The chemokine receptor CXCR4 and its cognate ligand CXCL12 recently have been proposed to regulate the directional trafficking and invasion of breast cancer cells to sites of metastases. However, effects of CXCR4 on the growth of primary breast cancer tumors and established metastases and survival have not been determined. We used stable RNAi to reduce expression of CXCR4 in murine 4T1 cells, a highly metastatic mammary cancer cell line that is a model for stage IV human breast cancer. Using noninvasive bioluminescence and magnetic resonance imaging, we showed that knockdown of CXCR4 significantly limited the growth of orthotopically transplanted breast cancer cells. Mice in which parental 4T1 cells were implanted had progressively enlarging tumors that spontaneously metastasized, and these animals all died from metastatic disease. Remarkably, RNAi of CXCR4 prevented primary tumor formation in some mice, and all mice transplanted with CXCR RNAi cells survived without developing macroscopic metastases. To analyze effects of CXCR4 on metastases to the lung, an organ commonly affected by metastatic breast cancer, we injected tumor cells intravenously and monitored cell growth with bioluminescence imaging. Inhibiting CXCR4 with RNAi, or the specific antagonist AMD3100, substantially delayed the growth of 4T1 cells in the lung, although neither RNAi nor AMD3100 prolonged overall survival in mice with experimental lung metastases. These data indicate that CXCR4 is required to initiate proliferation and/or promote survival of breast cancer cells in vivo and suggest that CXCR4 inhibitors will improve treatment of patients with primary and metastatic breast cancer.

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Increased stromal collagen deposition in human breast tumours correlates with metastases. We show that activation of the collagen I receptor DDR2 regulates Snail1 protein stability by stimulating ERK2 activity, in a Src-dependent manner. Activated ERK2 directly phosphorylates Snail1, leading to Snail1 nuclear accumulation, reduced ubiquitination, and increased protein half-life. DDR2-mediated stabilization of Snail1 promotes breast cancer cell invasion and migration in vitro, and metastasis in vivo. DDR2 expression was observed in the majority of human invasive ductal breast carcinomas studied, and was associated with nuclear Snail1 and absence of E-cadherin expression. We propose that DDR2 maintains Snail1 protein level and activity in tumor cells that have undergone EMT, thereby facilitating continued tumor cell invasion through collagen I-rich ECM by sustaining the EMT phenotype. As such, DDR2 could be an RTK target for the treatment of breast cancer metastasis.

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...

The Wnt/β-catenin signaling pathway is activated in breast cancer, a leading cause of cancer mortality in women. Because mutations in the key intracellular components of this pathway are rare, identifying the molecular mechanisms of aberrant Wnt activation in breast cancer is critical for development of pathway-targeted therapy. Here, we show that expression of the Wnt signaling coreceptor LRP6 is upregulated in a subpopulation of human breast cancers. LRP6 silencing in breast cancer cells reduces Wnt signaling, cell proliferation, and in vivo tumor growth. In vivo administration of an LRP6 antagonist, Mesd, markedly suppressed growth of MMTV-Wnt1 tumors without causing undesirable side effects. These results demonstrate that Wnt activation at the cell surface contributes to breast cancer tumorigenesis. Together, our studies highlight LRP6 as a potential therapeutic target in breast cancer, and introduce Mesd as a promising antitumor agent for treating breast cancer subtypes with Wnt activation at the cell surface.

T cell malignancies represent a group of hematologic cancers with high rates of relapse and mortality in patients for whom no effective targeted therapies exist. The shared expression of target antigens between chimeric antigen receptor (CAR) T cells and malignant T cells has limited the development of CAR-T because of unintended CAR-T fratricide and an inability to harvest sufficient autologous T cells. Here we describe a fratricide resistant ‘off-the-shelf’ CAR-T (or UCART7) that targets CD7+ T cell malignancies and, through CRISPR/Cas9 gene editing, lacks both CD7 and T cell receptor alpha chain (TRAC) expression. UCART7 demonstrates efficacy against human T cell acute lymphoblastic leukemia (T-ALL) cell lines and primary T-ALL in vitro and in vivo without the induction of xenogeneic GvHD. Fratricide resistant, allo-tolerant ‘off-the-shelf’ CAR-T represents a strategy for treatment of relapsed and refractory T-ALL and non-Hodgkin’s T cell lymphoma without a requirement for autologous T cells.

The clinical goal of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is to minimize GVHD while maintaining GvL. Here, we show that interferon ␥ receptor-deficient (IFN␥R ؊/؊ ) allogeneic Tconv, which possess normal alloreactivity and cytotoxicity, induce significantly less GVHD than wild-type (WT) Tconv. This effect is mediated by altered trafficking of IFN␥R ؊/؊ Tconv to GVHD target organs, especially the gastrointestinal (GI) tract. We show that the chemokine receptor CXCR3 is induced via IFN␥R-mediated signaling and partially contributes to the trafficking of WT Tconv to GVHD target organs. Indeed, CXCR3 ؊/؊ Tconv recapitulate the reduced GVHD potential of IFN␥R ؊ IntroductionAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment for patients with relapsed/refractory leukemia, and marrow failure states such as myelodysplasia and aplastic anemia. However, the infusion of allogeneic donor T cells (conventional T cells or "Tconv") for allo-HSCT results in 2 distinct biologic effects: graft-versus-host disease (GVHD), which may be mild, moderate, or life-threatening 1,2 ; and a beneficial graft-versus-leukemia (GvL) effect, which results in enhanced leukemia cell clearance. 3,4 Thus, the clinical goal in allo-HSCT is to prevent GVHD while maintaining the beneficial GvL effect. Recent studies have suggested that this might be achieved by infusing regulatory T cells (Tregs), which in some preclinical models suppress GVHD-causing alloreactive Tconv but have only limited effects on GvL-promoting alloreactive Tconv. [5][6][7][8] Unfortunately, Tregs exist in low frequency in the peripheral blood, are difficult to purify and expand, and after expansion are difficult to isolate because of the lack of cell-surface markers, all of which prevent their routine use in the clinic. Thus, alternative therapeutic approaches that do not require Tregs are needed.Interferon ␥ (IFN␥) is a well-known proinflammatory cytokine. Serum levels of IFN␥ after allo-HSCT have been correlated with the severity of GVHD and the treatment of murine allo-HSCT recipients with blocking antibodies to IFN␥ mitigates GVHD. [9][10][11][12] In addition, IFN␥ facilitates T cell-mediated GvL. 11 In contrast, several reports suggest that IFN␥ Ϫ/Ϫ T cells induce more severe GVHD, especially in the lung, than WT T cells when infused into WT MHC-mismatched recipients that are lethally irradiated, [10][11][12][13][14] suggesting that IFN␥ may also have anti-inflammatory properties.Possible mechanisms underlying this anti-inflammatory effect of IFN␥ on lung GVHD have been proposed by several groups. [14][15][16] First, donor T cell-derived IFN␥ prevents allogeneic donor T-cell trafficking and expansion in the lung by inducing PDL1 expression on host lung tissue. 14,15,17 Second, donor T cell-derived IFN␥ induces indoleamine 2,3-dioxygenase (IDO) expression in donor bone marrow-derived dendritic cells, which in turn suppress GVHD. 16 All of these observations suggest that GVHD and GvL can be regulated by modi...