Laura A. Paganessi

Mesenchymal stem cells from the Wharton's jelly of umbilical cord segments provide stromal support for the maintenance of cord blood hematopoietic stem cells during long-term ex vivo culture

BACKGROUND: Hematopoietic stem cells (HSCs) are routinely obtained from marrow, mobilized peripheral blood, and umbilical cord blood. Mesenchymal stem cells (MSCs) are traditionally isolated from marrow. Bone marrow–derived MSCs (BM‐MSCs) have previously demonstrated their ability to act as a feeder layer in support of ex vivo cord blood expansion. However, the use of BM‐MSCs to support the growth, differentiation, and engraftment of cord blood may not be ideal for transplant purposes. Therefore, the potential of MSCs from a novel source, the Whartons jelly of umbilical cords, to act as stromal support for the long‐term culture of cord blood HSC was evaluated.

Effective mobilization of hematopoietic progenitor cells in G-CSF mobilization defective CD26−/− mice through AMD3100-induced disruption of the CXCL12-CXCR4 axis

OBJECTIVE We previously reported that inhibition or loss of CD26 (DPPIV/dipeptidylpeptidase IV) results in a defect in normal mobilization of hematopoietic stem and progenitor cells induced by granulocyte-colony stimulating factor (G-CSF). This suggests that CD26 is a necessary component of the mobilization pathway. Our goal in this study was to determine whether mobilization can be induced by the CXCR4 antagonist AMD3100 in mice lacking CD26 (CD26(-/-)). MATERIALS AND METHODS Ten week old CD26(-/-) and C57BL/6 mice received a subcutaneous injection of AMD3100. One hour post-injection the mice were euthanized and peripheral blood and bone marrow were collected and evaluated. RESULTS AMD3100 mobilizes hematopoietic progenitors into the peripheral blood of CD26(-/-) and mice. CONCLUSIONS Our finding that AMD3100 rapidly mobilizes hematopoietic progenitor cells from the bone marrow into the periphery in CD26-deficient transgenic mice that otherwise exhibit a mobilization defect in response to G-CSF suggests that: (1) CD26 is downstream of G-CSF but upstream of the CXCL12-CXCR4 axis and (2) AMD3100 can be used as a single agent to mobilize hematopoietic stem and progenitor cells in normal donors or patients that have an intrinsic defect in their response to G-CSF treatment. Stem cell transplants are often the only curative treatment in some cancer patients. The ability to perform the transplantation and its success is dependent on the ability to mobilize adequate numbers of hematopoietic progenitor cells. The use of AMD3100 as a single agent would give patients or donors an additional option for a successful stem cell transplant.

Aggressive disease defined by cytogenetics is associated with cytokine dysregulation in CLL/SLL patients.

Early treatment of CLL/SLL does not impact survival‐reflecting limitations in detecting progression early and identifying asymptomatic patients likely to benefit from early treatment. Improved understanding of CLL/SLL biology would identify better prognostic/predictive markers. This study attempts to address these issues by determining the relationship between cytokine aberrations and poor clinical outcomes in CLL/SLL in the context of a genetic–based prognostic model. Fifty‐nine serum cytokines/chemokines were measured in 28 untreated CLL/SLL patients. Patients were stratified as GR or int/PR using cytogenetics. Comparison of CLL/SLL with 28 HCs revealed increased expression of Th2 cytokines (IL‐10, IL‐5, sIL‐2Rα; P≤0.01) and decreased levels of Th1 cytokines (IL‐17, IL‐23, IFN‐γ; P≤0.003). In a multivariate analysis of GR versus int/PR groups, differential expression of sIL‐2Rα maintained significance with increased expression in int/PR CLL/SLL. With median follow‐up of 54.3 months after diagnosis, four patients incurred disease progression, with an IL‐17/sIL‐2Rα model predicting need for treatment in all cases. In summary, specific cytokine signatures are associated with genetically defined aggressive disease and predict need for therapy. This suggests utility in detecting disease progression early, identifying those likely to incur a survival advantage with early treatment, and directing future therapy.

In vivo expansion of the megakaryocyte progenitor cell population in adult CD26-deficient mice

OBJECTIVE Megakaryopoiesis involves commitment of hematopoietic stem cells (HSC) toward the myeloid lineage in combination with the proliferation, maturation, and terminal differentiation of progenitors into megakaryocytes. The exact mechanism of megakaryocyte development from HSC is unknown, but growth factors such as thrombopoietin have been identified as critical. Additionally, it has been suggested that the chemokine CXCL12/stromal-cell derived factor-1α has a role in regulating megakaryopoiesis and thrombopoiesis. We recently reported the importance of the extracellular protease CD26 (dipeptidylpeptidase IV) in regulating HSC responses to CXCL12, as well as modulating HSC trafficking into and out of the bone marrow. However, the importance of CD26 for megakaryopoiesis has not been reported. We therefore compared megakaryocyte development between CD26-deficient (CD26(-/-)) mice and C57BL/6 control mice. MATERIALS AND METHODS Adult CD26(-/-) mice and C57BL/6 control mice were evaluated using blood differentials, histological analysis, flow cytometric analysis, and progenitor colony assays. RESULTS Bone marrow from CD26(-/-) mice has a significantly expanded megakaryocyte and megakaryocyte progenitor population compared to control C57BL/6 mice bone marrow. CONCLUSIONS Our results indicate that endogenous CD26 normally suppresses megakaryopoiesis and that loss of CD26 activity results in expansion of the megakaryocyte progenitor population in vivo. This suggests the potential use of CD26 inhibitors to improve megakaryocyte progenitor function and/or reconstitution of the megakaryocyte cell population.

CD26 protease inhibition improves functional response of unfractionated cord blood, bone marrow, and mobilized peripheral blood cells to CXCL12/SDF-1.

Hematopoietic stem cell transplantation (HSCT) is an important treatment option for patients with malignant and nonmalignant hematologic diseases. Methods to improve transplant efficiency are being explored with the intent to improve engraftment and immune reconstitution post-HSCT. A current approach under investigation involves treatment of donor cells with inhibitors that target the protease CD26, a negative regulator of the chemokine CXCL12/stromal cell-derived factor-1. CD26 inhibitor treatment has been shown to improve the functional response of CD34(+) cord blood (CB) cells, but not CD34(+) granulocyte colony-stimulating factor-mobilized peripheral blood stem cells, to CXCL12/stromal cell-derived factor-1. The effect of CD26 inhibitors on unfractionated CB, bone marrow, or granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cells has not been evaluated previously. We observed that although CB had greater CD26 expression than bone marrow or mobilized peripheral blood, treatment with a CD26 inhibitor (Diprotin A) resulted in increased responsiveness to stromal cell-derived factor-1 for all three mononuclear cell sources tested. This suggests that clinical therapeutic benefit might be gained by using CD26 inhibitors as a strategy to improve engraftment of unfractionated mobilized peripheral blood cells as well as CB cells.

Loss of CD26 Protease Activity in Recipient Mice during Hematopoietic Stem Cell Transplantation Results in Improved Transplant Efficiency

BACKGROUND: A firm understanding of the biology of hematopoietic stem and progenitor cell (HSC/HPC) trafficking is critical to improve transplant efficiency and immune reconstitution during hematopoietic stem cell transplantation (HSCT). Our earlier findings suggested that suppression of CD26 (dipeptidyl peptidase IV) proteolytic activity in the donor cell population can be utilized as a method for increasing transplant efficiency. However, factors in the recipient should not be overlooked, given the potential for the bone marrow (BM) microenvironment to regulate HSCT.

Bone Marrow and Peripheral Blood AML Cells Are Highly Sensitive to CNDAC, the Active Form of Sapacitabine

Achieving improvements in survival and reducing relapse remains a challenge in acute myelogenous leukemia (AML) patients. This study evaluated the in vitro efficacy of the active form of novel agent sapacitabine, CNDAC, compared to current chemotherapeutic drugs Ara-C and mitoxantrone using two AML cell lines, HL-60 (promyelocytic) and THP-1 (monocytic), as well as bone marrow (BM) and peripheral blood (PB) cells collected from AML patients. Cell lines were exposed to compound for 3–6 days and primary cells for 4 days. The viability of primary cells was additionally evaluated 3, 7, and 31 days after removal of tested compound to determine the durability of the response. Our studies indicate that CNDAC and mitoxantrone have a greater impact on viability than ara-C in primary AML cells and AML cell lines. CNDAC is more effective at reducing viability and inducing apoptosis than ara-C at equivalent concentrations in the THP-1 cell line, which is defined as displaying resistance to ara-C. As sapacitabine has shown in vivo activity at clinically achievable doses, future studies are warranted to assess the potential for combining it with ara-C and/or mitoxantrone, with an emphasis on cells and patients insensitive to ara-C treatment.

Abstract 4668: Patient AML cells and AML cell lines are highly sensitive to CNDAC, the active form of sapacitabine

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Achieving improvements in survival and reducing relapse remains a challenge in acute myelogenous leukemia (AML) patients. This study evaluated the in vitro efficacy of the active form of novel agent sapacitabine, 2′-C-Cyano-2′-deoxy-1-α-d-arabino-pentofuranosylcytosine (CNDAC, Cyclacel Ltd, Dundee, UK), as compared to current chemotherapeutic drugs Ara-C (Cytarabine) and Mitoxantrone (Mit, synthetic anthracenedione) using two AML cell lines, HL-60 (promyelocytic) and THP-1 (monocytic), as well as bone marrow (BM) and peripheral blood (PB) cells collected from 5 AML patients. Methods: Cells lines and AML patient cells were treated in vitro with Ara-C (1-100 µM), CNDAC (1-100 µM) or Mit (0.005-0.5 µM) for 4 days. During treatment, HL-60, THP-1, and PB AML cells were cultured in suspension. BM AML cells were co-cultured with M2-10B4 mouse stromal cells. Cell lines were assessed immediately. BM and PB AML cells were assessed after an additional 3, 7, or 31 days of co-culture on M2-10B4 cells. Treated cells were assessed for sensitivity (cell death by trypan blue and apoptosis by 7AAD/Annexin V) as compared to untreated cells and IC50 values (50% of maximum possible effective response) were calculated. Results: In HL-60 cells, the amount of cell death was greater with CNDAC compared to Ara-C at all doses tested (p≤0.05, n=3). In THP-1 cells, CNDAC and Mit, but not Ara-C, induced a significant apoptotic response. At a 10-fold lower seeding density, which correlates to higher proliferation rates, the response of THP-1 cells to Ara-C, CNDAC and Mit reached significance compared to untreated cells (p≤0.05, n=3). However, the IC50 values for the 3 drugs in THP-1 cells reflect the observation that this cell line is in essence resistant to AraC (IC50 = 7.77 µM) but sensitive to CNDAC (IC50 = 0.929 µM) and Mit (IC50 = 0.003 µM). Using PB AML cells, a significant response to 1 µM CNDAC and 0.005 µM Mit but not 1 µM Ara-C was observed, as compared to untreated cells at all days post drug removal (p≤0.05, n=5). A significantly greater apoptotic response to CNDAC was observed compared to Ara-C at low doses (p≤0.05, n=5). At higher doses, all 3 drugs induced significant cell death (p≤0.05, n=5). In BM AML cells, overall survival with 1 µM CNDAC or 0.005 µM Mit, but not 1 µM Ara-C, was significantly less than untreated cells 31 days post-treatment (p≤0.05, n=5). Higher doses induced cell death (p≤0.05, n=5) with all 3 drugs. Conclusion: Low dose CNDAC and Mitoxantrone induce a greater response than low dose Ara-C in patient AML cells and AML cell lines. CNDAC also exhibits a greater activity in cell lines (THP-1) that are less sensitive to Ara-C. The in vitro sensitivity of AML cells to CNDAC supports the ongoing clinical evaluation of sapacitabine in AML patients. Future studies are warranted to assess the potential for combining sapacitabine with Ara-C and/or Mitoxantrone, with an emphasis on cells and patients insensitive to Ara-C treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4668. doi:1538-7445.AM2012-4668