The Role of Post-Translational Modifications of Chemokines by CD26 in Cancer

Chemokines are a large family of small chemotactic cytokines that fulfill a central function in cancer. Both tumor-promoting and -impeding roles have been ascribed to chemokines, which they exert in a direct or indirect manner. An important post-translational modification that regulates chemokine activity is the NH2-terminal truncation by peptidases. CD26 is a dipeptidyl peptidase (DPPIV), which typically clips a NH2-terminal dipeptide from the chemokine. With a certain degree of selectivity in terms of chemokine substrate, CD26 only recognizes chemokines with a penultimate proline or alanine. Chemokines can be protected against CD26 recognition by specific amino acid residues within the chemokine structure, by oligomerization or by binding to cellular glycosaminoglycans (GAGs). Upon truncation, the binding affinity for receptors and GAGs is altered, which influences chemokine function. The consequences of CD26-mediated clipping vary, as unchanged, enhanced, and reduced activities are reported. In tumors, CD26 most likely has the most profound effect on CXCL12 and the interferon (IFN)-inducible CXCR3 ligands, which are converted into receptor antagonists upon truncation. Depending on the tumor type, expression of CD26 is upregulated or downregulated and often results in the preferential generation of the chemokine isoform most favorable for tumor progression. Considering the tight relationship between chemokine sequence and chemokine binding specificity, molecules with the appropriate characteristics can be chemically engineered to provide innovative therapeutic strategies in a cancer setting.

Monitoring the stabilities of a mixture of peptides by mass-spectrometry-based techniques

Biomolecular degradation plays a key role in proteostasis. Typically, proteolytic enzymes degrade proteins into smaller peptides by breaking amino acid bonds between specific residues. Cleavage around proline residues is often missed and requires highly specific enzymes for peptide processing due to the cyclic proline side-chain. However, degradation can occur spontaneously (i.e. in the absence of enzymes). In this study, the influence of the first residue on the stability of a series of penultimate proline containing peptides, with the sequence Xaa–Pro–Gly–Gly (where Xaa is any amino acid), is investigated with mass spectrometry techniques. Peptides were incubated as mixtures at various solution temperatures (70℃ to 90℃) and were periodically sampled over the duration of the experiment. At elevated temperatures, we observe dissociation after the Xaa–Pro motif for all sequences, but at different rates. Transition state thermochemistry was obtained by studying the temperature-dependent kinetics and although all peptides show relatively small differences in the transition state free energies (∼95 kJ/mol), there is significant variability in the transition state entropy and enthalpy. This demonstrates that the side-chain of the first amino acid has a significant influence on the stability of the Xaa–Pro sequence. From these data, we demonstrate the ability to simultaneously measure the dissociation kinetics and relative transition state thermochemistries for a mixture of peptides, which vary only in the identity of the N-terminal amino acid.

DPP4 (CD26) Truncation Of GM-CSF and IL-3 Alters Their Signaling and Functional Activity In Normal and Leukemic Hematopoietic Stem and Progenitor Cells

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.

CD26 Truncation of GM-CSF and IL-3 Alters Their Functional Interactions

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.

Abstract 613: Interactive Effect of Ace Inhibition and DPP4 Inhibition on Substance P-Stimulated T-PA Release

Dipeptidyl peptidase 4 (DPP4) inhibitors improve glycemic control in patients with T2DM by preventing the degradation of the incretin hormones. DPP4 inhibition prevents the degradation of other peptides with a penultimate proline or alanine, including the vasodilator substance P and the metabolite of bradykinin formed by its cleavage by aminopeptidase P. Substance P and bradykinin are normally inactivated by ACE. This study tested the hypothesis that during ACE inhibition, DPP4 inhibition would potentiate the effects of substance P. Ten healthy, lean (BMI<30 kg/m 2 ) subjects, age 24-60 years of old (2 Black, 8 White, 6 Female) participated in this randomized, double-blinded, placebo-controlled crossover study. On separate study days separated by at least one week, Subjects received sitagliptin 200 mg po or matching placebo. Substance P (2, 4, 8 pmol/min) and bradykinin (25, 50, 100 ng/min) were given via the brachial artery before and after administration of intra-arterial enalaprilat (0.33 mcg/min/100mL forearm volume). Enalaprilat potentiated the FBF and tissue plasminogen activator (t-PA) responses to bradykinin and there was no effect of sitagliptin (Figure). Neither ACE inhibition nor DPP4 inhibition altered the FBF flow response to substance P. In contrast, there was an interactive effect of ACE inhibition and DPP4 inhibition on t-PA release in response to substance P (p=0.026).

Inhibition/Depletion of CD26/DPPIV Enhances Survival Promoting Activity of SDF-1/CXCL12 and Myelosuppressive Activities of Inhibitory Chemokines on Hematopoietic Progenitor Cells.

Hematopoiesis is regulated by an interacting network of cells, cell surface proteins, and cytokines/chemokines. CD26 is a cell surface peptidase (Dipeptidylpeptidase IV) that cleaves the N-terminal dipeptide from various substrates, such as selected members of the chemokine family, at a penultimate proline or alanine residue. CD26 is expressed by hematopoietic stem (HSCs) and progenitor (HPCs) cells, as well as a number of other immature and mature cell types. By truncating the chemokine stromal derived factor-1 (SDF-1/CXCL12), CD26 has been implicated in modulation of chemotaxis, homing and mobilization of HSCs/HPCs. Inhibition of CD26 by small peptides such as Diprotin A, or deletion of CD26 (e.g. cells from CD26 −/− mice or siRNA induced decreases in CD26) manifests in enhanced chemotaxis of HSCs/HPCs to SDF-1/CXCL12, and enhanced homing/engraftment of HSCs in mice. We hypothesized that inhibition of CD26 would manifest in enhanced activities of other functions of SDF-1/CXCL12. Moreover, since CD26 truncates other chemokines, we reasoned that inhibition of CD26 would enhance hematopoietic activities of these other chemokines. SDF-1/CXCL12 enhances survival/anti-apoptosis of HSCs/HPCs. We now report that pretreating mouse bone marrow cells (BMCs) with Diprotin A, whether or not the cells were washed prior to plating, or use of CD26 −/− mouse BMCs in a delayed addition of growth factor setting resulted in enhanced survival of HPCs at concentrations of SDF-1/CXCL12 at least 100-fold less than that which would normally manifest survival enhancing activities. We also assessed the influence of CD26 inhibition or deletion on the myelosuppressive effects of chemokines with known inhibitory activity and those that had not previously shown this activity. Suppression/deletion of CD26 on mouse BMCs greatly enhanced the activity of myelosuppressive chemokines such as is MIP-1α/CCL3 and GCP-2/CXCL6, such that these chemokines demonstrated inhibition of colony formation by multi-cytokine stimulated CFU-GM, BFU-E, and CFU-GEMM, at concentrations of these chemokines at least 100-fold less than that possible without inhibition/deletion of CD26. However, CD26 inhibition/deletion did not change the activity of non-myelosuppressive chemokines such as MIP-1β/CCL4, RANTES/CCL5, or SDF-1/CXCL12 into suppressive molecules. Since survival of HPCs is related to the cell cycle status of HPCs, such that slow or non-cycling HPCs are less sensitive to stresses leading to apoptosis, the enhanced activity of SDF-1/CXCL12 and the myelosuppressive chemokines (which are specifically inhibitory to HPCs in S-phase of the cell cycle) may act as coordinating separate signals to protect HPCs from stress-induced apoptosis. These results demonstrate that CD26 plays a modulating role on different hematopoietic activities mediated by chemokines, and this role for CD26 should be taken into account when describing models of chemokine effects on hematopoiesis, and attempts at modifying these effects.