Valerie I. Brown

Synergistic interaction of p185c-neu and the EGF receptor leads to transformation of rodent fibroblasts

The protein product of the rodent neu oncogene, p185neu, is a tyrosine kinase with structural similarity to the epidermal growth factor receptor (EGFR). Transfection and subsequent overexpression of the human p185c-erbB-2 protein transforms NIH 3T3 cells in vitro. However, NIH 3T3 cells are not transformed by overexpressed rodent p185c-neu. NIH 3T3 transfectants overexpressing EGF receptors are not transformed unless incompletely transformed. Several groups have recently demonstrated EGF-induced, EGFR-mediated phosphorylation of p185c-neu. During efforts to characterize the interaction of p185c-neu with EGFR further, we created cell lines that simultaneously overexpress both p185c-neu and EGFR and observed that these cells become transformed. These observations demonstrate that two distinct, overexpressed tyrosine kinases can act synergistically to transform NIH 3T3 cells, thus identifying a novel mechanism that can lead to transformation.

Rapamycin is active against B-precursor leukemia in vitro and in vivo, an effect that is modulated by IL-7-mediated signaling.

A balance between survival and apoptotic signals regulates B cell development. These signals are tightly regulated by a host of molecules, including IL-7. Abnormal signaling events may lead to neoplastic transformation of progenitor B cells. Signal transduction inhibitors potentially may modulate these abnormal signals. Inhibitors of the mammalian target of rapamycin (mTOR) such as rapamycin have been used as immunosuppressive agents. We hypothesized that rapamycin might demonstrate activity against B-precursor acute lymphoblastic leukemia. We have found that rapamycin inhibited growth of B-precursor acute lymphoblastic leukemia lines in vitro, with evidence of apoptotic cell death. This growth inhibition was reversible by IL-7. One candidate as a signaling intermediate cross-regulated by rapamycin and IL-7 was p70 S6 kinase. Rapamycin also demonstrated in vivo activity in Eμ-ret transgenic mice, which develop pre-B leukemia/lymphoma: Eμ-ret transgenic mice with advanced disease treated daily with rapamycin as a single agent showed a >2-fold increase in length of survival as compared with symptomatic littermates who received vehicle alone. These results suggest that mammalian target of rapamycin inhibitors may be effective agents against leukemia and that one of the growth signals inhibited by this class of drugs in precursor B leukemic cells may be IL-7-mediated.

mTOR inhibitors are synergistic with methotrexate: an effective combination to treat acute lymphoblastic leukemia

We have previously demonstrated that mTOR inhibitors (MTIs) are active in preclinical models of acute lymphoblastic leukemia (ALL). MTIs may increase degradation of cyclin D1, a protein involved in dihydrofolate reductase (DHFR) synthesis. Because resistance to methotrexate may correlate with high DHFR expression, we hypothesized MTIs may increase sensitivity of ALL to methotrexate through decreasing DHFR by increasing turn-over of cyclin D1. We tested this hypothesis using multiple ALL cell lines and nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenografted with human ALL. We found MTIs and methotrexate were synergistic in combination in vitro and in vivo. Mice treated with both drugs went into a complete and durable remission whereas single agent treatment caused an initial partial response that ultimately progressed. ALL cells treated with MTIs had markedly decreased expression of DHFR and cyclin D1, providing a novel mechanistic explanation for a combined effect. We found methotrexate and MTIs are an effective and potentially synergistic combination in ALL.

Mammalian target of rapamycin inhibitors and their potential role in therapy in leukaemia and other haematological malignancies.

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that functions as a key regulator of cell growth, protein synthesis, and cell‐cycle progression through interactions with a number of signalling pathways, including PI3K/AKT, ras, TCL1, and BCR/ABL. Many haematological malignancies have aberrant activation of the mTOR and related signalling pathways. Accordingly, mTOR inhibitors, a class of signal transduction inhibitors that were originally developed as immunosuppressive agents, are being investigated in preclinical models and clinical trials for a number of haematological malignancies. Sirolimus and second‐generation mTOR inhibitors, such as temsirolimus and everolimus, are safe and relatively well‐tolerated, making them potentially attractive as single agents or in combination with conventional cytotoxics and other targeted therapies. Promising early clinical data suggests activity of mTOR inhibitors in a number of haematological diseases, including acute lymphoblastic leukaemia, chronic myeloid leukaemia, mantle cell lymphoma, anaplastic large cell lymphoma, and lymphoproliferative disorders. This review describes the rationale for using mTOR inhibitors in a variety of haematological diseases with a focus on their use in leukaemia.

Targeting the PI3K/AKT/mTOR signaling axis in children with hematologic malignancies.

The phosphatidylinositiol 3-kinase (PI3K), AKT, mammalian target of rapamycin (mTOR) signaling pathway (PI3K/AKT/mTOR) is frequently dysregulated in disorders of cell growth and survival, including a number of pediatric hematologic malignancies. The pathway can be abnormally activated in childhood acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), and chronic myelogenous leukemia (CML), as well as in some pediatric lymphomas and lymphoproliferative disorders. Most commonly, this abnormal activation occurs as a consequence of constitutive activation of AKT, providing a compelling rationale to target this pathway in many of these conditions. A variety of agents, beginning with the rapamycin analogue (rapalog) sirolimus, have been used successfully to target this pathway in a number of pediatric hematologic malignancies. Rapalogs demonstrate significant preclinical activity against ALL, which has led to a number of clinical trials. Moreover, rapalogs can synergize with a number of conventional cytotoxic agents and overcome pathways of chemotherapeutic resistance for drugs commonly used in ALL treatment, including methotrexate and corticosteroids. Based on preclinical data, rapalogs are also being studied in AML, CML, and non-Hodgkins lymphoma. Recently, significant progress has been made using rapalogs to treat pre-malignant lymphoproliferative disorders, including the autoimmune lymphoproliferative syndrome (ALPS); complete remissions in children with otherwise therapy-resistant disease have been seen. Rapalogs only block one component of the pathway (mTORC1), and newer agents are under preclinical and clinical development that can target different and often multiple protein kinases in the PI3K/AKT/mTOR pathway. Most of these agents have been tolerated in early-phase clinical trials. A number of PI3K inhibitors are under investigation. Of note, most of these also target other protein kinases. Newer agents are under development that target both mTORC1 and mTORC2, mTORC1 and PI3K, and the triad of PI3K, mTORC1, and mTORC2. Preclinical data suggest these dual- and multi-kinase inhibitors are more potent than rapalogs against many of the aforementioned hematologic malignancies. Two classes of AKT inhibitors are under development, the alkyl-lysophospholipids (APLs) and small molecule AKT inhibitors. Both classes have agents currently in clinical trials. A number of drugs are in development that target other components of the pathway, including eukaryotic translation initiation factor (eIF) 4E (eIF4E) and phosphoinositide-dependent protein kinase 1 (PDK1). Finally, a number of other key signaling pathways interact with PI3K/AKT/mTOR, including Notch, MNK, Syk, MAPK, and aurora kinase. These alternative pathways are being targeted alone and in combination with PI3K/AKT/mTOR inhibitors with promising preclinical results in pediatric hematologic malignancies. This review provides a comprehensive overview of the abnormalities in the PI3K/AKT/mTOR signaling pathway in pediatric hematologic malignancies, the agents that are used to target this pathway, and the results of preclinical and clinical trials, using those agents in childhood hematologic cancers.

Thymic Stromal-Derived Lymphopoietin Induces Proliferation of Pre-B Leukemia and Antagonizes mTOR Inhibitors, Suggesting a Role for Interleukin-7Rα Signaling

Understanding the pathogenesis of leukemia in the context of lymphopoiesis may reveal novel therapeutic targets. Previously, we have shown that mTOR inhibitors (MTI) show activity in vitro and in preclinical models of both human and murine precursor B acute lymphoblastic leukemia (pre-B ALL), inhibiting cell proliferation and inducing apoptosis. These MTI-mediated effects can be reversed by interleukin-7 (IL-7), an important regulator of early B-cell development. This observation led us to examine the contribution of signaling via the IL-7Ralpha chain, which is shared by the receptor complexes of IL-7 and thymic stromal-derived lymphopoietin (TSLP). TSLP is closely related to IL-7 and active in lymphopoiesis, but an effect of TSLP on leukemia cells has not been described. We examined the effect of TSLP on pre-B ALL cells and their response to MTIs. Here, we show that TSLP stimulates proliferation of pre-B ALL cell lines. TSLP also partially reverses the effects of MTI on proliferation, apoptosis, and ribosomal protein S6 and 4E-BP1 phosphorylation in cell lines, with similar biological effects seen in some primary human lymphoblast samples. These data show that TSLP can promote survival of pre-B ALL cells and antagonize the effects of MTIs. These findings suggest that IL-7Ralpha chain is responsible for transducing the survival signal that overcomes MTI-mediated growth inhibition in pre-B ALL. Thus, further exploration of the IL-7Ralpha pathway may identify potential therapeutic targets in the treatment of ALL. Our data illustrate that growth-factor-mediated signaling may provide one mechanism of MTI resistance.

Novel molecular and cellular therapeutic targets in acute lymphoblastic leukemia and lymphoproliferative disease

While the outcome for pediatric patients with lymphoproliferative disorders (LPD) or lymphoid malignancies, such as acute lymphoblastic leukemia (ALL), has improved dramatically, patients often suffer from therapeutic sequelae. Additionally, despite intensified treatment, the prognosis remains dismal for patients with refractory or relapsed disease. Thus, novel biologically targeted treatment approaches are needed. These targets can be identified by understanding how a loss of lymphocyte homeostasis can result in LPD or ALL. Herein, we review potential molecular and cellular therapeutic strategies that (i) target key signaling networks (e.g., PI3K/AKT/mTOR, JAK/STAT, Notch1, and SRC kinase family-containing pathways) which regulate lymphocyte growth, survival, and function; (ii) block the interaction of ALL cells with stromal cells or lymphoid growth factors secreted by the bone marrow microenvironment; or (iii) stimulate innate and adaptive immune responses.

Long-term protection from syngeneic acute lymphoblastic leukemia by CpG ODN-mediated stimulation of innate and adaptive immune responses.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and remains a major cause of mortality in children with recurrent disease and in adults. Despite observed graft-versus-leukemia effects after stem cell transplantation, successful immune therapies for ALL have proven elusive. We previously reported immunostimulatory oligodeoxynucleotides containing CpG motifs (CpG ODN) enhance allogeneic T(h)1 responses and reduce leukemic burden of primary human ALL xenografts. To further the development of CpG ODN as a novel ALL therapy, we investigated the antileukemia activity induced by CpG ODN in a transplantable syngeneic pre-B ALL model. CpG ODN induced early killing of leukemia by innate immune effectors both in vitro and in vivo. Mice were treated with CpG ODN starting 7 days after injection with leukemia to mimic a minimal residual disease state and achieved T cell-dependent remissions of more than 6 months. In addition, mice in remission after CpG ODN treatment were protected from leukemia rechallenge, and adoptive transfer of T cells from mice in remission conferred protection against leukemia growth. To our knowledge, this is the first demonstration that CpG ODN induce a durable remission and ongoing immune-mediated protection in ALL, suggesting this treatment may have clinical utility in patients with minimal residual disease.

Targeted Busulfan therapy with a steady-state concentration of 600-700 ng/mL in patients with sickle cell disease receiving HLA-identical sibling bone marrow transplant

Busulfan (BU) has a narrow therapeutic window and the average concentration of BU at steady state (Css) is critical for successful engraftment in children receiving BU as part of the preparative regimen for allogeneic transplants. Sixteen patients with sickle cell disease (SCD) underwent allogeneic bone marrow transplant (BMT) from HLA-identical siblings. The preparative regimen consisted of intravenous BU 0.8–1 mg/kg/dose for 16 doses, cytoxan (CY) of 50 mg/kg daily for four doses and equine anti-thymocyte globulin (ATG) 30 mg/kg daily for three doses. BU levels were adjusted to provide a total exposure Css of 600–700 ng/mL. The median age at the time of transplant was 6.2 years (range 1.2–19.3). Fourteen (87%) patients required adjustment of the BU dose to achieve a median Css of 652 ng/mL (range 607–700). All patients achieved neutrophil and platelet engraftment without significant toxicity. Median donor engraftment at the last follow-up was 100% (range 80–100). None of the patients experienced sickle cell-related complications post transplant. With a median follow-up of 3 years (range 1.3–9), the event-free survival (EFS) and overall survival (OS) are both 100%. We conclude that targeting of BU Css between 600 and 700 ng/mL in this regimen can result in excellent and sustained engraftment in young patients with SCD.

Transplant Outcomes for Children with T Cell Acute Lymphoblastic Leukemia in Second Remission: A Report from the Center for International Blood and Marrow Transplant Research

Survival for children with relapsed T cell acute lymphoblastic leukemia (T-ALL) is poor when treated with chemotherapy alone, and outcomes after allogeneic hematopoietic cell transplantation (HCT) is not well described. Two hundred twenty-nine children with T-ALL in second complete remission (CR2) received an HCT after myeloablative conditioning between 2000 and 2011 and were reported to the Center for International Blood and Marrow Transplant Research. Median age was 10 years (range, 2 to 18). Donor source was umbilical cord blood (26%), matched sibling bone marrow (38%), or unrelated bone marrow/peripheral blood (36%). Acute (grades II to IV) and chronic graft-versus-host disease occurred in, respectively, 35% (95% confidence interval [CI], 27% to 45%) and 26% (95% CI, 20% to 33%) of patients. Transplant-related mortality at day 100 and 3-year relapse rates were 13% (95% CI, 9% to 18%) and 30% (95% CI, 24% to 37%), respectively. Three-year overall survival and disease-free survival rates were 48% (95% CI, 41% to 55%) and 46% (95% CI, 39% to 52%), respectively. In multivariate analysis, patients with bone marrow relapse, with or without concurrent extramedullary relapse before HCT, were most likely to relapse (hazard ratio, 3.94; P = .005) as compared with isolated extramedullary disease. In conclusion, HCT for pediatric T-ALL in CR2 demonstrates reasonable and durable outcomes, and consideration for HCT is warranted.