Despite recent advances, treatment of leukemia is often not curative. New insights indicate that this may be attributable to a small population of therapy-resistant malignant cells with self-renewal capacity and the ability to generate large numbers of more differentiated leukemia cells. These leukemia-initiating cells are commonly referred to as Leukemia Stem Cells (LSCs). LSCs are regarded as the root of leukemia origin and leukemia recurrence after seemingly successful therapy. Not surprisingly therefore, contemporary leukemia research has focused on ways to specifically eliminate LSCs, leading to the identification of several promising anti-LSC strategies. Firstly, LSCs may be eliminated by antibody- or ligand-based cell surface delivery of therapeutics such as naked antibodies, immunotoxins, and immunocytokines. This approach exploits LSC-associated surface antigens, such as CD33, CD44, CD96, CD123 and CLL-1 for LSC-selective therapy and aims to spare normal hematopoietic stem cells. A second strategy aims to disrupt the interactions between LSCs and their highly specialized niche. These interactions appear to be pivotal for maintenance of the stem cell-like characteristics of LSCs. A third strategy centers on the selective modulation of aberrantly activated signaling pathways central to LSC biology. A fourth strategy, dubbed 'epigenetic reprogramming', aims to selectively reverse epigenetic alterations that are implicated in ontogeny and maintenance of LSCs. In this review, we will discuss the rationale for these LSCs-targeted strategies and highlight recent advances that may ultimately help pave the way towards selective LSCs-elimination.

Currently, the majorities of AML patients eventually relapse and develop refractory disease indicating that novel and preferably targeted approaches are urgently needed. recently, C-type Lectin-Like Molecule-1 (CLL-1) has been identified as a cell surface marker expressed in ~90% of the AML patients. Importantly, CLL-1 is expressed on AML blasts and CD34+/CD38AML stem cells, but not on normal hematopoietic stem cells. Moreover, this expression profile is retained under all conditions of treatment and disease. This prompted us to develop a novel approach for the targeted elimination of CLL-1+ AML and AML stem cells. To this end, a CLL-1-specific antibody fragment (scFvCLL-1) was genetically fused to soluble forms of the pro-apoptotic proteins TrAIL (sTrAIL) and FasL (sFasL), yielding fusion proteins scFvCLL-1:sTrAIL and scFvCLL-1:sFasL, respectively. Both fusion proteins potently induced CLL-1restricted apoptosis in AML cell lines and primary AML cells. Moreover, CD34+/ CD38AML stem cells were sensitive to treatment with scFvCLL-1:sTrAIL and scFvCLL-1:sFasL. In contrast, human CLL-1hepatocytes were fully resistant to treatment, whereas CLL-1+ monocytes showed moderate sensitivity to scFvCLL1:sFasL. In conclusion, treatment with scFvCLL-1:sTrAIL and scFvCLL-1:sFasL results in AML-restricted apoptosis including the CD34+/CD38AML stem cells. This novel approach may be of value for the treatment of AML

Gemtuzumab ozogamicin (GO, Mylotarg) is a targeted therapeutic agent in which an anti-CD33 antibody is chemically coupled to a highly cytotoxic calicheamicin derivative through a hydrolysable linker. GO has improved the treatment outcome for a subgroup of acute myeloid leukemia (AML) patients, but its use is associated with severe myelosuppression and hepatotoxicity. Here, we report on a novel anti-leukemia agent, designated scFvCD33:sTRAIL, in which an anti-CD33 single chain fragment of variable regions (scFv) antibody fragment is genetically linked to soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). Normal CD33-positive monocytes were fully resistant to prolonged treatment with scFvCD33:sTRAIL, whereas treatment with GO resulted in substantial cytotoxicity. The activity of scFvCD33:sTRAIL towards AML cells was up to 30-fold higher than GO. The CD33-restricted anti-leukemia activity of scFvCD33:sTRAIL remained stable during prolonged storage at 37 °C, whereas GO showed a rapid increase in CD33-independent cytotoxicity. Moreover, scFvCD33:sTRAIL showed potent anti-leukemia activity towards CD33+ CML cells when treatment was combined with the Bcr-Abl tyrosine kinase inhibitor, Gleevec. Importantly, ex vivo treatment of patient-derived CD33+ AML tumor cells with scFvCD33:sTRAIL resulted in potent apoptosis induction that was enhanced by valproic acid, mitoxantrone and 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG). Taken together, scFvCD33:sTRAIL is superior to GO in terms of tumor selectivity, activity and stability, warranting its further development for the treatment of CD33-positive leukemias.

Gemtuzumab ozogamicin (GO) is a calicheamicin-conjugated antibody directed against CD33, an antigen highly expressed on acute myeloid leukemic (AML) cells. CD33-specific binding triggers internalization of GO and subsequent hydrolytic release of calicheamicin. Calicheamicin then translocates to the nucleus, intercalates in the DNA structure and subsequently induces double-strand DNA breaks. GO is part of clinical practice for AML, but is frequently associated with severe side effects. Therefore, combination of GO with other therapeutics is warranted to reduce toxicity, while maximizing therapeutic selectivity. We hypothesized that the histone deacetylase inhibitor valproic acid (VPA) sensitizes AML cells to GO. VPA-induced histone hyperacetylation opens the chromatin structure, whereby the DNA intercalation of calicheamicin should be augmented. We found that clinically relevant concentrations of VPA potently augmented the tumoricidal activity of GO towards AML cell lines and primary AML blasts. Moreover, VPA treatment indeed augmented the DNA intercalation of calicheamicin and enhanced DNA degradation. Importantly, synergy was restricted to CD33-positive AML cells and did not require caspase activation. In conclusion, the synergistic proapoptotic activity of cotreatment of AML cells with VPA and GO indicates the potential value of this strategy for AML.

Agonistic anti-Fas antibodies and multimeric recombinant Fas ligand (FasL) preparations show high tumoricidal activity against leukemic cells, but are unsuitable for clinical application due to unacceptable systemic toxicity. Consequently, new antileukemia strategies based on Fas activation have to meet the criterion of strictly localized action at the tumor-cell surface. Recent insight into the FasL/Fas system has revealed that soluble homotrimeric FasL (sFasL) is in fact nontoxic to normal cells, but also lacks tumoricidal activity. We report on a novel fusion protein, designated scFvCD7:sFasL, that is designed to have leukemia-restricted activity. ScFvCD7:sFasL consists of sFasL genetically linked to a high-affinity single-chain fragment of variable regions (scFv) antibody fragment specific for the T-cell leukemia-associated antigen CD7. Soluble homotrimeric scFvCD7:sFasL is inactive and acquires tumoricidal activity only after specific binding to tumor cell-surface-expressed CD7. Treatment of T-cell acute lymphoblastic leukemia (T-ALL) cell lines and patient-derived T-ALL, peripheral T-cell lymphoma (PTCL), and CD7-positive acute myeloid leukemia (AML) cells with homotrimeric scFvCD7:sFasL revealed potent CD7-restricted induction of apoptosis that was augmented by conventional drugs, farnesyl transferase inhibitor L-744832, and the proteasome inhibitor bortezomib (Velcade; Millenium, Cambridge, MA). Importantly, identical treatment did not affect normal human peripheral-blood lymphocytes (PBLs) and endothelial cells, with only moderate apoptosis in interleukin-2 (IL-2)/CD3-activated T cells. CD7-restricted activation of Fas in T-cell leukemic cells by scFvCD7:sFasL revitalizes interest in the applicability of Fas signaling in leukemia therapy.

This in vitro study aimed at investigating whether full remineralization would occur in white spot lesions when the surface porosity was increased by acid-etching. The effect of fluoride was also investigated. Enamel blocks with in vitro produced white spot lesions were used. Group A was exposed to a remineralizing solution only. In group B, the lesions were etched with 35% phosphoric acid for 30 s, then treated as in group A. Group C was treated as group A + daily treatment with a fluoride toothpaste slurry (1,000 ppm) for 5 min. Group D was treated as group B + the daily fluoride treatment of group C. The remineralization was measured weekly with Quantitative Light-induced Fluorescence during the experimental period. After 10 weeks of remineralization, mineral profiles were assessed with transverse microradiography. The enamel fluorescence was partly regained. There were significant differences in the lesion depth, mineral content at the surface layer, and integrated mineral loss between the groups. Addition of fluoride accelerated the remineralization only in the beginning; in later stages the process leveled out and even reached a plateau in all the groups. It was concluded that full remineralization was not achieved by etching, by the addition of fluoride, nor by the combination of both treatments in this in vitro study.