Reshmi Parameswaran

Integrin alpha4 blockade sensitizes drug resistant pre-B acute lymphoblastic leukemia to chemotherapy

Bone marrow (BM) provides chemoprotection for acute lymphoblastic leukemia (ALL) cells, contributing to lack of efficacy of current therapies. Integrin alpha4 (alpha4) mediates stromal adhesion of normal and malignant B-cell precursors, and according to gene expression analyses from 207 children with minimal residual disease, is highly associated with poorest outcome. We tested whether interference with alpha4-mediated stromal adhesion might be a new ALL treatment. Two models of leukemia were used, one genetic (conditional alpha4 ablation of BCR-ABL1 [p210(+)] leukemia) and one pharmacological (anti-functional alpha4 antibody treatment of primary ALL). Conditional deletion of alpha4 sensitized leukemia cell to nilotinib. Adhesion of primary pre-B ALL cells was alpha4-dependent; alpha4 blockade sensitized primary ALL cells toward chemotherapy. Chemotherapy combined with Natalizumab prolonged survival of NOD/SCID recipients of primary ALL, suggesting adjuvant alpha4 inhibition as a novel strategy for pre-B ALL.

Combination of drug therapy in acute lymphoblastic leukemia with a CXCR4 antagonist

The bone marrow (BM) stromal niche can protect acute lymphoblastic leukemia (ALL) cells against the cytotoxicity of chemotherapeutic agents and is a possible source of relapse. The stromal-derived factor-1 (SDF-1)/CXCR4 axis is a major determinant in the crosstalk between leukemic cells and BM stroma. In this study, we investigated the use of AMD11070, an orally available, small-molecule antagonist of CXCR4, as an ALL-sensitizing agent. This compound effectively blocked stromal-induced migration of human ALL cells in culture and disrupted pre-established adhesion to stroma. To examine how to optimally use this compound in vivo, several combinations with cytotoxic drugs were tested in a stromal co-culture system. The best treatment regimen was then tested in vivo. Mice transplanted with murine Bcr/Abl ALL cells survived significantly longer when treated with a combination of nilotinib and AMD11070. Similarly, immunocompromised mice transplanted with human ALL cells and treated with vincristine and AMD11070 had few circulating leukemic cells, normal spleens and reduced human CD19+ cells in the BM at the termination of the experiment. These results show that combined treatment with AMD11070 may be of significant benefit in eradicating residual leukemia cells at locations where they would otherwise be protected by stroma.

A Functional Receptor for B-Cell–Activating Factor Is Expressed on Human Acute Lymphoblastic Leukemias

B-lineage acute lymphoblastic leukemia (ALL) arises by transformation of a progenitor (pre-B) cell. Cure rates in adults remain low and treatment is complicated by support provided by the microenvironment to the leukemic cells, indicating an urgent need to better understand the factors that promote their survival. B-cell-activating factor (BAFF) and its receptor BAFF-R are important for survival and growth of mature normal and malignant B cells but are not expressed on pre-B cells. Unexpectedly, all cells in the primary Philadelphia chromosome (Ph)-positive and Ph-negative ALL samples tested were positive for high BAFF-R cell surface expression. BAFF-R was fully competent to bind BAFF, and stimulation of the receptor activated both the classic and the noncanonical NF-kappaB pathways. Recombinant BAFF supported survival of the ALL cells in the absence of stroma, and it significantly attenuated the rate of apoptosis caused by exposure to nilotinib, a drug used therapeutically to treat Ph-positive ALLs. Surprisingly, BAFF mRNA and protein were also expressed in the same cells but BAFF was not shed into the medium. Our report is the first showing universal expression of BAFF-R by pre-B ALL cells and opens the possibility of blocking its function as an adjuvant therapeutic strategy.

The mesenchyme expresses T cell receptor mRNAs: Relevance to cell growth control

The mesenchyme plays a crucial regulatory role in organ formation and maintenance. However, comprehensive molecular characterization of these cells is lacking. We found unexpectedly that primary mesenchyme, as well as mesenchymal cell clones, express T cell receptor (TCR)αβ mRNAs, lacking the variable region. Immunological and genetic evidence support the expression of a corresponding TCRβ protein. Additionally, mRNAs encoding TCR complex components including CD3 and ζ chain are present. A relatively higher expression of the mesenchymal TCRβ mRNA by cultured mesenchymal cell clones correlates with fast growth, whereas poorly expressing cells are slow growers and are contact inhibited. The clones that express relatively higher amount of the TCR mRNA exhibit an increased capacity to form tumors in nude mice. However, the expression of this mRNA in the mesenchyme is not per se leading to tumorigenesis, as demonstrated by primary mesenchyme that does not form tumors in mice while expressing moderate amounts of the TCR transcripts. The expression of mesencymal TCRβ was confined to the G2/M phases of the cell cycle in the MBA-13 mesenchymal cell line. This cell cycle dependent expression, considered together with the correlation between growth properties and the level of TCR expression by cell clones, implies association of mesenchymal TCR with cell growth control.

Repression of GSK3 restores NK cell cytotoxicity in AML patients

Natural killer cells from acute myeloid leukaemia patients (AML-NK) show a dramatic impairment in cytotoxic activity. The exact reasons for this dysfunction are not fully understood. Here we show that the glycogen synthase kinase beta (GSK3β) expression is elevated in AML-NK cells. Interestingly, GSK3 overexpression in normal NK cells impairs their ability to kill AML cells, while genetic or pharmacological GSK3 inactivation enhances their cytotoxic activity. Mechanistic studies reveal that the increased cytotoxic activity correlates with an increase in AML-NK cell conjugates. GSK3 inhibition promotes the conjugate formation by upregulating LFA expression on NK cells and by inducing ICAM-1 expression on AML cells. The latter is mediated by increased NF-κB activation in response to TNF-α production by NK cells. Finally, GSK3-inhibited NK cells show significant efficacy in human AML mouse models. Overall, our work provides mechanistic insights into the AML-NK dysfunction and a potential NK cell therapy strategy.

O-acetylated N-acetylneuraminic acid as a novel target for therapy in human pre-B acute lymphoblastic leukemia

Removal of 9-O-acetyl residues from the cell surface N-acetylneuraminic acid makes ALL cells drug sensitive.

Effector-Mediated Eradication of Precursor B Acute Lymphoblastic Leukemia with a Novel Fc-Engineered Monoclonal Antibody Targeting the BAFF-R

B-cell activating factor receptor (BAFF-R) is expressed on precursor B acute lymphoblastic leukemia (pre-B ALL) cells, but not on their pre-B normal counterparts. Thus, selective killing of ALL cells is possible by targeting this receptor. Here, we have further examined therapeutic targeting of pre-B ALL based on the presence of the BAFF-R. Mouse pre-B ALL cells lacking BAFF-R function had comparable viability and proliferation to wild-type cells, but were more sensitive to drug treatment in vitro. Viability of human pre-B ALL cells was further reduced when antibodies to the BAFF-R were combined with other drugs, even in the presence of stromal protection. This indicates that inhibition of BAFF-R function reduces fitness of stressed pre-B ALL cells. We tested a novel humanized anti–BAFF-R monoclonal antibody optimalized for FcRγIII-mediated, antibody-dependent cell killing by effector cells. Antibody binding to human ALL cells was inhibitable, in a dose-dependent manner, by recombinant human BAFF. There was no evidence for internalization of the antibodies. The antibodies significantly stimulated natural killer cell–mediated killing of different human patient-derived ALL cells. Moreover, incubation of such ALL cells with these antibodies stimulated phagocytosis by macrophages. When this was tested in an immunodeficient transplant model, mice that were treated with the antibody had a significantly decreased leukemia burden in bone marrow and spleen. In view of the restricted expression of the BAFF-R on normal cells and the multiple anti–pre-B ALL activities stimulated by this antibody, a further examination of its use for treatment of pre-B ALL is warranted. Mol Cancer Ther; 13(6); 1567–77. ©2014 AACR.

Treatment of acute lymphoblastic leukemia with an rGel/BLyS fusion toxin

Acute lymphoblastic leukemia (ALL) is the most common malignancy affecting children and a major cause of mortality from hematopoietic malignancies in adults. A substantial number of patients become drug resistant during chemotherapy, necessitating the development of alternative modes of treatment. rGel (recombinant Gelonin)/BlyS (B-lymphocyte stimulator) is a toxin-cytokine fusion protein used for selective killing of malignant B-cells expressing receptors for B-cell-activating factor (BAFF/BLyS) by receptor-targeted delivery of the toxin, Gelonin. Here, we demonstrate that rGel/BLyS binds to ALL cells expressing BAFF receptor (BAFF-R) and upon internalization, it induces apoptosis of these cells and causes downregulation of survival genes even in the presence of stromal protection. Using an immunodeficient transplant model for human ALL, we show that rGel/BLyS prolongs survival of both Philadelphia chromosome-positive and negative ALL-bearing mice. Furthermore, we used AMD3100, a CXCR4 antagonist, to mobilize the leukemic cells protected in the bone marrow (BM) microenvironment and the combination with rGel/BLyS resulted in a significant reduction of the tumor load in the BM and complete eradication of ALL cells from the circulation. Thus, a combination treatment with the B-cell-specific fusion toxin rGel/BLyS and the mobilizing agent AMD3100 could be an effective alternative approach to chemotherapy for the treatment of primary and relapsed ALL.

AMD3100 sensitizes acute lymphoblastic leukemia cells to chemotherapy in vivo

Despite substantial advances in the treatment of acute lymphoblastic leukemia (ALL) during the past four decades, long-term survival remains at approximately 80% for children and 40% for adults. The lack of efficacy of treatment can be partly attributed to the protection provided to the leukemia cells by the bone marrow microenvironment. SDF-1α (CXCL12) is a cytokine produced by bone marrow stromal cells that stimulates the growth of pre-B cells. Its receptor, CXCR4, is expressed on mature and precursor hematopoietic cells. The CXCL12–CXCR4 interaction was shown to be essential for the earliest stages of B-cell development and is also involved in retaining pre-B cells in the bone marrow. Original interest in CXCR4 from a therapeutic point of view was focused on its role as a co-receptor for human immunodeficiency virus, but drugs that block CXCR4, such as AMD3100, can also be used for hematopoietic stem cell mobilization.1 Juarez et al.2, 3 reported that CXCR4 receptor-binding drugs inhibit CXCR4-mediated functions of pre-B ALL cells in vitro and can mobilize transplanted ALL cells into the circulation of mice. However, the effect of combined CXCR4 inhibition with chemotherapy on ALL remains to be determined. We previously showed that AMD3100 is able to block protection provided by a stromal layer to mouse transgenic Bcr/Abl P190 ALL cells treated with Imatinib.4 To test whether these results can be extended in vivo, we performed fluorescence-activated cell sorting analysis on the peripheral blood of leukemic P190 Bcr/Abl ALL (8093)-transplanted mice before and 2 h after intraperitoneal injection with saline or AMD3100, as it has been shown that a single bolus of AMD3100 can mobilize progenitor cells.1 Interestingly, there was a marked increase in circulating leukemic CD45.2+ (8093 ALL) cells after injection of AMD3100 (Figure 1a) but not in mobilized CD45.1+ C57BL/6 cells. This lack of effect on endogenous CD45+ cells may be caused by the suppression of normal hematopoiesis by the presence of large numbers of malignant lymphoblasts in the bone marrow at this stage. Using the same model, we also examined whether AMD3100 is able to repeatedly mobilize ALL cells in leukemic mice over a longer period of time. As shown in Figure 1b, AMD3100 was able to mobilize a similarly large number of cells into the circulation at days 1 and 10, indicating a sustained mobilizing effect of this drug. These results are consistent with those found in murine transplant models of acute promyelocytic leukemia and multiple myeloma .5, 6 To investigate whether their mobilization into the peripheral blood will make ALL cells more vulnerable to drug treatment, we next tested the combination effect of AMD3100 and nilotinib (AMN107) in vivo. Leukemia cells were allowed to proliferate and generate a substantial tumor burden before the start of treatment with phosphate-buffered saline, nilotinib, AMD3100, or a combination of nilotinib and AMD3100 (Figure 1c). As reported previously,7 nilotinib treatment prolonged the survival of mice. AMD3100 treatment alone had no significant beneficial or detrimental effect on survival (Figure 1c). Interestingly, combination therapy using AMD3100 and nilotinib led to significantly prolonged survival in this murine leukemia model compared with treatment with only nilotinib (P<0.05). Figure 1 Combination therapy using AMD3100 and nilotinib prolongs survival of murine ALL transplant recipients. C57Bl mice transplanted with 104 Bcr/Abl P190 ALL 8093 cells were allowed to develop full leukemia within 11–17 days. (a) Fold increase 2 h ... We verified that the AMD3100-mobilized cells included ALL cells by engrafting non-obese diabetic/severe combined immunodeficient mice with the human pre-B ALL cell line 697. As shown in Figure 2a, the increased white blood cells at 2 h after injection with AMD3100 (top) correlated with a significant increase in the peripheral blood of cells expressing human CD19 (bottom), a marker that is highly expressed in this ALL (>99%, data not shown). We next tested the combined treatment of chemotherapy and AMD3100 in vivo using primary human US7R, a Philadelphia chromosome-negative ALL. At 12 days after transplant with US7R, mice were started on treatment with phosphate-buffered saline, AMD3100, VDL (Vincristine, Dexamethasone, -asparaginase) or AMD3100 plus VDL. The untreated control group died rapidly 27 days post-leukemia injection (Figure 2b). Interestingly, mice treated with VDL plus AMD3100 (MST=61.5 days) survived significantly longer compared with those treated with VDL alone (MST=54 days; P=0.015) or AMD3100 alone (MST=27 days; P=0.0022). Treatment with AMD3100 appeared to be well tolerated, as indicated by continuous weight gain in the treatment groups (data not shown). Figure 2 Preclinical evaluation of AMD3100 as an adjuvant treatment for human drug-resistant ALL. (a) Mobilization of human ALL cells. At 12 days after transplant of human pre-B ALL 697 (6 × 106 cells/mouse) NOD/SCID/IL2Rγ−/− mice ... The concept of using mobilizing agents to bring ALL cells into the circulation in which they can be more effectively treated with other drugs had not been tested in vivo, although Juarez et al.2 did show that AMD3100 enhanced the cytotoxic and anti-proliferative effects of vincristine and dexamethasone in pre-B ALL cells in culture. In AML, AMD3465, a compound related to AMD3100, enhanced the anti-leukemic effects of chemotherapy and sorafenib in mouse transplant models.8 Use of AMD3100 with Ara-C or with bortezomib in acute promyelocytic leukemia or multiple myeloma also showed that combination treatment sensitized these cancer cells to the therapeutic drug in mouse models.5, 6 Moreover, there are currently ongoing Phase clinical I/II trials (http://clinicaltrials.gov identifier: NCT00512252) for the study of AMD3100 in relapsed or refractory acute myelogenous leukemia in combination with chemotherapy with mitoxantrone, etoposide and cytarabine. In view of the fact, that our studies were performed in models of very advanced primary ALL, in which the animals were allowed to accumulate a substantial tumor burden before treatment was initiated, combined with the fact that the human US7R cells are largely unresponsive to the therapeutic drug combination VDL (results not shown); the effect of AMD3100 combined with a second drug can be regarded as very promising. Thus, clinical trials that test the usefulness of combination treatment with CXCR4 antagonists for therapy of relapsed or high-risk ALL appear to be warranted.

Deficiency of Nuclear Factor-κB c-Rel Accelerates the Development of Autoimmune Diabetes in NOD Mice

The nuclear factor-κB protein c-Rel plays a critical role in controlling autoimmunity. c-Rel–deficient mice are resistant to streptozotocin-induced diabetes, a drug-induced model of autoimmune diabetes. We generated c-Rel–deficient NOD mice to examine the role of c-Rel in the development of spontaneous autoimmune diabetes. We found that both CD4+ and CD8+ T cells from c-Rel–deficient NOD mice showed significantly decreased T-cell receptor–induced IL-2, IFN-γ, and GM-CSF expression. Despite compromised T-cell function, c-Rel deficiency dramatically accelerated insulitis and hyperglycemia in NOD mice along with a substantial reduction in T-regulatory (Treg) cell numbers. Supplementation of isogenic c-Rel–competent Treg cells from prediabetic NOD mice reversed the accelerated diabetes development in c-Rel–deficient NOD mice. The results suggest that c-Rel–dependent Treg cell function is critical in suppressing early-onset autoimmune diabetogenesis in NOD mice. This study provides a novel natural system to study autoimmune diabetes pathogenesis and reveals a previously unknown c-Rel–dependent mechanistic difference between chemically induced and spontaneous diabetogenesis. The study also reveals a unique protective role of c-Rel in autoimmune diabetes, which is distinct from other T-cell–dependent autoimmune diseases such as arthritis and experimental autoimmune encephalomyelitis, where c-Rel promotes autoimmunity.