Cecilia Sheen

Targeting JAK1/2 and mTOR in murine xenograft models of Ph-like acute lymphoblastic leukemia

CRLF2 rearrangements, JAK1/2 point mutations, and JAK2 fusion genes have been identified in Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL), a recently described subtype of pediatric high-risk B-precursor ALL (B-ALL) which exhibits a gene expression profile similar to Ph-positive ALL and has a poor prognosis. Hyperactive JAK/STAT and PI3K/mammalian target of rapamycin (mTOR) signaling is common in this high-risk subset. We, therefore, investigated the efficacy of the JAK inhibitor ruxolitinib and the mTOR inhibitor rapamycin in xenograft models of 8 pediatric B-ALL cases with and without CRLF2 and JAK genomic lesions. Ruxolitinib treatment yielded significantly lower peripheral blast counts compared with vehicle (P < .05) in 6 of 8 human leukemia xenografts and lower splenic blast counts (P < .05) in 8 of 8 samples. Enhanced responses to ruxolitinib were observed in samples harboring JAK-activating lesions and higher levels of STAT5 phosphorylation. Rapamycin controlled leukemia burden in all 8 B-ALL samples. Survival analysis of 2 representative B-ALL xenografts demonstrated prolonged survival with rapamycin treatment compared with vehicle (P < .01). These data demonstrate preclinical in vivo efficacy of ruxolitinib and rapamycin in this high-risk B-ALL subtype, for which novel treatments are urgently needed, and highlight the therapeutic potential of targeted kinase inhibition in Ph-like ALL.

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.

Treatment with sirolimus results in complete responses in patients with autoimmune lymphoproliferative syndrome

We hypothesized that sirolimus, an mTOR inhibitor, may be effective in patients with autoimmune lymphoproliferative syndrome (ALPS) and treated patients who were intolerant to or failed other therapies. Four patients were treated for autoimmune cytopenias; all had a rapid complete or near complete response. Two patients were treated for autoimmune arthritis and colitis, demonstrating marked improvement. Three patients had complete resolution of lymphadenopathy and splenomegaly and all patients had a reduction in double negative T cells, a population hallmark of the disease. Based on these significant responses, we recommend that sirolimus be considered as second‐line therapy for patients with steroid‐refractory disease.

Identifying autoimmune lymphoproliferative syndrome in children with Evans syndrome: a multi-institutional study

Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of abnormal lymphocyte survival caused by dysregulation of the Fas apoptotic pathway. Clinical manifestations of ALPS include autoimmune cytopenias, organomegaly, and lymphadenopathy. These findings overlap with Evans syndrome (ES), defined by presence of at least 2 autoimmune cytopenias. We hypothesized a subset of patients with ES have ALPS and tested 45 children at 22 institutions, measuring peripheral blood double-negative T cells (DNTs) and Fas-mediated apoptosis. ALPS was diagnosed in 47% of patients tested. Markedly elevated DNTs (> or = 5%) were a strong predictor of ALPS (positive predictive value = 94%), whereas no patients with DNTs less than 2.5% had ALPS on apoptosis testing. Severity of cytopenias and elevated immunoglobulin levels also predicted ALPS. This is the largest published series describing children with ES and documents a high rate of ALPS among pediatric ES patients. These data suggest that children with ES should be screened for ALPS with DNTs.

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.

Statins are active in acute lymphoblastic leukaemia (ALL): a therapy that may treat ALL and prevent avascular necrosis.

The prognosis for children with acute lymphoblastic leukaemia (ALL) has substantially improved with the use of multi-agent therapy over the last few decades. Unfortunately, two opposing challenges remain in childhood ALL treatment: relapse and toxicity. One in five children with ALL relapse and many of these succumb to disease. One toxicity from ALL therapy that is particularly debilitating is avascular necrosis (AVN). A recent study suggested over 70% of children with ALL develop AVN, including 15–20% who develop symptomatic AVN (Kawedia et al, 2011). While the cause of AVN is multi-factorial, significant high risk factors include high cholesterol and treatment with corticosteroids. An ideal new agent for ALL treatment would be a drug that has single agent activity, can be effectively combined with existing cytotoxics, and can alleviate the toxic effects of existing agents. Statins, a class of cholesterol lowering agents, may fit this model. Statins are effective in preclinical models of a number of malignancies, including acute myeloid leukaemia (Burke & Kukoly, 2008). Pravastatin and simvastatin have been studied in preclinical models, demonstrating the ability to prevent steroid-induced AVN (Iwakiri et al, 2008). Based on these data, clinicians are using statins in patients off-label and multi-institutional cooperative group clinical trials are under development, moving pravastatin forward as an AVN preventing agent (Sala et al, 2007). We investigated six different statins for their effects against ALL in vitro, including their ability to inhibit proliferation, induce apoptosis, and be combined with cytotoxics. Statins used were atorvastatin, simvastatin, pravastatin, and mevastatin (LKT Laboratories, St. Paul, MN, USA), as well as, lovastatin and fluvastatin (Calbiochem, Gibbstown, NJ, USA). The following human ALL cell lines were used for these experiments: precursor-B cell ALL (Reh, Nalm 6, 380, and RS4-11) and precursor-T ALL (Jurkat, Molt4, CEM). All cell lines were purchased from American Type Culture Collection (Manassas, VA, USA) or Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). In order to determine the activity of statins against ALL cells, the seven different ALL cell lines were treated with six different statins at doses from 1 to 20 μmol/l. Dose ranges were based on other published work treating malignant cell lines with statins, targeting levels obtainable in patients (Burke & Kukoly, 2008; Gbelcova et al, 2008). Response to treatment was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, St. Louis, MO, USA) as previously described (Teachey et al, 2008). ALL cells were responsive to most agents (Fig 1A–D). Every ALL cell line was sensitive to fluvastatin, lovastatin, atorvastatin, and simvastatin, however, the degree of sensitivity varied between lines. All cell lines were resistant to pravastatin. The majority of cell lines (five of seven) were resistant to mevastatin. Fig 1 Statins inhibit ALL-cell proliferation. ALL cell lines were treated with six different statins for 72 h and proliferation was determined by MTT assay (Panels A–D and data not shown). ALL cells from all lines were sensitive to simvastatin, fluvastatin, ... The majority of statins, including fluvastatin, lovastatin, atorvastatin, and simvastatin are highly lipophilic substances. Pravastatin, in contrast, is hydrophilic (Gbelcova et al, 2008). Mevastatin is only slightly lipophilic (Neuhaus et al, 2002). Lipophilic statins can non-selectively diffuse into most cell types. Hydrophilic statins require active transport to enter a cell, limiting activity primarily to hepatocytes. Mevastatins permeability lies in between as it can diffuse into some cell types and not others; it is unable to diffuse into lymphocytes (Neuhaus et al, 2002). The differences in response of ALL cells to statins could be explained by lipid solubility. Simvastatin and pravastatin are the only agents proven to reverse AVN in preclinical models (Iwakiri et al, 2008). Other statins could have a similar benefit; however, they are untested. As simvastatin was one of the most potent statins against the ALL cell lines and pravastatin had no activity, we focused on simvastatin, assessing whether it could eliminate ALL cells as opposed to stopping cell growth, using four cells lines (Nalm6, CEM, Jurkat, and Molt4). We found that simvastatin caused cell death by inducing apoptosis in each cell line (Fig 2A, B). In order to confirm that these findings were not limited to cell lines, primary human ALL cells collected from three patients with pre-B ALL were treated with simvastatin, using a bone marrow stromal culture system supplemented with stem cell factor as previously described (Teachey et al, 2006). All three samples were sensitive to simvastatin with a dose response similar to the cell lines, as determined by 7-aminoactinomycin D staining. After treatment with 20 μmol/l simvastatin for 48 h, there were 52–58% fewer viable CD19+ lymphoblasts in treated wells as compared to control. After treatment for 7 d with 5 or 20 μmol/l of simvastatin, there were 70–81% and 94–98% fewer blasts in treated wells as compared with control, respectively. Using this culture system, it cannot formally be excluded that the statins do not have effects on the stromal cells as well. Fig 2 Mechanistic effects of statins on ALL. Cells were cultured with combinations of simvastatin (20 μmol/l) and mevalonolactone (10 μmol/l) for 72 h. Mevalonolactone converts to mevalonate once added to solution. ALL blasts were plated at ... In order to determine whether statins can be effectively combined with cytotoxic chemotherapy, ALL cells from the seven cell lines were tested with simvastatin in combination with three cytotoxic agents used to treat human ALL, vincristine (Hospira Inc., Lake Forest, IL, USA), doxorubicin (Bedford Labs, Bedford, OH, USA), and dexamethasone (American Reagent Inc., Shirley, NY, USA) (Fig 1E, F and data not shown). Using median effects analysis, we found that the majority of combinations were synergistic; however, some were additive in certain cell lines. Very slight antagonism was found when doxorubicin was combined with simvastatin at very high doses in one of six cell lines and when vincristine was combined with simvastatin at lower doses in one of six cell lines. Overall, these data suggest that simvastatin can be safely combined with cell cycle dependent and independent cytotoxic agents. Statins are 3-hydroxyl-3-meythlglutaryl-CoA (HMG-CoA) reductase inhibitors. To determine if the effects of statins against ALL cells were dependent on inhibiting HMG-CoA reductase, ALL cells were treated with simvastatin with and without mevalonate (mevalonic acid, mevalonolactone) (Gbelcova et al, 2008). HMG-CoA reductase converts HMG-CoA into mevalonate as part of the HMG-CoA reductase pathway in cholesterol biosynthesis. Providing mevalonolactone (Sigma-Aldrich), which converts to mevalonate in solution, directly to the ALL cells in vitro bypasses HMG-CoA reductase. Mevalonate was able to reverse the effects of simvastatin on ALL cells (Fig 2A, B). In AML, statins were demonstrated to have some anti-tumour activity through down-regulation of signalling through the Raf/MEK/ERK pathway (Wu et al, 2004). As targeting the Raf/MEK/ERK pathway is effective against ALL in preclinical models and as ERK-mediated phosphorylation of BIM can reduce ALL blast responsiveness to steroids, the effects of statins on ERK signalling were assessed (Rambal et al, 2009). Treatment with simvastatin decreased phosphorylation of ERK in a dose-dependent manner and had no effect on total ERK (Fig 2C). Mevalonate reversed these effects. In summary, statins have preclinical activity against ALL cells and can be effectively combined with chemotherapy. As this class of drug has the potential ability to reverse avascular necrosis, a devastating side effect of ALL therapy, statins hold the unique promise of treating a disease and reversing the side effects of other medications used in therapy. Nevertheless, not all statins are the same, and careful consideration should be made regarding the best agent to move forward in clinical trials.