The CXCR4-SDF-1 axis plays a central role in the trafficking and retention of normal and malignant stem cells in the bone marrow (BM) microenvironment. Here, we used a mouse model of acute promyelocytic leukemia (APL) and a small molecule competitive antagonist of CXCR4, AMD3100, to examine the interaction of mouse APL cells with the BM microenvironment. APL cells from a murine cathepsin G-PML-RAR␣ knockin mouse were genetically modified with firefly luciferase (APL luc ) to allow tracking by bioluminescence imaging. Coculture of APL luc cells with M2-10B4 stromal cells protected the leukemia cells from chemotherapy-induced apoptosis in vitro. Upon injection into syngeneic recipients, APL luc cells rapidly migrated to the BM followed by egress to the spleen then to the peripheral blood with death due to leukostasis by day 15. Administration of AMD3100 to leukemic mice induced a 1.6-fold increase in total leukocytes and a 9-fold increase of circulating APL blast counts, which peak at 3 hours and return to baseline by 12 hours. Treatment of leukemic mice with chemotherapy plus AMD3100 resulted in decreased tumor burden and improved overall survival compared with mice treated with chemotherapy alone. These studies provide a proof-of-principle for directing therapy to the critical tethers that promote AMLniche interactions. (Blood. 2009;113: 6206-6214) IntroductionHematopoietic stem cells (HSCs) reside in the bone marrow (BM) and interact with a highly organized microenvironment composed of a diverse population of stromal cells and an extracellular matrix rich in fibronectin, collagens, and various proteoglycans. The interaction between HSCs and the BM microenvironment is critical in regulating HSC processes such as trafficking, self-renewal, proliferation, and differentiation.Egress (mobilization) of stem cells from the BM to the peripheral blood can be induced by cytokines (G-CSF and GM-CSF), chemokines (Gro-␤ and IL-8), or by small molecule inhibitors of both CXCR4 and VLA-4. 1 Interaction between the chemokine, SDF-1, and its cognate receptor CXCR4 functions as a key regulator of stem cell mobilization and trafficking. 2,3 Constitutive secretion of SDF-1 by marrow stromal cells creates a gradient by which HSCs expressing the receptor CXCR4 home to and interact with its marrow niche. 4 In response to factors such as G-CSF, SDF-1 production is down-regulated by stromal cells that release HSCs into the peripheral circulation. 5 AMD3100 is a bicyclam molecule that potently, selectively, and reversibly antagonizes the binding of SDF-1 to CXCR4. 6 In multiple clinical studies, AMD3100 rapidly and effectively mobilizes HSCs into the peripheral circulation and is currently under development as a stem cell mobilization agent prior to high-dose chemotherapy for multiple myeloma, non-Hodgkin lymphoma, and other hematologic malignancies. [7][8][9] In acute myeloid leukemia (AML), the bone marrow microenvironment provides the primary site of minimal residual disease after chemotherapy. [10][11][12] Similar to normal HSCs, AM...

Regulatory T cells (Tregs IntroductionAllogeneic stem cell transplantation (SCT) represents the most effective treatment for patients with marrow failure states and other hematologic malignancies such as acute and chronic leukemias. One of the major complications of allogeneic SCT is graft-versushost disease (GVHD), caused by donor T cells reacting against host antigens. 1 This acute inflammatory reaction can be mild, moderate, or life-threatening especially in recipients of unrelated or human leukocyte antigen-mismatched stem cell products. 2 However, these same alloreactive donor T cells provide a beneficial graft-versusleukemia (GVL) effect, reducing the risk of leukemia relapse. 3,4 Therefore, the current clinical goal in treatment of GVHD is to preferentially suppress GVHD while maintaining GVL.Regulatory T cells (Tregs) are known to contribute to the maintenance of self-tolerance by regulating inflammatory responses and to suppression of autoimmunity and GVHD in mouse models. [5][6][7][8][9] The major population of Tregs is naturally occurring Tregs or nTregs. They are generated in the thymus and defined by CD4 ϩ CD25 ϩ FOXP3 ϩ . [5][6][7][8] Small number of Tregs can also be generated in the periphery from naive CD4 ϩ CD25 Ϫ T cells by T cell-receptor stimulation along with retinoic acid, TGF-␤, and IL-10. 10,11 Because Tregs can also mitigate GVHD by suppressing alloreactive donor T cells without sacrificing GVL in animal models, their use in the allogeneic transplantation setting provides a promising strategy to treat or mitigate GVHD. 9 However, circulating numbers of Tregs in peripheral blood are limited (5%-10% of CD4 ϩ T cells), and despite significant improvements in methodologies for in vitro purification of Tregs, the current protocols for in vitro Treg expansion are inefficient, costly, and time-consuming. 12-15 Furthermore, the lack of Treg-specific cell surface markers makes it impossible to purify Tregs expanded in vitro, and expanded Tregs often fail to maintain their suppressor function, 13,16 possibly due to the loss of expression of FOXP3 and/or chemokine receptors, such as CXCR3, 17 CCR6, 18 and CCR8 19 that facilitate trafficking of Tregs to sites of inflammation.FOXP3 is a forkhead box transcription factor exclusively expressed in nTregs. [5][6][7][8] Its mutations lead to autoimmune diseases due to the loss of functional nTregs and forced expression of FOXP3 in CD4 ϩ CD25 Ϫ T cells induces regulatory properties. 5,7,8,[20][21][22] These data suggest that Foxp3 is necessary and sufficient for functional nTregs. Recent reports demonstrated that the Foxp3 locus in both humans and mice is unmethylated in Tregs while heavily methylated and silenced in CD4 ϩ CD25 Ϫ T cells. [23][24][25] Dec and AzaC, analogues of 2Ј-deoxycytidine and cytidine, respectively, are hypomethylating agents that the FDA approved for the treatment of myelodysplastic syndromes. Dec can incorporate into replicating DNA, while AzaC incorporates primarily into RNA with some integration into DNA after 5-aza-ribonucleoti...

Transgenic mice expressing PML-RAR␣ in early myeloid cells under control of human cathepsin G regulatory sequences all develop a myeloproliferative syndrome, but only 15% to 20% develop acute promyelocytic leukemia (APL) after a latent period of 6 to 14 months. However, this transgene is expressed at very low levels in the bone marrow cells of transgenic mice. Because the transgene includes only 6 kb of regulatory sequences from the human cathepsin G locus, we hypothesized that sequences required for high-level expression of the transgene might be located elsewhere in the cathepsin G locus and that a knock-in model might yield much higher expression levels and higher penetrance of disease. We, therefore, targeted a human PML-RAR␣ cDNA to the 5 untranslated region of the murine cathepsin G gene, using homologous recombination in embryonic stem cells. This model produced a high-penetrance APL phenotype, with more than 90% of knock-in mice developing APL between 6 and 16 months of age. The latent period and phenotype of APL (including a low frequency of an interstitial deletion of chromosome 2) was similar to that of the previous transgenic model. Remarkably, however, the expression level of PML-RAR␣ in bone marrow cells or APL cells was less than 3% of that measured in the low-penetrance transgenic model. Although the explanation for this result is not yet clear, one hypothesis suggests that very low levels of PML-RAR␣ expression in early myeloid cells may be optimal for the development

Here we show that interruption of the VCAM-1/VLA-4 axis with a small molecule inhibitor of VLA-4, BIO5192, results in a 30-fold increase in mobilization of murine hematopoietic stem and progenitors (HSPCs) over basal levels. An additive affect on HSPC mobilization (3-fold) was observed when plerixafor (AMD3100), a small molecule inhibitor of the CXCR-4/SDF-1 axis, was combined with BIO5192. Furthermore, the combination of granulocyte colony-stimulating factor (G-CSF), BIO5192, and plerixafor enhanced mobilization by 17-fold compared with G-CSF alone. HSPCs mobilized by BIO5192 or the combination of BIO5192 and plerixafor mobilized long-term repopulating cells, which successfully engraft and expand in a multilineage fashion in secondary transplantation recipients.

We have recently reported that interferon gamma receptor deficient (IFNγR−/−) allogeneic donor T cells result in significantly less graft-versus-host disease (GvHD) than wild-type (WT) T cells, while maintaining an anti-leukemia or graft-versus-leukemia (GvL) effect after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We demonstrated that IFNγR signaling regulates alloreactive T cell trafficking to GvHD target organs through expression of the chemokine receptor CXCR3 in alloreactive T cells. Since IFNγR signaling is mediated via JAK1/JAK2, we tested the effect of JAK1/JAK2 inhibition on GvHD. While we demonstrated that pharmacologic blockade of JAK1/JAK2 in WT T cells using the JAK1/JAK2 inhibitor, INCB018424 (Ruxolitinib), resulted in a similar effect to IFNγR−/− T cells both in vitro (reduction of CXCR3 expression in T cells) and in vivo (mitigation of GvHD after allo-HSCT), it remains to be determined if in vivo administration of INCB018424 will result in preservation of GvL while reducing GvHD. Here, we report that INCB018424 reduces GvHD and preserves the beneficial GvL effect in two different murine MHC-mismatched allo-HSCT models and using two different murine leukemia models (lymphoid leukemia and myeloid leukemia). In addition, prolonged administration of INCB018424 further improves survival after allo-HSCT and is superior to other JAK1/JAK2 inhibitors, such as TG101348 or AZD1480. These data suggest that pharmacologic inhibition of JAK1/JAK2 might be a promising therapeutic approach to achieve the beneficial anti-leukemia effect and overcome HLA-barriers in allo-HSCT. It might also be exploited in other diseases besides GvHD, such as organ transplant rejection, chronic inflammatory diseases and autoimmune diseases.