In vitro and in vivo chemosensitizing activity of LFM-A13, a dual-function inhibitor of Bruton's tyrosine kinase and polo-like kinases, against human leukemic B-cell precursors

The present study documents the chemosensitizing anti-leukemic activity of the leflunomide metabolite (LFM) analog, LFM-A13, a dual-function inhibitor of Bruton's tyrosine kinase (BTK) and Polo-like kinases (PLK), against human leukemic B-cell precursors. The results in 135 xenografted NOD/SCID mice regarding the anti-leukemic activity of GMP-grade LFM-A13, obtained with only 4-days of LFM-A13 therapy at nontoxic dose levels corresponding to 1-20% of its NOAEL (no observable advserse effect level), alone or in combination with the standard chemotherapy drug vincristine, demonstrate the potential of LFM-A13 as a new anti-leukemic drug candidate. All 82 LFM-A13-treated mice, including those receiving a combination of vincristine + LFM-A13 at the highest dose level of LFM-A13, tolerated their treatments well without weight loss, diarrhea, lethargy/ paralysis, other signs of morbidity, or mortality. The present study provides preclinical proof-of-principle for the development of LFM-A13 as a new chemosensitizing and apoptosis-promoting anti-leukemic agent and lends support to the hypothesis that the chemoresistance of relapsed B-cell precursor acute lymphoblastic leukemia (BCP-ALL) can be overcome by using LFM-A13 in combination with chemotherapy. Also presented are the results of a comprehensive meta-analysis of the overexpression of genes for LFM-A13 targeted kinases and their downstream effector molecules in B-lineage lymphoid malignancies utilizing the Oncomine database.     

Chemoprevention of colorectal cancer by targeting Janus kinase 3 with a rationally designed small molecule inhibitor

The effects of the rationally designed JAK3 inhibitor JANEX-1 on the development of intestinal tumors in the APC(min) mouse model of familial adenomatous polyposis were examined. At a non-toxic dose level, >4 times lower than its day 30 LD(10), JANEX-1 was highly effective in preventing intestinal tumor development in Min mice, resulting in markedly improved survival outcomes. JAK3 inhibitors may, therefore, be useful in the chemoprevention of colorectal cancer. Here, an overview regarding the potential of JANEX-1 as a chemopreventive agent is provided.     

Radiobiological features of human pluripotent bone marrow progenitor cells (CFU-GEMM)

The purpose of this study was to evaluate the radiobiologic features of human pluripotent bone marrow progenitor cells (CFU-GEMM; colony forming unit-granulocyte-erythroid-macrophage/monocyte-megakaryocyte). Experiments were performed using fresh bone marrow cells as well as bone marrow cells stimulated with recombinant granulocyte-macrophage-colony stimulating factor (rGM-CSF) to increase the CFU-GEMM pool. The D0 values for CFU-GEMM in normal bone marrow samples (n = 9) ranged from 30.9 cGy to 85.7 cGy (mean +/- SE = 54.4 cGy +/- 6.2 cGy) and the D0 value of the composite radiation survival curve was 56.9 cGy, indicating that CFU-GEMM were acutely sensitive to the lethal effects of ionizing radiation. There was no distinct shoulder on the single dose radiation survival curves with Dq values ranging from -29.6 cGy to 4.4 cGy, and no increase in CFU-GEMM survival was observed when the radiation was fractionated. Hence, CFU-GEMM were unable to repair sublethal radiation damage. These findings confirm and extend previous studies on the radiobiologic features of human hematopoietic progenitor cell populations.     

Increased efficacy in selective elimination of leukemic cell line clonogenic cells by a combination of monoclonal antibodies BA-1, BA-2, BA-3 + complement and mafosfamid (ASTA Z 7557)

Studies were undertaken to examine the efficacy of combining monoclonal antibodies BA-1, BA-2, BA-3 + complement with a stabilized derivative of cyclophosphamide (mafosfamid) for elimination of leukemic cell line clonogenic cells. In a series of experiments conducted with leukemic cell lines a combination of two cycles of treatment with BA-1, 2, 3 + complement combined with 25 micrograms/ml-1 mafosfamid was superior to either modality alone in killing clonogenic leukemic cells. Identical treatment of normal bone marrow had no effect on the monopotent stem cells CFU-E and CFU-GM, or the pluripotent stem cell CFU-MIX, but some inhibition of the monopotent stem cell CFU-MK did occur. These results suggest that consideration be given to incorporation of BA-1, 2, 3 + complement and mafosfamid combination treatment in autologous bone marrow transplantation for acute lymphoblastic leukemia.     

Effects of the Intermolecular Toxin-Monoclonal Antibody Linkage on the In Vivo Stability, Immunogenicity and Anti-Leukemic Activity of B43 (Anti-CD19) Pokeweed Antiviral Protein Immunotoxin

We have successfully constructed highly potent and selective anti-CD19 PAP immunotoxins using each of the three crosslinking agents, SPDP, LC-SPDP, or SMPT, to generate an intermolecular bridge between the B43 MoAb and PAP toxin moieties. These immunotoxins were selectively immunoreactive with and cytotoxic against CD19' B-lineage ALL cells. In this report, we compared (a) in vivo chemical, immunological, and biological stability, (b) in uiuo immunogenicity, and (c) in vivo anti-leukemic activity of various B43-PAP immunotoxin constructs. Our data recommend the use of SPDP and SMPT rather than LC-SPDP for generation of B43(anti-CD19)-PAP immunotoxins as clinical anti-leukemic agents. To our knowledge, this is the first comparative analysis of the in vivo pharmacokinetic features, immunogenicity, and anti-leukemic activity of anti-CD19 PAP immunotoxins that were prepared with different heterobifunctional crosslinking agents.     

Isolation and characterization of pokeweed antiviral protein mutations in Saccharomyces cerevisiae: identification of residues important for toxicity.

Pokeweed antiviral protein (PAP), a 29-kDa protein isolated from Phytolacca americana inhibits translation by catalytically removing a specific adenine residue from the 28S rRNA of eukaryotic ribosomes. PAP has potent antiviral activity against many plant and animal viruses, including human immunodeficiency virus. We describe here development of a positive selection system to isolate PAP mutants with reduced toxicity. In vitro translation in the presence or absence of microsomal membranes shows that PAP is synthesized as a precursor and undergoes at least two different proteolytic processing steps to generate mature PAP. The PAP cDNA was placed under control of the galactose-inducible GAL1 promoter and transformed into Saccharomyces cerevisiae. Induction of PAP expression was lethal to yeast. The PAP expression plasmid was mutagenized and plasmids encoding mutant PAP genes were identified by their failure to kill S. cerevisiae. A number of mutant alleles were sequenced. In one mutant, a point mutation at Glu-177 inactivated enzymatic function in vitro, suggesting that this glutamic acid residue is located at or near the catalytic site. Mutants with either point mutations near the N terminus or a nonsense mutation at residue 237 produced protein that was enzymatically active in vitro, suggesting that the toxicity of PAP is not due solely to enzymatic activity. Toxicity of PAP appears to be a multistep process that involves possibly different domains of the protein.     

Interleukin 7 receptor engagement stimulates tyrosine phosphorylation, inositol phospholipid turnover, proliferation, and selective differentiation to the CD4 lineage by human fetal thymocytes.

The purposes of this study were to elucidate the effects of recombinant human interleukin 7 (rhIL-7) on proliferation as well as differentiation of human fetal thymocytes and to analyze the biochemical nature of the IL-7 receptor-linked transmembrane signal. In the absence of costimulants, rhIL-7 stimulated the in vitro proliferation and colony formation of CD4+CD8+ double-positive immature fetal thymocytes. Furthermore, rhIL-7 promoted partial differentiation of immature thymocytes with a selective advantage for the development of CD4+CD8- single-positive thymocytes. Our observations suggest that IL-7 likely has an important regulatory role during the earliest stages of human T-cell ontogeny. Stimulation of fetal thymocytes with rhIL-7 resulted in enhanced tyrosine phosphorylation of three distinct phosphoproteins with molecular masses of 72, 98, 123, and 190 kDa and induced a rapid and biphasic increase in the production of inositol 1,4,5-trisphosphate, which was inhibitable by the tyrosine protein kinase inhibitor genistein. Thus, the transmembrane signal triggered by engagement of the IL-7 receptor is intimately linked to a functional tyrosine protein kinase pathway and stimulates the inositol phospholipid turnover and proliferation, as well as selective differentiation to the CD4 lineage, by human fetal thymocytes.