CCL11/Eotaxin M HPGSI... a,b IL-27 α H FPRPP… a,b CCL12/MCP-5 M GPDAV... a,b IL-27 α M FPTDP… a,b CCL16/HCC-4/LEC/LCC-1/LMC H QPKVP… a,b IL-28A H VPVAR… a,b CCL19/CKβ11/MIP-3β/ELC/Exodus-3 M GANDA… a,b IL-28B H VPVAR… a,b CCL22/MDC/STCP-1 H GPYGA… a,b IL-28A M DPVPR… a,b CCL22/MDC/STCP-1 M GPYGA… a,b IL-28B M DPVPR… a,b CCL26/Eotaxin-3/MIP-4α M HPGSI… a,b IL-29 H GPVPT… a,b CCL27/CTACK/ILC/ESKINE M LPLPS… a,b

Chemokines/Cytokines with putative penultimate (Alanine/Proline) truncation site for DPP4

Modulatory Factors with putative penultimate (Alanine/Proline) truncation site for DPP4 (part 1)

Modulatory Factors Peptide Species N-terminus Peptide Species N-terminus Adipsin (propeptide) H PPRGR… a,b GDF-1 H DAEPV… a,b Adipsin (propeptide) M QPRGR…a,b GDF-3 H AAIPV…a,b α-1 microglobulin H GPVPT… a,b GDF-3 M AAISV… a,b α-1 microglobulin M DPAST… a,b GDF-5 H APLAT… a,b ANGPTL4 (isoform a) H GPVQS…a,b GDF-5 M APLAN… a,b ANGPTL4 (isoform b) H GPVQS…a,b GDF 6 H TAFAS… a,b ANGPTL6 H RAGAP…a,b GDF 6 M TAFAS… a,b BDNF (propeptide) H APMKE…a,b GDF-7 H TALAG… a,b BDNF (propeptide) M APMKE...a,b GDF-7 M TALAG… a,b bFGF H PALPE… a,b GDF-8 H GPVDL… a bFGF M PALPE… a,b GDNF (propeptide) H SPDKQ…a,b Bradykinin H RPPGF…a,b GDNF (propeptide) M SPDKQ… a,b Bradykinin M RPPGF… a,b GHRH H YADAI… a,b BMP-4 H SPKHH…a,b GLP-1 (7-36) H HAEGT… a,b BMP-4 M SPKHH…a,b GLP-2 H HADGS… a,b BMP-5 H AANKR… a,b GRP H VPLPA…a,b BMP-5 M AASKR… a,b GRP M APVST… a,b c1qTNF5 H SPPLD… a,b HCG α H APDVQ… a,b c1qTNF5 M SPPLD…a,b HCG α M LPDGD… a,b Chromogranin H LPVNS… a,b IGF-1 isoform 1 H GPETL… a,b Chromogranin M LPVNS…a,b IGF-1 isoform 2 H GPETL… a,b DKK 3 H APAPT…a,b IGF-1 isoform 3 H GPETL… a,b DNER H NPVPA… a,b IGF-1 isoform 4 H GPETL… a,b Enterostatin H APGPR... a,b IGF-1 isoform 1 M GPETL… a,b Enterostatin M APGPR…a,b IGF-1 isoform 2 M GPETL… a,b

IGF-1 isoform 3 M GPETL…a,b IGF-1 isoform 4 M GPETL… a,b IGF-1 isoform 5 M GPETL… a,b

Modulatory Factors with putative penultimate (Alanine/Proline) truncation site for DPP4 (part 2)

Modulatory Factors Peptide Species N-terminus Peptide Species N-terminus Inhibin alpha chain (propeptide) H HALGG…a,b RBP3 H GPTHL… a,b Inhibin alpha chain (propeptide) M HAVGG… a,b RBP3 M GPTHL…a,b Inhibin beta E chain H TPTCE… a,b Somatotropin isoform 1 H FPTIP… a,b Inhibin beta E chain M TPTCE… a,b Somatotropin isoform 2 H FPTIP… a,b Lactoferrin M KATTV… a,b Somatotropin isoform 3 H FPTIP… a,b leptin H VPIQK… a,b Somatotropin isoform 4 H FPTIP… a,b leptin M VPIQK… a,b Somatotropin M FPAMP… a,b LIF H SPLPI… a,b Transferrin H VPDKT… a,b LIF M SPLPI… a,b Transferrin M VPDKT… a,b LTα H LPGVG… a,b Trypsinogen H APFDD… a,b Neurotrophin-3 H YAEHK... a,b Vasostatin-1 H LPVNS… a,b Neurotrophin-3 M YAEHK… a,b Vasostatin-2 H LPVNS… a,b Notch 3 H APPCL… a,b VEGF A isoform i H APMAE… a,b Notch 3 M APPCL… a,b VEGF A isoform k H APMAE…a,b NPY H YPSKP… a,b VEGF A isoform l H APMAE… a,b NPY M YPSKP… a,b VEGF A isoform m H APMAE… a,b Oncostatin M H AAIGS… a,b VEGF A isoform n H APMAE… a,b Osteopontin H IPVKQ… a,b VEGF A isoform o H APMAE… a,b Osteopontin M LPVKV… a,b VEGF A isoform p H APMAE… a,b PDGFD M TPQRA…a,b VEGF A isoform 1 M APTTE…a,b Peptide YY H YPIKP… a,b VEGF A isoform 2 M APTTE… a,b Peptide YY M YPAKP… a,b VEGF A isoform 3 M APTTE… a,b Prolactin H LPICP…a,b Wnt 9b M AAYFG… a,b Prolactin M LPICS…a,b Wnt 10a H MPRSA... a,b

Wnt 10a M VPRSA… a,b

Peptides Species N-terminus Peptides Species N-terminus

CXCL1 H ASVAT… a,b ANGPT2 M YSNFR… a,b

CXCL3 H ASVVT… a,b BDNF H HSDPA… a,b

CXCL7 H SSTKG… a,b BDNF M HSDPA… a,b

CXCL7 M KSDGM… a,b DLL3 M HSFGP… a,b

CCL1 H KSMQV… a,b DLL4 M GSGIF… a,b

CCL1 M KSMLT… a,b DKK 3 M PSPTV… a,b

CCL20 H ASNFD… a,b FZD6 H HSLFT… a,b

CCL20 M ASNYD… a,b FZD6 M HSLFT… a,b

IL-3 M ASISG… a,b Glucagon M HSQGT… a,b

IL-17E M VSLRI… a,b Glucagon M HSQGT… a,b

IFN-β H MSYN… a,b GM2A H SSFSW… a,b

LTα M LSGVR… a,b Beta NGF H SSSHP… a,b

TGF-β1 H LSTCK… a,b Beta NGF M SSTHP… a,b

TGF-β1 M LSTCK… a,b PACAP (1-27) H HSDGI… a,b

TNF-β M LSGVR… a,b PACAP (1-38) H HSDGI… a,b

TNF (Membrane Form) H MSTES… a,b PDGFC M ESNLS… a,b

TNF (Membrane Form) M MSTES… a,b PDGFC H ESNLS… a,b

DEK H MSASA… a,b Wnt 8a M ASAWS… a,b

DEK M MSAAA… a,b Wnt 8b H WSVNN… a,b

Factors with putative truncation site (Serine) for DPP4

Potential Models for DPP4 Activity

Lab Members

Former Lab Members

Scott Cooper, M.S. Research Associate

Giao Hangoc, D.V.M. Research Associate

Charlie Mantel, B.S. Research Associate

Hal E Broxmeyer, Ph.D.

Man Ryul Lee, PhD Post Doc

Xuan Ou, Ph.D Post Doc

Heather O’Leary, Ph.D Post Doc

Sunanda Basu , Ph.D.. Sara Rohrabaugh Young-June Kim, Ph.D. Ying Liu , Ph.D.

Tammi Taylor, Ph.D. Timothy Campbell, M.D., Ph.D. Kent W. Christophersen II, PhD John Kinzfogl, Ph,D. Myung Kwan Han, PhD

Hee-Don Chae, Ph.D.

Steven Messina-Graham Grad Student

Xinxin Huang, PhD Post Doc

Maegan Capitano, Ph.D Post Doc