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.

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.

The mammalian target of rapamycin inhibitor RAD001 (everolimus) synergizes with chemotherapeutic agents, ionizing radiation and proteasome inhibitors in pre-B acute lymphocytic leukemia.

BACKGROUND: Despite incremental improvements in outcomes for patients with acute lymphoblastic leukemia, significant numbers of patients still die from this disease. Mammalian target of rapamycin inhibitors have shown potential in vitro and in vivo as therapeutic agents against a range of tumors including acute lymphoblastic leukemia. DESIGN AND METHODS: Flow cytometry was used to evaluate drug-induced cell death in acute lymphoblastic leukemia cell lines and patients' samples. Human xenografts in immunocompromised mice were used to assess the in vivo effects of selected combinations. Pharmacological inhibitors and lentiviral small interfering ribonucleic acid knock-down of p53 were used to investigate the mechanism of cell killing involved. RESULTS: Synergistic interactions between RAD001 and cytotoxic agents were demonstrated in vitro and in vivo, with increased caspase-dependent killing. RAD001 suppressed p53 and p21 responses, while suppression of p53 did not prevent killing, indicating p53 independence. RAD001 and cytotoxic agents activated the JUN N-terminal kinase pathway and the combination further increased JUN N-terminal kinase activation. JUN N-terminal kinase inhibition reduced synergistic cell killing by cytotoxic agents and RAD001 in pre-B acute lymphoblastic leukemia cell lines and patients' samples. Bortezomib and MG132, which activate the JUN N-terminal kinase pathway, also synergized with RAD001 in killing pre-B acute lymphoblastic leukemia cells. Killing was greater when RAD001 was combined with proteasome inhibitors than with cytotoxic drugs. CONCLUSIONS: These observations suggest that combining mammalian target of rapamycin inhibitors with conventional chemotherapy or selected novel agents has the potential to improve clinical responses in patients with pre-B acute lymphoblastic leukemia.

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.

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.

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.

Expression of CD44, but not CD44v6, predicts relapse in children with B cell progenitor acute lymphoblastic leukemia lacking adverse or favorable genetics.

Although significant progress has been made in the treatment of childhood acute lymphoblastic leukemia (ALL) the prognosis following relapse is still poor. Additional prognostic indicators are needed to better target treatment and thereby improve the treatment of these patients. We have previously demonstrated an association between poor outcome and CD44v6 expression in a heterogeneous cohort of patients. Others have shown by microarray analysis that CD44 expression in diagnostic samples is linked with relapse risk. In this study, we examined CD44 and CD44v6 protein expression by flow cytometry and CD44v6 mRNA expression by quantitative RT-PCR in diagnostic samples from 97 pediatric patients with ALL. We found that CD44 protein expression was associated with disease relapse and was independent of age and WCC. In contrast, high CD44v6 expression was not associated with relapse. These findings may assist in the process of refining prognostic groups for children with ALL.

The O-antigen gene cluster of Escherichia coli O55:H7 and identification of a new UDP-GlcNAc C4 epimerase gene.

Escherichia coli O55 is an important antigen which is often associated with enteropathogenic E. coli clones. We sequenced the genes responsible for its synthesis and identified genes for O-antigen polymerase, O-antigen flippase, four enzymes involved in GDP-colitose synthesis, and three glycosyltransferases, all by comparison with known genes. Upstream of the normal O-antigen region there is a gne gene, which encodes a UDP-GlcNAc epimerase for converting UDP-GlcNAc to UDP-GalNAc and is essential for O55 antigen synthesis. The O55 gne product has only 20 and 26% identity to the gne genes of Pseudomonas aeruginosa and E. coli O113, respectively. We also found evidence for the O55 gene cluster's having evolved from another gene cluster by gain and loss of genes. Only three of the GDP-colitose pathway genes are in the usual location, the other two being separated, although nearby. It is thought that the E. coli O157:H7 clone evolved from the O55:H7 clone in part by transfer of the O157 gene cluster into an O55 lineage. Comparison of genes flanking the O-antigen gene clusters of the O55:H7 and O157:H7 clones revealed one recombination site within the galF gene and located the other between the hisG and amn genes. Genes outside the recombination sites are 99.6 to 100% identical in the two clones, while most genes thought to have transferred with the O157 gene cluster are 95 to 98% identical.