A Potential Global Role for Dipeptidylpeptidase 4
(DPP4/CD26) and Its Inhibition In The Regulation of
Hematopoiesis and Ot...
Disclosure: Hal E. Broxmeyer, Ph.D.
Financial interests to disclose
Consulting:
Cord Use - a public cord blood banking com...
Since our initial scientific and clinical studies,
Broxmeyer et al Proc. Natl. Acad. Science USA 86:3828-3832,
1989
Gluckm...
Goal
Understanding biology of HSC to enhance
transplantation engraftment:
• CD26/Dipeptidylpeptidase (DPP) 4 influence
on ...
CD26/DPPIV (dipeptidylpeptidase IV)
cleaves dipeptides from the N-terminus
after a proline or an alanine
CD26/DPPIV
N- X1 ...
Modulation of Hematopoietic
Stem Cell Homing and
Engraftment by CD26
K.W. Christophersen, Giao Hangoc,
Charlie Mantel and ...
CD26/Dipeptidylpeptidase IV Negatively
Regulates Colony Stimulating Factor
Activity and Stress Hematopoiesis
Hal E. Broxme...
In addition to SDF-1/CXCL12 and a number of other
chemokines, there are other cytokines
that have putative CD26 truncation...
Cytokine
Cytokine +
DPPIV(18 hrs)
Cytokine +
Diprotin A +
DPPIV (18 hrs)
GM-CSF IL-3
m/z14100 14600
Intensity
160
0
<14471...
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 Dipr...
Influence of Diprotin A (DPA) Pretreatment of Mouse Bone Marrow
or Human Cord Blood on EPO Stimulation of BFU-E colony
For...
• DPP4-truncated CSFs were much less active
than full length CSFs
• Truncated CSFs blocked the activity of their own
full ...
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...
Effects of Full Length and Truncated rmuGM-CSF, Alone and in Combination,
on Absolute Numbers of Hematopoietic Progenitor ...
Equilibrium receptor binding kinetic
analysis using the factor dependent human
cell line, TF-1, as well as primary purifie...
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 ex...
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
%Chang...
%Increaseinmeanflourescence
(mean±SE)
0
20
40
60
80
100
120
0
20
40
60
80
100
120
Truncated
Full Length
pJAK2
pSTAT5
GRAY ...
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
...
CFU-GM
Progenitors/femur(thousands)
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
20
40
60
80
100
120
140
CFU-GM
Days Post 5FU
Progenitors/femur(thousands)
D0 D7 D10 D21
3.0X(<0.001)
1.8X(<0.004)
9.5X(<...
Control
Sitagliptin
(oral)
0
20
40
60
80
1 2 4 6
p=<0.001
p=<0.001
p=<0.001
p=<0.001
Primary Transplant
(Competitive)
Mont...
In-vivo inhibition to enhance engraftment of
single-unit cord blood transplants in adults with
high-risk hematological mal...
Sherif Farag M.D.
Sitagliptin: A clinical DPP-4 inhibitor
• Specific DPP-4 inhibitor
• Only available PO
• FDA approved for treatment of typ...
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/da...
Comparison to NYBC Data on
engraftment of single UCB Units
Expected Outcome:
< 80% engraftment by day 77
50% engraft by da...
Activity of Plasma DPPIV (RCD CB Unit Transplant)*
* Maximum inhibition occurred 2-4 hrs after dosing, with DPPIV activity...
Conclusions
• DPP4 enzyme activity in patients receiving
sitagliptin was suboptimal
• Opportunities to further improve the...
Influence of DPP4 Truncation on
Negative Regulators of
Hematopoiesis
Table 1. DPPIV Cleavage Sites on Human Chemokines
Chemokines N-terminus…
CCL2 / MCP-1 QPDAINAPVTCCYNFT
CCL3 / MIP1α APLAAD...
(0.01)
(0.1)
(1)
(10)
CXCL4 (100)
(0.01)
(0.1)
(1)
(10)
CXCL10 (100)
(0.01)
(0.1)
(1)
(10)
CXCL9 (100)
(0.01)
(0.1)
(1)
(1...
In Vitro
• Truncated chemokine is inactive as a
myelosuppressive molecule
• Truncated chemokine blocks
myelosuppression by...
i) CCL3
Absolute Number Progenitors
per Femur (in thousands)
0
10
20
30
40
50
60
*
Control
Full Length (FL)
Truncated (TR)...
DEK
• Mammalian nuclear phosphoprotein originally
identified as a fusion protein resulting from a t (6;9)
translocation of...
DEK
• DEK can leave the cell, and in paracrine fashion, be
taken up by another cell, traffic to the nucleus of that
cell a...
DEK Regulates Hematopoietic Stem
Engraftment and Progenitor Cell Proliferation
Broxmeyer, H.E., Kappes, F., Mor-Vakin, N.,...
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 (-/-...
DEK is myelosuppressive in vitro for
colony formation by mouse CFU-GM,
BFU-E, and CFU-GEMM
a) CFU-GM
b) BFU-E
c) CFU-GEMM
0 20 40 60 80 100
0.041
NS
NS
0.010
0.001
0.023
0.001
0.003
0.009
0.001
0.001
0.015
0.002
0...
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 Rec...
A Role For DEK in Stem/Progenitor Cell Biology
Broxmeyer, H.E., Mor-Vaknin, N., Kappes, F.,
Legendre, M., Saha, A.K., Ou, ...
0 20 40 60 80
Control
FL DEK (50nM)
“ (10nM)
“ (1nM)
TR DEK (50nM)
“ (25nM)
FL DEK (50nM)+TR DEK (25nM)
“ + " (12.5nM)
“ +...
Potential Role for Dipeptidylpeptidase (DPP) 4
in Regulation of Many Different Cell and
Tissue Systems
Implications of DPP...
Chemokines Cytokines
Peptide Species N-terminus Peptide Species N-terminus
CXCL1 /GRO-α M APIAN… a,b Epo H APPRL… a,b
CXCL...
Modulatory Factors with putative penultimate (Alanine/Proline) truncation site for DPP4
(part 1)
Modulatory Factors
Peptid...
Modulatory Factors with putative penultimate (Alanine/Proline) truncation site for DPP4
(part 2)
Modulatory Factors
Peptid...
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 H...
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...
A Potential Global Role for Dipeptidylpeptidase 4 (DPP4/CD26) and Its Inhibition In The Regulation of Hematopoiesis and Ot...
A Potential Global Role for Dipeptidylpeptidase 4 (DPP4/CD26) and Its Inhibition In The Regulation of Hematopoiesis and Ot...
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A Potential Global Role for Dipeptidylpeptidase 4 (DPP4/CD26) and Its Inhibition In The Regulation of Hematopoiesis and Other Cell Systems

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

  1. 1. 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
  2. 2. 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
  3. 3. 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
  4. 4. 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
  5. 5. 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
  6. 6. 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
  7. 7. 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
  8. 8. 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.
  9. 9. Cytokine Cytokine + DPPIV(18 hrs) Cytokine + Diprotin A + DPPIV (18 hrs) GM-CSF IL-3 m/z14100 14600 Intensity 160 0 <14471> m/z14100 14600 Intensity 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 15200 Intensity 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
  10. 10. 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)
  11. 11. 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
  12. 12. • 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)
  13. 13. Effects of CD26 -/- on Cytokine Actions In Vivo.
  14. 14. 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
  15. 15. 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 HPCperFemur(x103)%inS-Phase Control GMCSF:Full Length (FL) GMCSF:Truncated (T) GMCSF:FL+T
  16. 16. 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.
  17. 17. 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 %Changeinmeanfluorescence (mean+SE) 0 10 20 30 40 50 60 70 80 90 100 N = 3 expts %Changeinmeanfluorescence (mean+SE) 0 10 20 30 40 50 60 70 80 90 100 N = 3 expts %Changeinmeanfluorescence (mean+SE) 0 10 20 30 40 50 60 70 80 90 100 %Changeinmeanfluorescence (mean+SE) 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)
  18. 18. 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 %Changeinmeanfluorescence (mean+SE) 0 10 20 30 40 50 60 70 80 90 100 15 minutes 5 minutes 30 minutes 30 minute ratios %Changeinmeanfluorescence (mean+SE) 0 10 20 30 40 50 60 70 80 90 100 N = 3 expts %Changeinmeanfluorescence (mean+SE) 0 10 20 30 40 50 60 70 80 90 100 N = 3 expts %Changeinmeanfluorescence (mean+SE) 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)
  19. 19. %Increaseinmeanflourescence (mean±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
  20. 20. Effects of CD26/DPPIV -/- In Vivo on Recovery from Stress (radiation and drugs)
  21. 21. 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 RelativeLightUnits DPPIV Activity in Plasma and Cell Lysates of Mice Pre- and Post- Radiation
  22. 22. CFU-GM Progenitors/femur(thousands) 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-/-
  23. 23. 0 20 40 60 80 100 120 140 CFU-GM Days Post 5FU Progenitors/femur(thousands) 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-/-
  24. 24. Control Sitagliptin (oral) 0 20 40 60 80 1 2 4 6 p=<0.001 p=<0.001 p=<0.001 p=<0.001 Primary Transplant (Competitive) Months (post transplant) %Donor(CD45.2)Chimerism 9 p=<0.001 Effect of Oral Administration of DPP4 Inhibitor (Sitagliptin) to Lethally Irradiated Recipient Mice on Engraftment of Mouse Bone Marrow Cells
  25. 25. 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
  26. 26. Sherif Farag M.D.
  27. 27. 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
  28. 28. 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
  29. 29. 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 Cumulativeincidence ofneutrophilengraftment Median days to engraftment 21 days
  30. 30. 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 PlasmaCD26Activity(%ofpre-doselevel)
  31. 31. 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
  32. 32. Influence of DPP4 Truncation on Negative Regulators of Hematopoiesis
  33. 33. Table 1. DPPIV Cleavage Sites on Human Chemokines Chemokines N-terminus… CCL2 / MCP-1 QPDAINAPVTCCYNFT CCL3 / MIP1α APLAADTPTACCFSYT CCL4 / MIP-1β APMGSDPPTACCFSY CCL5 / RANTES SPYSSDTTPCCFAYIAR CXCL4 / PF-4 EAEEDGDLQCLCVKT CXCL5 / ENA-74 AAVLRELRCVCLQTTQ CXCL5 / ENA-78 (non-cleavable) AGPAAAVLRELRCVCL CXCL6 / GCP-2 GPVSAVLTELRCTCLR CXCL8 / IL-8 SAKELRCQCIKTYSKP CXCL8 / IL-8 (non-cleavable) AVLPRSAKELRCQCIK CXCL9 / MIG TPVVRKGRCSCISTNQ CXCL10 / IP-10 VPLSRTVRCTCISISNQ XCL1 / Lymphotactin VGSEVSDKRTCVSLTT
  34. 34. (0.01) (0.1) (1) (10) CXCL4 (100) (0.01) (0.1) (1) (10) CXCL10 (100) (0.01) (0.1) (1) (10) CXCL9 (100) (0.01) (0.1) (1) (10) CXCL6 (100) (0.01) (0.1) (1) (10) CCL3 (100) Control Medium Percent Inhibition Bl/6 +/+ 0 20 40 60 Bl/6+/+ with Diprotin A 0 20 40 60 Bl/6 CD26 -/- 0 20 40 60 Chemokine (ng/ml)
  35. 35. In Vitro • Truncated chemokine is inactive as a myelosuppressive molecule • Truncated chemokine blocks myelosuppression by that full length molecule
  36. 36. i) CCL3 Absolute Number Progenitors per Femur (in thousands) 0 10 20 30 40 50 60 * Control Full Length (FL) Truncated (TR) FL+TR 0 10 20 30 0 5 ii) CXCL8 iii) CXCL9 0 10 20 30 40 50 CFU-GM 0 10 BFU-E CFU-GEMM CFU-GM * ** * * * 0 20 40 60 80 100 * 0 20 40 60 80 100 Percent HPC in S-Phase 0 20 40 60 80 100 CFU-GM * * * *** CFU-GM BFU-E CFU-GEMM CFU-GM BFU-E CFU-GEMM CFU-GM BFU-E CFU-GEMM
  37. 37. 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
  38. 38. 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
  39. 39. 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
  40. 40. 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
  41. 41. DEK is myelosuppressive in vitro for colony formation by mouse CFU-GM, BFU-E, and CFU-GEMM
  42. 42. a) CFU-GM b) BFU-E c) CFU-GEMM 0 20 40 60 80 100 0.041 NS NS 0.010 0.001 0.023 0.001 0.003 0.009 0.001 0.001 0.015 0.002 0.004 0.011 0.002 0.013 NS 0.001 0.001 0.001 0.003 NS NS 0.001 0.001 0.001 Epo+SCF Epo+GM-CSF+ IL3+SCF IL3+SCF GM-CSF GM-CSF+SCF 0 25 50 75 Epo Epo+SCF Control DEK (100nM) DEK (1nM) DEK (10nM) Human Cord Blood Colony Numbers Epo+GM-CSF+ IL3+SCF
  43. 43. 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
  44. 44. 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 %DonorCellChimerism inCompetitiveRepopulatingAssay a) b)
  45. 45. 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
  46. 46. 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)
  47. 47. 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
  48. 48. 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
  49. 49. 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
  50. 50. 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
  51. 51. 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
  52. 52. Potential Models for DPP4 Activity
  53. 53. 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 RohrabaughYoung-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
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