Kevin G. Waddick

Biotherapy of B-cell precursor leukemia by targeting genistein to CD19-associated tyrosine kinases

B-cell precursor (BCP) leukemia is the most common form of childhood cancer and the second most common form of acute leukemia in adults. Human BCP leukemia was treated in a severe combined immunodeficient mouse model by targeting of the tyrosine kinase inhibitor Genistein (Gen) to the B cell-specific receptor CD19 with the monoclonal antibody B43. The B43-Gen immunoconjugate bound with high affinity to BCP leukemia cells, selectively inhibited CD19-associated tyrosine kinases, and triggered rapid apoptotic cell death. At less than one-tenth the maximum tolerated dose more than 99.999 percent of human BCP leukemia cells were killed, which led to 100 percent long-term event-free survival from an otherwise invariably fatal leukemia. The B43-Gen immuno-conjugate might be useful in eliminating leukemia cells in patients who have failed conventional therapy.

BTK as a mediator of radiation-induced apoptosis in DT-40 lymphoma B cells.

Brutons tyrosine kinase (BTK) is a member of the SRC-related TEC family of protein tyrosine kinases (PTKs). DT-40 lymphoma B cells, rendered BTK-deficient through targeted disruption of the btk gene by homologous recombination knockout, did not undergo radiation-induced apoptosis, but cells with disrupted lyn or syk genes did. Introduction of the wild-type, or a SRC homology 2 domain or a plecstrin homology domain mutant (but not a kinase domain mutant), human btk gene into BTK-deficient cells restored the apoptotic response to radiation. Thus, BTK is the PTK responsible for triggering radiation-induced apoptosis of lymphoma B cells, and its kinase domain is indispensable for the apoptotic response.

Innovative treatment programs against cancer. II. Nuclear factor-κB (NF-κB) as a molecular target

Abstract Nuclear factor-κB (NF-κB) activity affects cell survival and determines the sensitivity of cancer cells to cytotoxic agents as well as to ionizing radiation. Preventing the protective function of NF-κB may result in chemo- and radio-sensitization of cancer cells. Therefore, NF-κB has emerged as one of the most promising molecular targets in rational drug design efforts of translational cancer research programs.

Engagement of the CD19 receptor on human B-lineage leukemia cells activates LCK tyrosine kinase and facilitates radiation-induced apoptosis.

As presently reported, both ionizing radiation and engagement of the CD19 receptor are capable of inducing apoptosis in B-lineage acute lymphoblastic leukemia (ALL) cells. In both instances, activation of tyrosine kinases appears to be a proximal and mandatory step, since it can be prevented by the tyrosine kinase inhibitor genistein. This common biochemical signaling pathway involves the rapid activation of the Src family tyrosine kinase LCK (p56lck), which is physically associated with the CD19 receptor, and enhanced tyrosine phosphorylation of multiple substrates leading to stimulation of phosphoinositide turnover, and activation of protein kinase C. Importantly, engagement of the CD19 receptor promoted radiation-induced apoptosis in radiation-resistant B-lineage ALL cells in a cell type-specific fashion. Our results prompt the hypothesis that clonogenic B-lineage ALL blasts with an inherent or acquired resistance to radiation could be radiosensitized in clinical settings using anti-CD19 MoAb B43 or its homoconjugate as adjuncts.

Innovative treatment programs against cancer. I. Ras oncoprotein as a molecular target.

Modulation of Ras function may provide a novel means by which cancer cells with oncogenic mutations can be sensitized to chemotherapeutic or radiotherapeutic regimens. Moreover, cancer cells without ras oncogene mutations can also be eliminated by compounds that interfere with the mevalonate pathway, which is more fundamental to mitogenesis because it allows the synthesis of sterol and nonsterol lipids and without which many Ras-related proteins and nuclear lamins would not be prenylated and functional.

Radiation sensitivity of human B-lineage lymphoid precursor cells

We studied the radiation sensitivity of eight immunophenotypically distinct B-lineage lymphoid precursor cell (LPC) lines of acute lymphoblastic leukemia (ALL) or fetal liver origin corresponding to discrete developmental stages of human B-cell ontogeny. The radiation sensitivity of B-lineage LPC showed a temporal association with the distinct stages of development. FL112 and FL114 fetal liver pro-B cells (Stage 0 B-lineage LPC) with germline immunoglobulin heavy chain (IgH) genes but rearranged T-cell receptor gamma (T gamma) genes (DO of FL112 = 80.3 cGy, DO of FL114 = 50.2 cGy), REH ALL pre-pre-B cells (Stage I B-lineage LPC) with rearranged IgH and T gamma genes (DO = 66.1 cGy), and NALM-6 ALL pre-pre-B/pre-B cells (Stage II B-lineage LPC) (DO = 50.5 cGy) corresponding to the earliest three stages of human B-lymphocyte development were the most radiation sensitive B-lineage LPC populations. By comparison, KM-3 ALL pre-B (Stage III B-lineage LPC) (DO = 194.7 cGy), HPB-NULL ALL pre-B (Stage IV B-lineage LPC) (DO = 134.6 cGy), and sIgM+ RAJI/NAMALWA early B (Stage Va/b B-lineage LPC) cell lines (DO of RAJI = 144.0 cGy, DO of NAMALWA = 165.5 cGy) corresponding to the later stages of human B-lymphocyte development were much more radiation resistant. These results indicate that the radiation sensitivity of B-lineage LPC decreases during maturation within the B-lineage lymphoid precursor pathway. By comparison, the S-phase index (% of S-phase cells as determined by DNA flow cytometry) or proliferation index (% S + G2M), cellular protein content, intracellular glutathione (GSH) level, glutathione-S-transferase (GST) activity, intracellular pH, or free cytoplasmic calcium concentration did not correlate with the radiation sensitivity of the B-lineage LPC.

A Recombinant fusion toxin targeted to the granulocyte-Macrophage colony-Stimulating factor receptor

Human granulocyte-macrophage colony stimulating factor (GMCSF) and its high affinity receptor function to regulate the proliferation and differentiation of myeloid lineage hematopoietic cells, and may participate in the pathogenesis of many malignant myeloid diseases. We have used genetic engineering based on the elucidated molecular structures of human granulocyte-macrophage colony-stimulating factor and diphtheria toxin (DT) to produce a recombinant fusion toxin, DTctGMCSF, that targets diphtheria toxin to high affinity GMCSF receptors expressed on the surface of blast cells from a large fraction of patients with acute myeloid leukemia (AML). DTctGMCSF was specifically immunoreactive with antidiphtheria toxin and anti-GMCSF antiseras, and exhibited the characteristic catalytic activity of diphtheria toxin, catalyzing the in vitro ADP-ribosylation of purified elongation factor 2. The cytotoxic effects of DTctGMCSF were examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-tetrazolium (MTT) bromide assay of cell viability and in vivo assays of protein synthesis inhibition. DTctGMCSF were specifically cytotoxic to human leukemia cell lines bearing high affinity receptors for human GMCSF with IC50 of 10(-9) to 10(-11) M. It was not toxic to mammalian hematopoietic cell lines lacking human GMCSF (hGMCSF) receptors. In receptor positive cells, cytotoxicity can be specifically blocked by a large excess of hGMCSF, confirming that its cytotoxicity is mediated through the hGMCSF receptor. THough DTctGMCSF inhibited granulocyte-macrophage colony formation by committed myeloid progenitor cells (CFU-GM), it did not significantly affect erythroid burst formation by committed erythroid progenitor cells (BFU-E), or mixed granulocyte-erythroid-macrophage-megakaryocyte colony formation by pluripotent multilineage progenitor cells (CFU-GEMM). DTctGMCSF holds promise for the treatment of myeloid lineage malignancies, and is a useful reagent to study hematopoiesis.

Granulocyte-Macrophage colony-Stimulating factor receptor-Targeted therapy of chemotherapy- and radiation-Resistant human myeloid leukemias

Contemporary therapies for acute myeloid leukemia (AML) commonly fail to cure patients because of the emergence of drug resistance. Drug resistance in AML is multifactorial but can be associated with the overexpression of transmembrane transporter molecules, including P-glycoprotein (Pgp) or the multidrug resistance-associated protein (MRP), or associated with inactivation of the p53 tumor suppressor gene, as well as overexpression of the anti-apoptotic protein bcl-2. We are investigating if novel recombinant biotherapeutics can circumvent these resistance mechanisms to effectively treat refractory AML. To target the lethal action of diphtheria toxin (DT) to high affinity granulocyte-macrophage colony-stimulating factor (GMCSF) receptors on AML blasts, we have produced a recombinant chimeric fusion toxin, DTctGMCSF. Since DTctGMCSF enters and kills its target cells by unique mechanisms (GMCSF-receptor binding and protein synthesis inhibition) and is not similar in structure to Pgp or MRP substrates, we postulated that it would be an active agent against therapy-resistant AML. DTctGMCSF was selectively cytotoxic (IC50 1-10ng/ml) to GMCSF-receptor positive AML cells expressing the Pgp- or MRP-associated multi-drug resistant phenotypes, despite high level resistance to conventional chemotherapeutic agents. DTctGMCSF also efficiently killed AML cells deficient in p53 expression, as well as radiation-resistant AML cells and mixed lineage leukemia cells expressing high levels of bcl-2. In addition, DTctGMCSF killed > 99% of primary leukemic progenitor cells from therapy-refractory AML patients under conditions that we have previously found to not adversely affect the proliferative capacity or differentiation of pluripotent normal hematopoietic progenitor cells. DTctGMCSF may prove useful in treating myeloid leukemias that are otherwise resistant to a wide range of conventional therapies.

In Vivo Radiosensitizing Effects of Recombinant Interleukin 6 on Radiation Resistant BCL-1 B-Lineage Leukemia Cells in a Murine Syngeneic Bone Marrow Transplant Model System

The ability of total body irradiation (TBI) to eradicate clonogenic leukemia cells from B-lineage acute lymphoblastic leukemia patients prior to bone marrow transplantation (BMT) is greatly hampered by their inherent or acquired radiation resistance. The radiorefractory nature of these cells is believed to contribute to the high relapse rate subsequent to TBI and BMT in patients with B-lineage acute lymphoblastic leukemia (ALL). A method by which clonogenic leukemia cells could be radiosensitized in vivo could be clinically beneficial. In the present study, we used a highly radiation resistant subclone of the murine B-lineage leukemia cell line BCL-1 in a syngeneic BMT model to investigate if any of the B-cell stimulatory cytokines interleukin 2, interleukin 4, interleukin 5, or interleukin 6 could have radiosensitizing effects. All untreated BALB/c mice (N = 33) inoculated with 1 x 10(6) BCL-1 cells died of disseminated leukemia within 24 days with a median survival of 13.3 days. TBI (700 cGy = LD100/30 for BALB/c mice) followed by syngeneic BMT (N = 70) extended the median survival to 23.6 days (P < 0.001 by log-rank test). A single intraperitoneal bolus injection of 100 ng, 500 ng, or 2500 ng recombinant murine interleukin 6(rmIL-6) 2-4 hours before TBI extended the median survival to 32.5 days, 31.0 days, and 30.5 days, respectively (P < 0.01 by log-rank test for all dose groups). The improved survival was not due to any direct anti-leukemic activity of rmIL-6 and all control BALB/c mice (N = 15) that received the same doses of rmIL-6 but did not undergo TBI and BMT died of BCL-1 leukemia within 28 days with a median survival of 13.6 days. In contrast to rmIL-6, recombinant murine interleukin 5 (rmIL-5) had minimal radiosensitizing effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of Tyrosine Phosphorylation in Radiation-Induced Cell Cycle-Arrest of Leukemic B-Cell Precursors at the G2-M Transition Checkpoint

Here we provide experimental evidence that ionizing radiation induces inhibitory tyrosine phosphorylation of the p34cdc2 kinase in human leukemic B-cell precursors. Herbimycin A markedly reduced tyrosine phosphorylation of p34cdc2 in irradiated leukemic B-cell precursors, thereby preventing radiation-induced cell cycle arrest at the G2-M transition checkpoint. Thus, tyrosine phosphorylation is directly responsible for the inactivation of p34cdc2 in irradiated human leukemic B-cell precursors and activation of protein tyrosine kinases is a proximal and mandatory step in radiation-induced G2-arrest arrest at the G2-M checkpoint. Human WEE1 kinase isolated from unirradiated or irradiated leukemic B-cell precursors had minimal tyrosine kinase activity towards p34cdc2. We detected no increase of human WEE1 kinase activity after radiation of leukemic B-cell precursors, as measured by (a) autophosphorylation, (b) tyrosine phosphorylation of a synthetic peptide derived from the p34cdc2 amino-terminal region or (c) recombinant human p34cdc2-cyclin B complex. Thus the signaling pathway leading to inhibitory tyrosine phosphorylation of p34cdc2 and G2-arrest in irradiated human leukemic B-cell precursors functions independent of p49 WEE1 HU and enzymes which augment the tyrosine kinase activity of p49 WEE 1HU.