Sphingosine kinase 2 supports the development of BCR/ABL-independent acute lymphoblastic leukemia in mice.

BACKGROUND: Sphingosine kinase (SphK) 2 has been implicated in the development of a range of cancers and inhibitors of this enzyme are currently in clinical trial. We have previously demonstrated a role for SphK2 in the development of acute lymphoblastic leukemia (ALL). METHODS: In this and our previous study we use mouse models: in the previous study the disease was driven by the proto-oncogene BCR/ABL1, while in this study cancer risk was elevated by deletion of the tumor suppressor ARF. RESULTS: Mice lacking ARF and SphK2 had a significantly reduced incidence of ALL compared mice with wild type SphK2. CONCLUSIONS: These results show that the role of SphK2 in ALL development is not limited to BCR/ABL1 driven disease extending the potential use of inhibitors of this enzyme to ALL patients whose disease have driver mutations other than BCR/ABL1.

Regulation of the bone marrow microenvironment by G-CSF: Effects of G-CSF on acute lymphoblastic leukaemia.

It has been suggested that disruption of the lymphoid niche by G-CSF may be of therapeutic benefit to patients with acute lymphoblastic leukaemia. We used a xenograft model to determine the effect of G-CSF on ALL progression in a minimal residual disease setting. Consistent with the effects on normal murine B cell progenitors, G-CSF slowed disease in the majority of ALL xenografts tested, suggesting that G-CSF may provide benefits beyond neutrophil recovery for ALL patients. However, two of eight xenografts demonstrated accelerated disease progression. G-CSF could be detrimental for these patients due to expansion of the malignant clone.

Bone Marrow Graft-Versus-Host Disease in Major Histocompatibility Complex-Matched Murine Reduced-Intensity Allogeneic Hemopoietic Cell Transplantation.

BACKGROUND: Most clinical allogeneic hemopoietic cell transplants (alloHCT) are now performed using reduced-intensity conditioning (RIC) instead of myeloablative conditioning (MAC); however, the biology underlying this treatment remains incompletely understood. METHODS: We investigated a murine model of major histocompatibility complex-matched multiple minor histocompatibility antigen-mismatched alloHCT using bone marrow (BM) cells and splenocytes from B6 (H-2) donor mice transplanted into BALB.B (H-2) recipients after RIC with fludarabine of 100 mg/kg per day for 5 days, cyclophosphamide of 60 mg/kg per day for 2 days, and total body irradiation (TBI). RESULTS: The lowest TBI dose capable of achieving complete donor chimerism in this mouse strain combination was 325 cGy given as a single fraction. Mice that underwent RIC had a reduced incidence and delayed onset of graft-versus-host disease (GVHD) and significantly prolonged survival compared with MAC-transplanted recipients (TBI of 850 cGy plus cyclophosphamide of 60 mg/kg per day for 2 days). Compared with syngeneic controls, RIC mice with GVHD showed evidence of BM suppression, have anemia, reduced BM cellularity, and showed profound reduction in BM B cell lymphopoiesis associated with damage to the endosteal BM niche. This was associated with an increase in BM CD8 effector T cells in RIC mice and elevated blood and BM plasma levels of T helper1 cytokines. Increasing doses of splenocytes resulted in increased incidence of GVHD in RIC mice. CONCLUSIONS: We demonstrate that the BM is a major target organ of GVHD in an informative clinically relevant RIC mouse major histocompatibility complex-matched alloHCT model by a process that seems to be driven by CD8 effector T cells.

Extracellular molecules in hematopoietic stem cell mobilisation.

Hematopoietic stem cells are a remarkable resource currently used for the life saving treatment, hematopoietic stem cell transplantation. Today, hematopoietic stem cells are primarily obtained from mobilized peripheral blood following treatment of the donor with the cytokine G-CSF, and in some settings, chemotherapy and/or the CXCR4 antagonist plerixafor. The collection of hematopoietic stem cells is contingent on adequate and timely mobilization of these cells into the peripheral blood. The use of healthy donors, particularly when unrelated to the patient, requires mobilization strategies be safe for the donor. While current mobilization strategies are largely successful, adequate mobilization fails to occur in a significant portion of donors. Understanding the mechanisms involved in the egress of stem cells from the bone marrow provides opportunities to further improve the process of collecting hematopoietic stem cells. Here, the role extracellular components of the blood and bone marrow in the mobilization process are discussed.

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization.

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

A Phase I/II Study of the mTOR Inhibitor Everolimus in Combination with HyperCVAD Chemotherapy in Patients with Relapsed/Refractory Acute Lymphoblastic Leukemia.

PURPOSE: Previous studies suggest a potential therapeutic role for mTOR inhibition in lymphoid malignancies. This single-center phase I/II study was designed to test the safety and efficacy of the mTOR inhibitor everolimus in combination with HyperCVAD chemotherapy in relapsed/refractory acute lymphoblastic leukemia (ALL). EXPERIMENTAL DESIGN: Twenty-four patients were treated; 15 received everolimus 5 mg/day and 9 received 10 mg/day with HyperCVAD. RESULTS: The median age of patients was 25 years (range, 11-64) and median number of prior treatments was 2 (range, 1-7). Grade 3 mucositis was the dose-limiting toxicity and the maximum tolerated everolimus dose was 5 mg/day. Responses included complete remission (CR) in 6 patients (25%), CR without platelet recovery (CRp) in 1 (4%), and CR without recovery of counts (CRi) in 1 (4%), for an overall response rate of 33%. In addition, partial response (PR) was noted in 2 patients (8%). Seven of 11 patients treated in first salvage achieved CR/CRp (64%). The median OS was 29 weeks for patients in first salvage versus 15 weeks for patients in second salvage and beyond (P ≤ 0.001). A response was noted in 5 of 10 (50%) heavily pretreated T-ALL patients (median of 4 prior salvage regimens). Everolimus significantly inhibited phosphorylation of S6RP, but this did not correlate with response. No significant decreases in p4EBP1 and pAkt levels were noted. Responders had higher everolimus dose-adjusted area under the curve (P = 0.025) and lower clearance (P = 0.025) than nonresponders. CONCLUSIONS: The combination of HyperCVAD and everolimus is well tolerated and moderately effective in relapsed ALL, specifically T-ALL.

Efficacy of dual PI-3K and mTOR inhibitors in vitro and in vivo in acute lymphoblastic leukemia.

The major regulators of human acute lymphoblastic leukemia (ALL) cell growth and survival mediate their effects through the phosphoinositide 3-kinase (PI-3K)/mammalian target of rapamycin (mTOR) pathway. We have shown that the mTOR inhibitor everolimus extended survival in a non-obese diabetic/severe combined immune-deficient (NOD/SCID) mouse xenograft model of human ALL. Since PI-3K has mTOR dependent and independent functions we examined the effect of the dual PI-3K/mTOR inhibitors BEZ235 and BGT226. These agents inhibited the proliferation of ALL cell lines with a three log greater potency than everolimus. However, the induction of cell death differed, with BGT226 being cytotoxic in the low micromolar range while a two log higher concentration of BEZ235 was required to produce the same effect. While all three agents extended the survival of NOD/SCID mice engrafted with human ALL, the responses of individual xenografts varied. Although differential phosphorylation of AKT on Ser(473) and Thr(308) in response to everolimus exposure was observed, this did not entirely explain the different in vivo responses to the drugs. Our data suggests that while dual PI-3K/mTOR inhibitors may improve therapeutic outcomes for a subset of ALL patients, patient selection will be important, with some patients likely to respond better to single mTOR inhibition.

G-CSF: From granulopoietic stimulant to bone marrow stem cell mobilizing agent.

G-CSF was among the first cytokines to be identified and rapidly transitioned into clinical medicine. Initially used to promote the production of neutrophils in patients with chemotherapy-induced neutropenia it helped to revolutionize the delivery of cancer therapy. Its ability to mobilize hematopoietic stem cells from the bone marrow into the blood was subsequently exploited, changing the face of hematopoietic stem cell transplantation. Today the knowledge gained in unraveling the mechanisms of stem cell mobilization by G-CSF is being explored as a means to increase chemosensitivity in hematological malignancies. This review provides a brief history of G-CSF and then focuses on recent advances in our understanding of G-CSF-induced stem cell mobilization and the potential clinical application of this knowledge in chemo-sensitization.

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.

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.

Sphingosine kinase 2 promotes acute lymphoblastic leukemia by enhancing MYC expression.

Sphingosine kinase 2 (SK2) may have utility as a prognostic marker in inflammatory diseases such as cancer in which it has been rationalized as a candidate therapeutic target. Here, we show that SK2 has an oncogenic role in acute lymphoblastic leukemia (ALL) by influencing expression of MYC. Genetic ablation of SK2 impaired leukemia development in a mouse model of ALL and pharmacologic inhibition extended survival in mouse xenograft models of human disease. SK2 attenuation in both the settings reduced MYC expression in leukemic cells, with reduced levels of acetylated histone H3 within the MYC gene associated with reduced levels of MYC protein and expression of MYC-regulated genes. Our results demonstrated that SK2 regulates MYC, which has a pivotal role in hematologic malignancies, providing a preclinical proof of concept for this pathway as a broad-based therapeutic target in this setting.

Role of sphingosine 1-phosphate in trafficking and mobilization of hematopoietic stem cells.

PURPOSE OF REVIEW: The 'mobilization' of hematopoietic stem cells (HSCs) out of the bone marrow and into the peripheral blood is used clinically to obtain HSCs for transplantation. Although generally successful, mobilization protocols remain imperfect and the mechanisms involved are not fully understood. This review discusses the latest findings in respect to the mechanisms involved in the egress of HSCs from the bone marrow into the circulation and the potential for these recent developments to improve mobilization procedures. RECENT FINDINGS: It has recently become apparent that the bioactive lipid sphingosine 1-phosphate (S1P) plays an active role in attracting HSCs into the peripheral blood. S1P is the first factor identified that provides a chemoattractant gradient promoting the movement of HSCs into the peripheral blood. Drugs that mimic S1P are available with others in development, raising the possibility of increasing the strength of the egress signal and thereby improving the efficacy of mobilization procedures. SUMMARY: S1P is the first egress factor described for HSCs, but the details of the underlying biology are only just emerging. Although manipulating the S1P axis to enhance mobilization protocols is an exciting possibility, much needs to be learned before improvements in mobilization strategies can be realized.

Oncogenic properties of sphingosine kinases in haematological malignancies.

The sphingosine kinases (SphKs) have relatively recently been implicated in contributing to malignant cellular processes with particular interest in the oncogenic properties of SPHK1. Whilst SPHK1 has received considerable attention as a putative oncoprotein, SPHK2 has been much more difficult to study, with often conflicting data surrounding its role in cancer. Initial studies focused on non-haemopoietic malignancies, however a growing body of literature on the role of sphingolipid metabolism in haemopoietic malignancies is now emerging. This review provides an overview of the current state of knowledge of the SphKs and the bioactive lipid sphingosine 1-phosphate (S1P), the product of the reaction they catalyse. It then reviews the current literature regarding the roles of S1P and the SphKs in haemopoietic malignancies and discusses the compounds currently available that modulate sphingolipid metabolism and their potential and shortcomings as therapeutic agents for the treatment of haematological malignancies.

B-lymphopoiesis is stopped by mobilizing doses of G-CSF and is rescued by overexpression of the anti-apoptotic protein Bcl2.

Osteoblasts are necessary to B lymphopoiesis and mobilizing doses of G-CSF or cyclophosphamide inhibit osteoblasts, whereas AMD3100/Plerixafor does not. However, the effect of these mobilizing agents on B lymphopoiesis has not been reported. Mice (wild-type, knocked-out for TNF-α and TRAIL, or over-expressing Bcl-2) were mobilized with G-CSF, cyclophosphamide, or AMD3100. Bone marrow, blood, spleen and lymph node content in B cells was measured. G-CSF stopped medullar B lymphopoiesis with concomitant loss of B-cell colony-forming units, pre-pro-B, pro-B, pre-B and mature B cells and increased B-cell apoptosis by an indirect mechanism. Overexpression of the anti-apoptotic protein Bcl2 in transgenic mice rescued B-cell colony forming units and pre-pro-B cells in the marrow, and prevented loss of all B cells in marrow, blood and spleen. Blockade of endogenous soluble TNF-α with Etanercept, or combined deletion of the TNF-α and TRAIL genes did not prevent B lymphopoiesis arrest in response to G-CSF. Unlike G-CSF, treatments with cyclophosphamide or AMD3100 did not suppress B lymphopoiesis but caused instead robust B-cell mobilization. G-CSF, cyclophosphamide and AMD3100 have distinct effects on B lymphopoiesis and B-cell mobilization with: 1) G-CSF inhibiting medullar B lymphopoiesis without mobilizing B cells in a mechanism distinct from the TNF-α-mediated loss of B lymphopoiesis observed during inflammation or viral infections; 2) CYP mobilizing B cells but blocking their maturation; and 3) AMD3100 mobilizing B cells without affecting B lymphopoiesis. These results suggest that blood mobilized with these three agents may have distinct immune properties.

Plerixafor (AMD3100) induces prolonged mobilization of acute lymphoblastic leukemia cells and increases the proportion of cycling cells in the blood in mice.

The CXCR4 antagonist Plerixafor (AMD3100) induces the rapid mobilization of hematopoietic stem and progenitor cells into the blood in mice and humans. AMD3100 similarly induces the mobilization of human acute lymphoblastic leukemia (ALL) cells into the blood in mice. In this study, the temporal response of pre-B ALL cells to AMD3100 was compared with that of normal hematopoietic progenitor cells (HPC) using an NOD/SCID xenograft model of ALL and BALB/c mice, respectively. ALL cells remained in the circulation up to 6 hours after AMD3100 administration, by which time normal HPCs were no longer detectable. AMD3100 also increased the proportion of actively cycling ALL cells in the peripheral blood. Together, these data suggest that ALL cells are more sensitive to the effects of bone marrow disruption than normal progenitors. Using the NOD/SCID xenograft model, we demonstrated that AMD3100 increased the efficacy of the cell cycle specific drug vincristine, resulting in reduced disease levels in the blood and spleens of animals over 3 weeks and extended the survival of NOD/SCID mice with ALL. These data demonstrate that mobilizing agents can increase the therapeutic effect of cell cycle dependent chemotherapeutic agents.

Disparate in vivo efficacy of FTY720 in xenograft models of Philadelphia positive and negative B-lineage acute lymphoblastic leukemia.

Most patients with acute lymphoblastic leukemia (ALL) respond well to standard chemotherapy-based treatments. However a significant proportion of patients, particularly adult patients, relapse with the majority dying of leukemia. FTY720 is an immunosuppressive drug that was recently approved for the treatment of multiple sclerosis and is currently under pre-clinical investigation as a therapy for a number of hematological malignancies. Using human ALL xenografts in NOD/SCIDγc(-/-) mice, we show for the first time that three Ph(+) human ALL xenografts responded to FTY720 with an 80 ± 12% (p = 0.048) reduction in overall disease when treatment was commenced early. In contrast, treatment of mice with FTY720 did not result in reduced leukemia compared to controls using four separate human Ph(-) ALL xenografts. Although FTY720 reactivated PP2A in vitro, this reactivation was not required for death of Ph(-) ALL cells. The plasma levels of FTY720 achieved in the mice were in the high nanomolar range. However, the response seen in the Ph(+) ALL xenografts when treatment was initiated early implies that in vivo efficacy may be obtained with substantially lower drug concentrations than those required in vitro. Our data suggest that while FTY720 may have potential as a treatment for Ph(+) ALL it will not be a useful agent for the treatment of Ph(-) B-ALL.

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.

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.