Research Milestones from Johns Hopkins Pediatric Oncologists

Kenneth J. Cohen, M.D., M.B.A.

Brain tumors have the highest case mortality rate of any tumors as a group in children. Significant advances have been made in surgical and supportive care, but with less evident benefits in therapy. A variety of novel therapeutic agents and approaches are being studied in the hope of finding new approaches to treatment that are efficacious and ideally less toxic to the developing brain. The development of novel therapeutics is being driven by a growing translational initiative in cooperation with the neuropathology and neurosurgical sciences group.

Donald Small, M.D., Ph.D.

My laboratory is interested in how the normal signal transduction pathway goes awry and leads to the development of leukemia and lymphoma. Most of our efforts are concentrated on FLT3, which codes for a type III receptor tyrosine kinase normally restricted to expression by CD34+hematopoietic stem/progenitor cells. The gene is aberrantly expressed by most B-lineage acute lymphoblastic leukemia and acute myeloid leukemia patients’ blast cells, most of which co-express FLT3 ligand. In addition, about one-third of patients with AML express a mutated form of the receptor, which results in its constitutive activation in the absence of ligand.

We are studying the anti-apoptotic and antidifferentiation signals transduced by the activated receptor in leukemic cells. We are also studying the control elements that are responsible for regulating FLT3 expression. We have discovered several small-molecule tyrosine kinase inhibitors that interfere with FLT3 kinase activity fairly specifically. These inhibitors are able to induce cell death in cells transformed with the constitutively activated FLT3 while not interfering with the normal proliferative pathways in these cells. Thus, these new drugs are true examples of molecularly targeted therapy. We have moved one of these drugs into the clinics in patients with relapsed/refractory AML with FLT3 mutations and have seen clinical responses in these patients. We have studied how FLT3 inhibitors best combine with chemotherapeutics and have used this data to design the next generation clinical trial now under way at Johns Hopkins and more than 20 other institutions. We continue to perform correlative and surrogate assays on material from the patients on these trials to try to understand the factors that determine response to this new class of agents. We have also demonstrated that infant leukemias and others with high- level expression of wild-type FLT3 respond in vitro to these inhibitors. This forms the basis for the preclinical work to support trials of these drugs in leukemias other than FLT3-mutant AML

Allen R. Chen, M.D., Ph.D., M.H.S.

Children with metastatic or recurrent malignancies have a poor prognosis despite initially chemoresponsive disease. There is a steep linear relationship between the dose of many cytotoxic agents and the log tumor cell kill. For agents whose major dose-limiting toxicity is myelosuppression, hematopoietic stem cell rescue may permit five to 10 times dose escalation, which would produce several times more cell killing. The Children’s Cancer Group has demonstrated in a prospective, randomized, controlled study in patients with high-risk neuroblastoma that purged autologous bone marrow transplantation (BMT) produces superior three-year event-free survival (EFS), compared with conventional dose-intensive chemotherapy as consolidation in first response. Although prospective randomized trials have yet to be completed for other pediatric solid tumors, autologous BMT appears to produce survival at least equivalent to longer courses of intensive conventional chemotherapy, and the major risk remains tumor progression. Therefore, additional agents and modalities that are not cross-resistant with current therapies are needed. Dr. Chen is investigating tandem transplantation and autologous GVHD as alternative approaches to provide improved control of pediatric solid tumors.

In allogeneic BMT, nonablative preparative regimens can produce engraftment with less preparative-regimen-related toxicity, but at a price of more graft versus.host disease. In fact, GVHD is a major limitation of the success of BMT for all indications, but its morbidity and mortality are offset in patients with hematologic malignancies by an improved graft-versus-leukemia effect. Therefore, for patients with nonmalignant diseases, such as inherited hematologic and metabolic diseases that present during childhood, preventing GVHD is particularly important. Rodent models indicate that cyclophosphamide administered at a critical interval after mismatched BMT can specifically destroy alloreactive T cell clones, and Dr. Leo Luznik’s trial in adult patients undergoing haploidentical BMT for hematologic malignancies supports a role for cyclophosphamide treatment after graft infusion to produce tolerance. Dr. Chen will test whether cyclophosphamide administered after graft infusion will allow long-term engraftment without GVHD in immune-competent patients with hemoglobinopathies receiving nonmyeloablative BMT.

Curt I. Civin, M.D.

Dr. Civin's research is focused on the cell and molecular biology of the stem and progenitor cells that form the normal blood and immune systems, and the malignant counterparts of these stem-progenitor cells, the leukemias. Current translational projects include using lentiviral transduction of Fas ligand to genetically engineer primary hematopoietic stem cells and human embryonic stem cells (presidentially approved) that will resist rejection, and hematopoietic growth regulatory genes to develop laboratory models that will elucidate the molecular steps in the development of leukemias from normal human stem-progenitor cells. In more basic studies, the Civin lab has comprehensively described which genes are active and functional in human hematopoietic stem-progenitor cells, human embryonic stem cells, mesenchymal stem cells, and leukemic stem cells, using microarrays and serial analysis of gene expression. Lab members are now performing functional genomic studies, in stem-progenitor cells, of genes selected from this repertoire. Finally, the Civin lab has developed models to study development of blood and endothelial cells from human embryonic stem-cell lines.

Alan D. Friedman, M.D.

The genes encoding the AML1 or CBFß subunits of CBF are commonly involved in translocations associated with acute myeloid leukemia or pediatric B-lineage acute lymphoblastic leukemia. Dr. Friedman’s group has demonstrated that the resulting fusion oncoproteins, including CBFß-SMMHC and AML1-ETO, inhibit G1 to S cell-cycle progression by inhibiting endogenous CBF activities. He has gone on to show that proteins that stimulate G1, in particular cdk4, cylcin D2, c-Myc, or E7, prevent cell-cycle inibition by CBF oncoproteins in cell lines, and his group has collaborated with that of Dr. Curt Civin to show that CBFß-SMMHC and E7 cooperate to induce acute leukemias in murine marrow. In the presence of such additional mutations, Dr. Friedman postulates that CBF oncoproteins contribute to transformation by inhibiting differentiation or stimulating survival. Dr. Friedman’s group has also identified a segment of the CBFß-SMMHC myosin domain critical for its activities, and his group has initiated a cell-based drug screen to identify candidate agents that could prove therapeutic for leukemias expressing CBFß-SMMHC or AML1-ETO.

C/EBPa is a key regulator of myelopoiesis and is mutated in another subset of AML patients. Dr. Friedman’s group was the first to demonstrate that C/EBP proteins are expressed in early myeloid cells but not in erythroid or lymphoid cells. He found that C/EBPa predominated in immature myeloid cells and demonstrated that an inducible form of C/EBPa drives differentiation and independently induces a delayed G1/S cell- cycle arrest in the 32D cl3 cell line. Dr. Friedman’s group has also found that a dominant-inhibitory form of C/EBPa blocks myeloid differentiation and that C/EBPa contributes to granulocytic development by inducing the PU.1 and C/EBPe genes. In addition, Dr. Friedman has found that C/EBPa directly interacts with NF-kB to induce bcl-2 and inhibit apoptosis in myeloid leukemias and has correlated C/EBPa and bcl-2 levels in a subset of AML cases. He postulates that targeting the C/EBPa:NF-kB interaction may prove of therapeutic benefit in AML and potentially in other malignancies co-expressing these proteins.

David M. Loeb, M.D., Ph.D.

Dr. Loeb is currently the Director of the Musculoskeletal Tumor Program.  This program provides comprehensive, multi-disciplinary care to children and adults with bone and soft tissue tumors.  Dr. Loeb is also the institutional principle investigator with SARC, the Sarcoma Alliance for Research through Collaboration, a multi-institutional consortium of sarcoma centers focused on early phase clinical trials for sarcoma patients.  Dr. Loeb is also an active member of the pediatric bone marrow transplantation team, with a particular interest in providing novel high dose therapies to sarcoma patients of all ages.

Dr. Loeb is the principle investigator on two institutional clinical trials for osteosarcoma patients using a drug called Quadramet.  Quadramet is a targeted radiopharmaceutical agent that is designed to deliver radiation to osteosarcoma lesions while sparing surrounding normal tissue.  One of these studies is a dose finding study, with a goal of identifying a dose of Quadramet that will allow this agent to be incorporated into a novel chemo-radiotherapy treatment protocol for patients with osteosarcoma metastatic to bone at diagnosis.  The other study uses a very high dose of Quadramet for the treatment of patients with high risk osteosarcoma.  Because the sole significant toxicity of this therapy is myelosuppression, autologous peripheral blood stem cell support is utilized to allow treatment with higher doses of radiation.

In addition to these studies, Dr. Loeb is also involved in a new clinical trial evaluating tandem autologous peripheral blood stem cell transplantation using a novel preparative regimen including an oral chemotherapy drug, temozolomide, for patients with high risk solid tumors, including Ewing sarcoma.  In the laboratory, Dr. Loeb is actively engaged in research aimed at identifying and characterizing Ewing sarcoma stem cells.  Cancer stem cells are thought to be inherently resistant to chemotherapy and are thought to cause most cases of refractory or relapsed disease.  A thorough understanding of the biology of these important cells will allow the development of therapies targeted at this critical component of most tumors.

Elias T. Zambidis, M.D., Ph.D.

Dr. Zambidis is interested in the developmental biology of normal and malignant hematopoietic stem cells. Our approach to studying the cellular and molecular mechanisms of human hematopoiesis relies on the genetic manipulation and differentiation of both embryonic and adult pluripotent stem cells. Current translational projects exploit the use of human embryonic stem cells (hESC) derived from both fertilized and somatic cell nuclear transfer (SCNT)-derived sources for the derivation, culture, and expansion of human HSPC. Our strategy for identifying early HSPC relies on the general hypothesis that a human hemangioblast (bipotential progenitor of HSPC and endothelium) stem cell gives rise to the entire human hematopoietic system and can be derived and expanded from differentiating hESC. We are currently probing the roles that the caudalizing homeodomain factor CDX4 and the bHLH transcription factor SCL/TAL1 play in orchestrating the initiation of human embryonic hematopoiesis by directing the formation of human hemangioblasts from hESC. hESC-derived blood progenitors are important not only for studies in human lympho-hematopoietic development and the understanding of the developmental origins of pediatric leukemia, but also possibly for clinical HSPC transplantation. Additional projects also focus on the role Notch signaling plays on the expansion and differentiation of early lymph-hematopoietic progenitors derived from hESC.