Junjie Fang

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.

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.

CD34 selection as a stem cell purging strategy for neuroblastoma: preclinical and clinical studies.

BACKGROUND The suitability of CD34 selection for purging peripheral blood progenitor cells (PBPC) collected from patients with neuroblastoma (NB) has been called into question, largely because of reports of detection of low levels of CD34 on the surface of some NB cell lines and tumors. PROCEDURE We used three approaches to address the issue of purging of NB from stem cell specimens and possible labeling of NB: 1) Flow cytometric detection of CD34 on NB cell lines. We assessed CD34 expression using a panel of anti-CD34 monoclonal antibodies (MoAbs) including 9C5, 12.8, and QBend10 and showed no increase in labeling over secondary-only control. 2) Spiking experiments with the Isolex 50 system. NB cell lines were used to contaminate aliquots of PBPC collections, after which the products were purified using the Isolex 50. Purging of NB was assessed by quantitative multiplex RT-PCR (TaqMan system) using a tumor-specific transcript, GAGE. We demonstrated >2 logs of tumor cell depletion from these specimens. 3) Analysis of clinical specimens. PBPC pre- and post-CD34 selection were analyzed from patients treated on the CHP-594 transplant trial. RESULTS In nine specimens selected using the Ceprate LC CD34 selection system where tumor was detectable by immunocytochemistry preselection, we observed >2.4 to >4.6 logs of NB purging after selection. We then analyzed 23 aliquots of PBPC infused into patients post-CD34 selection and compared them to the product preselection; 20/23 specimens showed depletion of NB, although some level of GAGE message was observed in most post-CD34 selection specimens. CONCLUSION These data show that purging of NB from PBPC specimens using CD34 selection is feasible, yielding infused products that are negative at the level of ICC but often positive at the level of RT-PCR.

Role of the BCR complex in B cell development, activation, and leukemic transformation

A primary focus of signal transduction in B cells, from the pre-B cell to the mature B cell, is the B cell receptor complex. Here we describe work demonstrating the importance of signaling via the pre-B cell receptor complex (pre-BCR) to the pre-B cell transition, the central checkpoint in B-cell development. We have shown tht pre-BCR complex components Igα and Igβ are critical to allowing the pre-B cell to move through thistransition, but may not be required for allelic exclusion. Pre-BCR expression also directly affects the response of leukemic cells to steroid treatment, suggesting that signals initiated by the pre-BCR complex may present therapeutic targetsin acute leukemia. Additionally, interleukin-7 may also modulate the response of leukemic cells arising from early B-cell stages to treatment. This observation has lead directly to proposals to test drugs which may antagonize early B-cell growth signals, such as rapamycin, in acute lymphoid leukemia.

Successful Purging of Stem Cell Products Using CD34 Selection

Purging of tumor cells from stem cell products used to support autologous transplant procedures to treat solid tumors may decrease the risk of relapse. Concerns have been raised about the use of the only widely available technique for stem cell purging, CD34 selection, for purging products collected from patients with neuroblastoma (NB), largely because of reports of detection of low levels of CD34 on the surface of some NB cell lines and tumors. We have used 3 approaches to address the issue of purging of NB from stem cell specimens and possible CD34 labeling of NB. 1. Flow cytometric detection of CD34 on NB cell lines. We assessed CD34 expression using a panel of anti-CD34 monoclonal antibodies including 9C5, 12.8 and QBend10 and showed no increase in labeling over secondary-only control. 2. Spiking experiments with the Isolex 50 system. NB cell lines were used to contaminate aliquots of stem cell collections, after which the products were purified using the Isolex 50. Purging of NB was assessed by quantitative multiplex RT-PCR (Taqman system) using a tumor-specific transcript, GAGE. We demonstrated <2 logs of tumor cell depletion from these specimens. 3. Analysis of clinical specimens. Stem cell pre- and post-CD34 selection were analyzed from patients treated on a tandem transplant trial for NB. In nine specimens selected using the Ceprate LC CD34 selection system where tumor was detectable by immunocytochemistry pre-selection, we observed >2.4 to >4.6 logs of NB purging after selection. We then analyzed 23 aliquots of stem This research was supported in part by the University of Pennsylvania Cancer Center (SG), by the Benacerraf/Frei Clinical Investigator Award, Dana-Farber Cancer Institute (LD) and the Fiftieth Anniversary Program for Scholars in Medicine, Harvard Medical School (LD). The abbreviations used are: FITC, fluorescein isothiocyanate; GAPDH, Glyceraldehyde 3 phosphate dehydrogenase; ICC, immunocytochemistry; MoAbs, monoclonal antibodies; PE, phycoerythrin; RT-PCR, reverse transcriptase-polymerase chain reaction; PBMC, peripheral blood mononuclear cells.

Angiogenesis Inhibitor TNP-470 During Bone Marrow Transplant and in Minimal Residual Disease

High-dose therapy with stem cell rescue is a treatment option for patients with advanced solid tumors. Although this approach has promise for some pediatric cancers, especially neuroblastoma, it is limited by the risk of relapse post transplant, as well as concern about possible reinfused tumor cells in autologous stem cell products. Anti-angiogenic agents given during and after recovery from highdose therapy with stem cell rescue may decrease the risk of relapse. Additionally, such agents may have their greatest therapeutic effect when administered at a point of minimal residual disease, rather than to treat bulky tumor. TNP-470 is an anti-angiogenic agent now in clinical trials. We have tested TNP-470 in the treatment of neuroblastoma in a mouse xenograft model, and demonstrate that it is most effective when given at lowest disease burden. Additionally, and to assess the feasibility of using anti-angiogenic agents during the period of posttransplant hematopoietic recovery, we have developed a model of stem cell transplant in mice. Mice were treated with TNP-470 and assessed for survival and engraftment. Both treated and control mice demonstrated reliable multilineage engraftment as well as normal lymphoid maturation with no excess mortality in the treated group. This indicates that inhibitors of angiogenesis do not adversely impact engraftment after stem cell transplantation and suggests a clinical study design in which such agents might best be employed in the immediate after stem cell transplant.