Rana Baraz

CXCR4 antagonists mobilize childhood acute lymphoblastic leukemia cells into the peripheral blood and inhibit engraftment.

The role of CXCL12 in the bone marrow (BM) homing and growth of B-cell progenitor acute lymphoblastic leukemia (ALL) has been established. However, the effect of modulating CXCL12/CXCR4 interactions on the retention of ALL cells within the supportive BM microenvironment and the expansion and dissemination of ALL cells in vivo has not been examined. We used mouse models of human childhood and murine leukemia and specific peptide and small molecule CXCR4 antagonists to examine the importance of CXCL12/CXCR4 in the development of leukemia in vivo. CXCR4 antagonists mobilized ALL cells into the peripheral blood (PB). Extended administration of CXCR4 antagonists to mice with leukemia resulted in a reduction in the number of leukemic cells in the PB and spleens of animals compared to control treated animals in three of the five cases tested. There was also a marked reduction in the dissemination of ALL cells to extramedullary sites including liver and kidney in all cases where this occurred. Considering the inhibitory effect of stromal layers on the activity of chemotherapeutic agents and the interactive effect of CXCL12 antagonists with chemotherapeutic agents in vitro, this raises the possibility of using these agents to potentiate the effects of current chemotherapy regimens.

Potentiating effects of RAD001 (Everolimus) on vincristine therapy in childhood acute lymphoblastic leukemia

Despite advances in the treatment of acute lymphoblastic leukemia (ALL), the majority of children who relapse still die of ALL. Therefore, the development of more potent but less toxic drugs for the treatment of ALL is imperative. We investigated the effects of the mammalian target of rapamycin inhibitor, RAD001 (Everolimus), in a nonobese diabetic/severe combined immunodeficiency model of human childhood B-cell progenitor ALL. RAD001 treatment of established disease increased the median survival of mice from 21.3 days to 42.3 days (P < .02). RAD001 together with vincristine significantly increased survival compared with either treatment alone (P < .02). RAD001 induced a cell-cycle arrest in the G(0/1) phase with associated dephosphorylation of the retinoblastoma protein, and reduced levels of cyclin-dependent kinases 4 and 6. Ultrastructure analysis demonstrated the presence of autophagy and limited apoptosis in cells of RAD001-treated animals. In contrast, cleaved poly(ADP-ribose) polymerase suggested apoptosis in cells from animals treated with vincristine or the combination of RAD001 and vincristine, but not in those receiving RAD001 alone. In conclusion, we have demonstrated activity of RAD001 in an in vivo leukemia model supporting further clinical development of target of rapamycin inhibitors for the treatment of patients with ALL.

Sphingosine-1-phosphate facilitates trafficking of hematopoietic stem cells and their mobilization by CXCR4 antagonists in mice

CXCL12 and VCAM1 retain hematopoietic stem cells (HSCs) in the BM, but the factors mediating HSC egress from the BM to the blood are not known. The sphingosine-1-phosphate receptor 1 (S1P(1)) is expressed on HSCs, and S1P facilitates the egress of committed hematopoietic progenitors from the BM into the blood. In the present study, we show that both the S1P gradient between the BM and the blood and the expression of S1P(1) are essential for optimal HSC mobilization by CXCR4 antagonists, including AMD3100, and for the trafficking of HSCs during steady-state hematopoiesis. We also demonstrate that the S1P(1) agonist SEW2871 increases AMD3100-induced HSC and progenitor cell mobilization. These results suggest that the combination of a CXCR4 antagonist and a S1P(1) agonist may prove to be sufficient for mobilizing HSCs in normal donors for transplantation purposes, potentially providing a single mobilization procedure and eliminating the need to expose normal donors to G-CSF with its associated side effects.

Defective p38 Mitogen-Activated Protein Kinase Signaling Impairs Chemotaxic but not Proliferative Responses to Stromal-Derived Factor-1α in Acute Lymphoblastic Leukemia

The chemokine stromal-derived factor-1alpha (SDF-1alpha) regulates leukemic cell motility and proliferation; however, the importance of these functions in the growth and dissemination of leukemia is unclear. We examined SDF-1alpha-mediated responses of cells from 27 cases of acute lymphoblastic leukemia (ALL). Although cells from the majority of cases showed chemotactic and proliferative responses to SDF-1alpha, a subset of cases did not undergo chemotaxis in response to SDF-1alpha, while still demonstrating dependence on SDF-1alpha for proliferation in stroma-supported cultures. This chemotactic defect was associated with an absence of phosphorylation of p38 mitogen-activated protein kinase (MAPK) induced by SDF-1alpha, and of SDF-1alpha-induced augmentation of beta(1) integrin-mediated adhesion. Signaling through phosphoinositide 3-kinase and MEK was not affected. No correlation was observed between CXCR4 expression and chemotactic function, in vitro migration into bone marrow stromal layers, and engraftment of leukemic cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. This study suggests that signaling through p38 MAPK is required for ALL cell chemotaxis but not for proliferation, and that the loss of a chemotactic response to SDF-1alpha does not impede engraftment in NOD/SCID mice.

CXCR4 mediates the homing of B cell progenitor acute lymphoblastic leukaemia cells to the bone marrow via activation of p38MAPK

The mechanisms regulating the migration of leukaemic cells between the blood and bone marrow compartments remain obscure, but are of fundamental importance for the dissemination of the disease. This study investigated the in vivo homing of human B cell progenitor acute lymphoblastic leukaemia (ALL) cells to the femoral bone marrow of non‐obese diabetic severe combined immunodeficient (NOD/SCID) mice. It was demonstrated that patient ALL cells use the chemokine axis, chemokine (CXC motif) receptor 4 (CXCR4)/ chemokine (CXC motif) ligand 12 (CXCL12), to home to the femoral marrow. CXCL12‐mediated signalling through p38 mitogen‐activated protein kinase (MAPK) was required for optimal homing. In contrast, the homing of normal peripheral blood CD34+ cells and the cytokine‐dependent CD34+ cell line Mo7e was independent of p38MAPK, consistent with the dependence of these cells, as well as normal CD34+ CD19+ B cell progenitors, on PI‐3K/AKT signalling. Altogether, our data provide clarification of the direct role of CXCL12 in the bone marrow homing of ALL cells and demonstrate unique signalling molecule usage that may have therapeutic implications for this disease.

RAD001 (everolimus) induces dose-dependent changes to cell cycle regulation and modifies the cell cycle response to vincristine

More than 50% of adults and ∼20% of children with pre-B acute lymphoblastic leukemia (ALL) relapse following treatment. Dismal outcomes for patients with relapsed or refractory disease mandate novel approaches to therapy. We have previously shown that the combination of the mTOR inhibitor RAD001 (everolimus) and the chemotherapeutic agent vincristine increases the survival of non-obese diabetic/severe combined immuno-deficient (NOD/SCID) mice bearing human ALL xenografts. We have also shown that 16 μM RAD001 synergized with agents that cause DNA damage or microtubule disruption in pre-B ALL cells in vitro. Here, we demonstrate that RAD001 has dose-dependent effects on the cell cycle in ALL cells, with 1.5 μM RAD001 inhibiting pRb, Ki67 and PCNA expression and increasing G0/1 cell cycle arrest, whereas 16 μM RAD001 increases pRb, cyclin D1, Ki67 and PCNA, with no evidence of an accumulation of cells in G0/1. Transition from G2 into mitosis was promoted by 16 μM RAD001 with reduced phosphorylation of cdc2 in cells with 4 N DNA content. However, 16 μM RAD001 preferentially induced cell death in cells undergoing mitosis. When combined with vincristine, 16 μM RAD001 reduced the vincristine-induced accumulation of cells in mitosis, probably as a result of increased death in this population. Although 16 μM RAD001 weakly activated Chk1 and Chk2, it suppressed strong vincristine-induced activation of these cell cycle checkpoint regulators. We conclude that RAD001 enhances chemosensitivity at least in part through suppression of cell cycle checkpoint regulation in response to vincristine and increased progression from G2 into mitosis.

Para-NO-aspirin inhibits NF-κB and induces apoptosis in B-cell progenitor acute lymphoblastic leukemia

Although patients with acute lymphoblastic leukemia (ALL) usually achieve complete remission, disease relapse is common and difficult to treat. Para-NO-aspirin (para-NO-ASA) is a novel drug with demonstrated efficacy against a number of solid tumors and most recently chronic lymphocytic leukemia. In this study, we used ALL cell lines to assess the effects on cell viability by flow cytometry and investigated the mechanism of cell death using chemical inhibitors of key molecules and assessed the effects by flow cytometry, electrophoretic mobility shift assay, Western blotting, and quantitative reverse transcription polymerase chain reaction. Para-NO-ASA induced cell death in the pre-B ALL cell lines in association with increased reactive oxygen species, and suppression of nuclear factor-κB (NF-κB) activity. Chemical inhibitors of NF-κB similarly induced apoptosis in ALL cells, suggesting a role for suppression of NF-κB in para-NO-ASA-induced cell death. Modulation of NF-κB was not via regulation of IκB but potentially through suppression of ROCK1 and loss of reduced glutathione. Our results demonstrate that para-NO-ASA potently induces apoptosis in B-lineage ALL cells via a reactive oxygen species-dependent mechanism that is associated with suppression of NF-κB activity.

mTOR inhibition by everolimus in childhood acute lymphoblastic leukemia induces caspase-independent cell death.

Increasingly, anti-cancer medications are being reported to induce cell death mechanisms other than apoptosis. Activating alternate death mechanisms introduces the potential to kill cells that have defects in their apoptotic machinery, as is commonly observed in cancer cells, including in hematological malignancies. We, and others, have previously reported that the mTOR inhibitor everolimus has pre-clinical efficacy and induces caspase-independent cell death in acute lymphoblastic leukemia cells. Furthermore, everolimus is currently in clinical trial for acute lymphoblastic leukemia. Here we characterize the death mechanism activated by everolimus in acute lymphoblastic leukemia cells. We find that cell death is caspase-independent and lacks the morphology associated with apoptosis. Although mitochondrial depolarization is an early event, permeabilization of the outer mitochondrial membrane only occurs after cell death has occurred. While morphological and biochemical evidence shows that autophagy is clearly present it is not responsible for the observed cell death. There are a number of features consistent with paraptosis including morphology, caspase-independence, and the requirement for new protein synthesis. However in contrast to some reports of paraptosis, the activation of JNK signaling was not required for everolimus-induced cell death. Overall in acute lymphoblastic leukemia cells everolimus induces a cell death that resembles paraptosis.

Silencer of Death Domains Controls Cell Death through Tumour Necrosis Factor-Receptor 1 and Caspase-10 in Acute Lymphoblastic Leukemia

Resistance to apoptosis remains a significant problem in drug resistance and treatment failure in malignant disease. NO-aspirin is a novel drug that has efficacy against a number of solid tumours, and can inhibit Wnt signaling, and although we have shown Wnt signaling to be important for acute lymphoblastic leukemia (ALL) cell proliferation and survival inhibition of Wnt signaling does not appear to be involved in the induction of ALL cell death. Treatment of B lineage ALL cell lines and patient ALL cells with NO-aspirin induced rapid apoptotic cell death mediated via the extrinsic death pathway. Apoptosis was dependent on caspase-10 in association with the formation of the death-inducing signaling complex (DISC) incorporating pro-caspase-10 and tumor necrosis factor receptor 1 (TNF-R1). There was no measurable increase in TNF-R1 or TNF-α in response to NO-aspirin, suggesting that the process was ligand-independent. Consistent with this, expression of silencer of death domain (SODD) was reduced following NO-aspirin exposure and lentiviral mediated shRNA knockdown of SODD suppressed expansion of transduced cells confirming the importance of SODD for ALL cell survival. Considering that SODD and caspase-10 are frequently over-expressed in ALL, interfering with these proteins may provide a new strategy for the treatment of this and potentially other cancers.