Ioannis D. Bonovolias

Multilevel targeting of hematopoietic stem cell self-renewal, differentiation and apoptosis for leukemia therapy

Human leukemias are considered clonal hematological malignancies initiated by chromosomal aberrations or epigenetic alterations occurring at the level of either pluripotent hematopoietic stem cells (HSCs) or early multipotent progenitors (MPPs). Leukemic cells are transformed, immortalized, actively proliferating cells that are still able to differentiate into cells resembling mature blood cells. Future therapies of leukemias require identification of molecular targets involved in hematopoiesis under normal and leukemic conditions and detailed understanding of the interactions between normal hematopoietic and leukemic cells within the bone marrow micro-environment. This review presents the basic aspects of hematopoiesis and highlights multilevel exploitable targets for leukemia therapy. These include HSC niche components, signaling pathways (SCF/c-kit-R, EPO-R-JAK2/STAT, Wnt, Notch, HOX), inducer-receptor interactions, superfine chromatin structure modifications, fused transcription factors, microRNAs and signaling of cell death through the Bcl-2 apoptotic switch (BH3-only proteins). The classes of therapeutics developed or being under development to eradicate human leukemias include novel antimetabolites, DNA hypomethylating agents, histone deacetylation inhibitors (HDACIs), retinoids and other inducers of differentiation, targeted monoclonal antibodies raised against cell surface proteins, pro-apoptotic receptor agonists (PARAs), BH3 peptidomimetics, cell cycle inhibitors, siRNAs and perhaps microRNAs. Some of these agents induce terminal differentiation while others promote cell cycle arrest and apoptosis in leukemia cells. At last but not least, this article describes the mechanisms of removal of damaged/harmful cells from organs since impairment in clearance of such cells can lead to autoimmune disorders by self-antigens.

Toward the Development of Innovative Bifunctional Agents To Induce Differentiation and To Promote Apoptosis in Leukemia: Clinical Candidates and Perspectives

Although the outcome of therapy for leukemia has improved over the years, mainly in younger patients, less than a third of adults with acute myeloid leukemia (AML), for example, are cured by current treatments, a fact stressing the need for new therapeutic approaches. Since leukemias are considered disorders of self-renewal, differentiation, and apoptosis of hematopoietic stem cells (HSCs) and/or their early progenitors, the treatment of leukemia is rapidly changing from conventional chemotherapy toward a more innovative individualized and targeted therapy. The discovery of leukemia stems cells (LSCs) in the late 1990s as a minor fraction within the subpopulation of hematopoietic cells and the compelling research efforts initiated thereafter have clearly shown that many malignancies are maintained via stemlike cells having the capacity for indefinite self-renewal. This LSC hypothesis has established the notion that the emergence of drug resistance and the clinical relapse of leukemias following an initial remission induced by cytotoxic or targeted therapy agents is related to acquired mutations of LSCs. Therefore, eradication of LSCs is considered necessary for the radical treatment of leukemias. As a matter of fact, novel exploitable targets for leukemia therapy emerged including enzymes like tyrosine kinases involved in signal transduction pathways, genes encoding proteins that regulate apoptosis and differentiation of malignant cells, celllineage transcriptional factors, angiogenesis factors, and unique proteins driving the cell cycle machinery. Interestingly, within the group of antileukemia agents exist small molecule drugs like tyrosine kinase inhibitors, proteasome inhibitors, farnesyl transferase inhibitors, hypomethylating agents, histone deacetylase inhibitors, mTOR targeting agents, bcl-2 inhibitors, and inhibitors of cyclin-dependent kinases (Figure 1). This paper is a comprehensive overview of the scientific efforts made to develop novel antileukemia therapeutics by presenting chemical, pharmacological, and pharmacogenomic data obtained during preclinical and clinical assessment of these agents.Furthermore, the designated synthesis of newmedicines inducing differentiation, cell cycle arrest, and/or promoting apoptosis along with multitargeted therapeutics will be also discussed. Such novel agents can be used in combination with other agents modulating different signaling pathways and molecular targets within the leukemia cells to overcome the emergence of drug resistance. This information can then be discussed from a pharmacogenomic view of antileukemia therapeutics. Individual genetic variations recorded in antileukemia drug therapy can be critical for personalized medicine and their clinical exploitation can achieve better pharmacotherapy outcomes.

Intracellular delivery of full length recombinant human mitochondrial L-Sco2 protein into the mitochondria of permanent cell lines and SCO2 deficient patient's primary cells

Mutations in human SCO2 gene, encoding the mitochondrial inner membrane Sco2 protein, have been found to be responsible for fatal infantile cardioencephalomyopathy and cytochrome c oxidase (COX) deficiency. One potentially fruitful therapeutic approach for this mitochondrial disorder should be considered the production of human recombinant full length L-Sco2 protein and its deliberate transduction into the mitochondria. Recombinant L-Sco2 protein, fused with TAT, a Protein Transduction Domain (PTD), was produced in bacteria and purified from inclusion bodies (IBs). Following solubilisation with l-arginine, this fusion L-Sco2 protein was transduced in cultured mammalian cells of different origin (U-87 MG, T24, K-562, and patients primary fibroblasts) and assessed for stability, transduction into mitochondria, processing and impact on recovery of COX activity. Our results indicate that: a) l-Arg solution was effective in solubilising recombinant fusion L-Sco2 protein, derived from IBs; b) fusion L-Sco2 protein was delivered successfully via a time- and concentration-dependent process into the mitochondria of human U-87 MG and T24 cells; c) fusion L-Sco2 protein was also transduced in human K-562 cells, transiently depleted of SCO2 transcripts and thus COX deficient; transduction of this fusion protein led to partial recovery of COX activity in such cells; d) [(35)S]Methionine-labelled fusion L-Sco2 protein, produced in a cell free transcription/translation system and incubated with intact isolated mitochondria derived from K-562 cells, was efficiently processed to yield the corresponding mature Sco2 protein, thus justifying the potential of the transduced fusion L-Sco2 protein to successfully activate COX holoenzyme; and finally, e) recombinant fusion L-Sco2 protein was successfully transduced into the mitochondria of primary fibroblasts derived from SCO2/COX deficient patient and facilitated recovery of COX activity. These findings provide the rationale of delivering recombinant proteins via PTD technology as a model for therapeutic approach of mitochondrial disorders.

Hemin counteracts the repression of Bcl-2 and NrF2 genes and the cell killing induced by imatinib in human Bcr-Abl(+) CML cells.

Imatinib is a targeted selective inhibitor of chimaeric Bcr-Abl tyrosine kinase developed for effective therapy of chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL) patients. Unfortunately, evidence now exists to indicate that a portion of such patients treated with imatinib acquire resistance and subsequently relapse. To understand the heterogeneous basis of imatinib resistance, we have investigated the possible mechanism(s) via which hemin, a key regulator of hematopoiesis that is converted to heme intracellularly, renders CML cells less susceptible to imatinib. Hemin at 30-90 aM protected a substantial proportion (>40%) of human Bcr-Abl(+) CML cells (K-562 and KU-812) from imatinib-induced cell killing by increasing the imatinib IC50 value, reducing DNA damage, and promoting erythroid differentiation. RT-PCR assessment of RNA transcripts encoded by human GAPDH, Ggamma-globin, Bcr-Abl, HO-2, Hpr-6, CEBPa, Bcl-2a, Bcl-2b, and Nrf2 genes revealed that hemin selectively counteracted the repression of antiapoptotic Bcl-2a, Bcl-2b, and Nrf2 genes in imatinib-treated cells. These genes are markedly repressed by imatinib alone in human K-562 CML cells. Hemin, however, had no detectable effect on the expression of the Bcr-Abl gene. Moreover, inhibition of de novo heme biosynthesis by succinyl-acetone enhanced the killing effect of imatinib. These data clearly indicate that: (a) cellular heme resulted from de novo biosynthesis and hemin uptake alters the developmental stage of human Bcr-Abl(+) CML cells and their susceptibility to imatinib; (b) cellular heme counteracts the ability of imatinib to repress Bcl-2 and Nrf2 gene expression; and (c) inhibitors of de novo biosynthesis can be developed and combined with imatinib to enhance its antileukemic activity.

Imatinib inhibits the expression of SCO2 and FRATAXIN genes that encode mitochondrial proteins in human Bcr–Abl+ leukemia cells

Imatinib mesylate (IM/Gleevec®), a selective inhibitor of chimeric Bcr-Abl tyrosine kinase, was developed as a first line drug to treat CML and ALL Ph(+) patients. Earlier studies have shown that hemin counteracts the IM-induced cell killing in human K-562 CML cells. In this study, we investigated whether IM disrupts the heme-dependent Cytochrome c Oxidase (COX) Biosynthesis and Assembly Pathway (HDCBAP) in Bcr-Abl(+) and Bcr-Abl(-) cells by affecting the expression of key-genes. Cells were exposed to IM and evaluated at time intervals for cell growth, cell death, expression of various genes by RT-PCR analysis as well as Sco2 mature protein levels by western blot analysis and COX enzymatic activity. IM at 1 μM induced extensive cell growth inhibition and cell death as well as marked suppression of the expression of SCO2 and FRATAXIN (FXN) genes in human K-562 and KU-812 Bcr-Abl(+) CML cells. IM also reduced the protein level of mature Sco2 mitochondrial protein as well as COX activity in these cell lines. However, treatment of human MOLT-4 Bcr-Abl(-) cells with 1μM and even with higher concentrations (4×10(-5)M) of IM neither reduced the expression of SCO2 and FXN genes nor suppressed the protein level of mature Sco2 protein and COX activity. Our findings indicate that SCO2 and FXN genes, involved in HDCBAP, are repressed by IM in human Bcr-Abl(+) CML cells and may represent novel target sites in leukemia therapy.