ALBERT

Description: Dipeptidylpeptidase (DPP) 4 has the potential to truncate proteins with a penultimate alanine, proline, or other selective amino acids at the N-terminus. DPP4 truncation of certain chemokines, colony-stimulating factors, and interleukins have recently been linked to regulation of hematopoietic stem/progenitor cells, more mature blood cells, and other cell types. We believe that the potential role of DPP4 in modification of many regulatory proteins, and their subsequent effects on numerous stem/progenitor and other cell-type functions has not been adequately appreciated. This review addresses the potential implications of the modifying effects of DPP4 on a large number of cytokines and other growth-regulating factors with either proven or putative DPP4 truncation sites on hematopoietic cells, and subsequent effects of DPP4-truncated proteins on multiple aspects of steady-state and stressed hematopoiesis, including stem/progenitor cell, and more mature cell, function.

Description: DPP4 (CD26) is a dipeptidyl peptidase that functions by enzymatically cleaving the penultimate proline, alanine or select other amino acids such as serine of proteins, resulting in functional alterations of the protein. We recently published that many cytokines, chemokines and growth factors have putative DPP4 truncations sites and that DPP4 specifically was able to truncate some colony stimulating factors such as GM-CSF and IL-3 with resultant blunting of their activity. However, the mechanism of action of the truncated factors is still unknown and requires further investigation. The expression, and activity, of DPP4 is relevant in normal and malignant hematopoiesis as we have data showing that CD34+ umbilical cord blood cells (UCB) as well as Acute Myelogenous Leukemia (AML) patient samples express active DPP4. Further, specific inhibition of DPP4 increases homing and engraftment of both human UCB and mouse bone marrow cells after transplantation in mice indicating the therapeutic potential of DPP4 activity altering compounds. Due to its potential importance in disease states, and their subsequent treatment, it is relevant to study how the activity of DPP4 alters the functions of the molecules it cleaves, and subsequently their interactions with each other. DPP4 can cleave the penultimate proline of GM-CSF and IL-3 resulting in truncated forms which have blunted colony stimulating factor activity for hematopoietic progenitor cells (HPC). Since GM-CSF and IL-3 receptors share a common receptor beta chain, we investigated if DPP4 truncation of GM-CSF (TGM) or IL-3 (T3) could inhibit the receptor binding and functional activity of the full length (FL) alternate compound (i.e TGM inhibition of FL3 activity or T3 inhibition of FLGM activity) in the factor dependent TF-1 cell line, UCB cells and in in vivo mouse studies. We determined using TF-1 and UCB that both T3 and TGM bound to the receptors with higher affinity than their FL forms and could blunt the receptor binding of the FLGM and FL3. Additionally, TGM and T3 decreased colony formation induced by either FLGM or FL3 in both TF-1, UCB, and primary AML patient cell samples. Strikingly, this inhibition of colony formation did not require a 1:1 ratio of the full length to truncated forms of these cytokines. Rather, approximately 4-10 fold less truncated molecules could be used to efficiently inhibit the colony formation activity of the full length form, even across molecules. In vivo injection of FL, T, or a mixture of FL/T or T/T factors into DPP4 activity knockout mice followed by colony assays showed the TGM and T3 suppresed the effect of FLGM or FL3 on progenitor cell numbers per femur and diminished cycling of hematopoietic progenitor cells as detected by high specificity tritiated thymidine kill assay. Proteomic analysis of the effects of full length and truncated factors (FLGM, FL3, TGM, T3) were performed with TF-1 cells where we detected differential protein regulation by the full length vs truncated factors. After 24 hour treatment with 10ng/ml of FLGM or TGM, TF-1 cells displayed statistically significant (p 〈 .05) differences in 26 proteins of which 17 were higher in the FL vs the T, and 9 higher in the T vs FL treated groups. These proteins included, but were not limited to, cell cycle proteins such as CDK6, HDAC6, as well as signal transduction proteins and redox control proteins such as STAM1 and Glutaredoxin. Additionally, alterations in protein phosphphorylation were detected for TF-1 cells treated for 15 or 30 min with the full length vs truncated IL-3 and GM-CSF proteins. Interestingly, the protein expression or phosphorylation levels were not always decreased by the truncated protein compared to the full length. In some cases, the truncated molecules induced an increase in the protein expression or phosphorylation. These data suggest interesting roles for full length and truncated GM-CSF and IL-3 in both normal and malignant hematopoiesis. Further investigation into the regulation of DPP4, and the roles that full length and truncated factors play during normal and malignant hematopoiesis, are important and will allow for a better understanding of the signficance of DPP4 activity during steady state, stressed, and disease hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Description: Hematopoietic stem cells (HSC) reside in hypoxic niches (~1-4% O2), however, HSC studies are consistently performed using cells isolated in ambient air (~20% O2), regardless of subsequent processing in low oxygen tension. We recently published that by collecting/processing stem cells in physiologically native conditions of hypoxia, with all procedures performed inside a hypoxic chamber (3% O2), we enhance the recovery of phenotypic, and functional, self-renewing long-term repopulating HSC (LT-HSC) with concomitantly decreasing numbers of progenitor cells. This occurs by inhibiting damage due to brief exposure of mouse bone marrow (BM) or human cord blood (CB) cells to ambient oxygen (a phenomenon we term Extra Physiologic Oxygen Shock/Stress (EPHOSS)) which we, in part, mechanistically linked to mitochondrial permeability transition pore (MPTP), Reactive Oxygen Species (ROS) and cyclophilin D. This data suggests that true numbers of HSCs, and the transplantation potency of BM and CB, have been consistently underestimated due to rapid differentiation of LT-HSCs in ambient air (EPHOSS), but the broad effects of EPHOSS on stem cell phenotype are unknown. We hypothesized that Dipeptidyl Peptidase 4 (DPP4) may be altered by EPHOSS and involved in the effects of EPHOSS on HSC. We showed that DPP4, a serine peptidase whose enzymatic activity leads to the N terminal cleavage of select penultimate amino acids of proteins, alters homing and engraftment of HSC and the number of cytokines, chemokines and growth factors that have putative DPP4 truncation sites have been dramatically underestimated. Functional and mechanistic roles of full length (FL) versus DPP4 truncated (T) factors, the ability of DPP4 T proteins to induce signaling that FL factors cannot, and the effects of EPHOSS on DPP4 expression/activity, and vice versa, have not been investigated and may have yet unappreciated clinical application. Here we present novel data demonstrating that mouse bone marrow harvested in air in the presence of Diprotin A, a DPP4 inhibitor, or from DPP4 K/O mice, results in a significant increase in the number of phenotypic LT-HSC (p=.017), suggesting that inhibition of DPP4 can diminish the loss of phenotypic LT-HSC due to EPHOSS. Further, the percentage of DPP4+ cells is significantly increased in primitive fractions of mouse bone marrow and human cord blood (LSK ~15% DPP4+, LSKCD150+ ~40%DPP4+, CD34+CD38- of CB ~10% DPP4+, CD34+CD38-CD45RA-CD90+CD49F+ ~40% DPP4+, p=.007), the numbers of DPP4+ cells are additionally enhanced 15- 20% when cells (BM and hCB) are isolated in hypoxia, especially in the LT-HSC fraction (Air 40% DPP4+ Hypoxia 60% DPP4+, p=.005). However, DPP4 activity on lineage- bone marrow harvested in hypoxia showed a 2 fold decrease (p=.005) compared to lineage- cells harvested in air. Interestingly, this increase in the number of DPP4+ cells in hypoxia is not recapitulated when mouse BM is harvested in the presence a Cyclosporin A, a cylophilin D inhibitor, (even though the increase in numbers of LT-HSC is preserved similarly to that in hypoxia) suggesting an alternative mechanism for modulation of DPP4 other than inhibition of mitochondrial ROS/MPTP. Unexpectedly, LT-HSC ROS levels (both mitochondrial and total) were not diminished in groups with decreased DPP4 activity (DPA or DPP4 K/O) harvested in air despite the blunting of EPHOSS leading to maintenance of the phenotypic LT-HSC increase over air harvest alone. These data suggest that pathways in addition to ROS, such as DPP4 expression/activity, may be influencing LT-HSC function after, and sensitivity to, EPHOSS as well as being modulated by EPHOSS. Further investigation of these collaborative pathways may facilitate increased HSC collections to enhance HSC transplantation. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1240 CD26 (DPPIV) is a dipeptidyl peptidase that functions by enzymatically cleaving the penultimate proline or alanine of proteins, resulting in functional alterations. The expression and activity of CD26 is relevant in many disease states including obesity and cancer. Modulation of CD26 activity has been shown to increase homing and engraftment of both cord blood and bone marrow cells after transplant indicating the impressive therapeutic potential of CD26 activity altering compounds. Due to its importance in disease states and their subsequent treatments, it is relevant to study how the activity of CD26 alters the functions of the molecules it cleaves, and subsequently their interactions with each other. Mass spectrometry data from our laboratory has shown that CD26 can cleave the penultimate proline of GM-CSF resulting in the truncated form which has blunted signaling and function. Additional data has recently confirmed that CD26 can cleave IL-3 and results in its diminished function as well. Further, and more importantly, since GM-CSF and IL-3 are members of the IL-3 receptor family, and share a common receptor beta chain, we investigated if CD26 truncation of GM-CSF (TGM) or IL-3 (T3) could inhibit the functional activity of the full length (FL) alternate compound (i.e TGM inhibition of FL IL-3 activity or T3 inhibition of FL GM-CSF activity) in the TF-1 cell line and cord blood cells. We determined that both T3 and TGM could inhibit the colony formation induced by either FL GM-CSF or FL IL-3. This inhibition of function correlated with alterations in reactive oxygen species (ROS) levels that mimicked the truncated versions of either GM-CSF or IL-3 even in the presence of the full length molecules. Strikingly, this inhibition of colony formation did not require a 1:1 ratio of the full length to truncated. Rather, approximately 4–10 fold less truncated could be used to efficiently inhibit the colony formation activity of the full length, even across molecules. Interestingly, the ratio of T3 needed to block the full length GM-CSF (1.25ng/ml T3: 10ng/ml FL GM-CSF) was less than the amount of TGM needed to block the full length IL-3 (2.5ng/ml TGM: 10ng/ml FL IL-3) suggesting that T3 is better at blocking FL GM-CSF than TGM is at blocking FL IL-3. However, the ratios of truncated needed to block the function of self FL molecules are identical for both GM-CSF and IL-3 (1.25ng/ml truncated: 10ng/ml FL). Signaling and receptor binding studies were performed for GM-CSF with TF-1 and CD34+cord blood cells, and showed that the truncated GM-CSF inhibited the Stat-5 and JAK2 signaling of FL GM-CSF at less than a 1:1 (10ng/ml FL: 1.25 ng/ml TGM) ratio. Receptor binding studies found that TGM bound to the GM-CSF receptor more efficiently than the FL form but concentrations required to produce 50% maximum inhibition of binding (IC50) is 8-fold lower for TGM compared to FL-GM-CSF, indicating that T-GM-CSF is a better competitor for binding, than is FL-GM-CSF itself suggesting that this may be how TGM is blocking the effects of FL GM-CSF, and potentially IL-3, in our model. Finally, cells treated with TGM had diminished respiratory and glycolytic rates compared to those treated with full-length cytokine. These data provide the first evidence of relevant interactions, with functional consequences, of the importance of full length and CD26 truncated cytokines across molecules. Disclosures: Broxmeyer: CordUse: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy.

Description: Dipeptidylpeptidase 4 (DPP4) is a serine peptidase with enzymatic activity leading to the N terminal cleavage of select penultimate amino acids of proteins. We previously published that the number of cytokines, chemokines and growth factors that have putative DPP4 truncation sites have been dramatically underestimated. Functional and mechanistic roles of full length (FL) versus DPP4 truncated (T) factors, as well as the ability of DPP4 T proteins to induce signaling that FL factors can not, have not been previously investigated and may have yet unappreciated clinical application. Here we present novel data demonstrating the here to fore unknown ability of DPP4 cleavage of proteins to alter not only cellular function, but intracellular signaling of growth factors leading to miRNA expression, phosphorylation, and global induction of proteins that the FL form of the protein does not induce. Additionally, and unexpectedly, a 1:1 mixture of the DPP4 T protein and their FL counterpart leads to both overlapping signaling between the FL and T, as well as unique signaling, that is not induced by the FL or T protein alone. This suggests multifaceted, important, and currently unappreciated roles that the DPP4 truncation of proteins may play in the regulation of normal and malignant hematopoiesis as well as other physiologic and pathophysiologic states. From functional data, utilizing the human factor-dependent TF-1 cell line, as well as primary samples from human cord blood (CB) and patients with Acute Myeloid Leukemia (AML), we observed that DPP4 truncation of GM-CSF (T-GM-CSF) and IL-3 (T-IL-3) results in decreased colony stimulating factor (CSF) activity in normal and malignant hematopoietic progenitor cells (HPC). Receptor binding studies confirmed that both T-GM-CSF and T-IL-3 have enhanced receptor affinity compared to their FL form and each can compete to blunt the receptor binding of either FL-GM-CSF or FL-IL-3. In vivo studies demonstrated that either exogenously added T-GM-CSF or T-IL-3 suppressed the effects of exogenously added FL-GM-CSF or FL-IL-3 on progenitor cell numbers per femur and diminished HPC cycling. Investigation of FL vs T mediated signaling alterations was done with TF-1 cells using proteomic and bioinformatic analysis of miRNA expression, and phosphorylation/global protein induction. Both T-GM-CSF and T-IL-3 induced unique signaling that FL-GM-CSF and FL-IL-3 did not, as well as signaling that overlapped with their FL counterparts (Figure 1, Venn Diagram example of global induction of proteins). Additionally, treatment with a 1:1 ratio of the FL/T proteins resulted in both distinctive, and common, signaling compared to that detected with treatment of only FL or T molecules, thus revealing the complexity of the signaling interactions and heretofore unknown activities of DPP4 truncated proteins (Figure 1). Fisher's exact test or B-H multiple testing correction in IPA software were used to statistically generate comparisons of molecules associated with specific signaling induction, and differential as well as overlapping signaling for all GM-CSF vs IL-3 groups. As examples, for both the GM-CSF and IL-3 groups, molecules associated with PI3K/Akt signaling were increased in T and 1:1 groups vs treatment with FL molecules. In contrast, molecules affiliated with CDC42 signaling were induced by treatment with a 1:1 ratio of FL:T molecules but not in the FL or T groups alone for both GM-CSF and IL-3. Interestingly, phosphorylated proteins associated with Flt3 and IL-9 signaling in hematopoietic progenitors was increased in the T and 1:1 groups for GM-CSF and IL-3 compared to the FL groups. Conversely, phosphorylated molecules involved in nucleic acid metabolism were induced by the FL, T and 1:1 of the GM-CSF groups but only by the FL version of IL-3. Further, phosphorylated molecules associated with antigen presentation were only detected in the FL IL-3 and 1:1 IL-3 groups, and molecules functionally associated with carbohydrate metabolism and cellular therapeutics were only detected in the T and 1:1 groups for both GM-CSF and IL-3, and not in the FL stimulated groups. Collectively, these data substantiate that further investigation into the roles and regulation of DPP4, in normal and malignant hematopoiesis, will allow for a better understanding of the significance, and potential clinical utility, of DPP4 activity altering compounds as well as DPP4 truncated molecules. Figure 1 Figure 1. Disclosures Broxmeyer: CordUse: Membership on an entity's Board of Directors or advisory committees.