Purpose: The ubiquitously expressed transmembrane glycoprotein CD47 delivers an anti-phagocytic (do not eat) signal by binding signal-regulatory protein a (SIRPa) on macrophages. CD47 is overexpressed in cancer cells and its expression is associated with poor clinical outcomes. TTI-621 (SIRPaFc) is a fully human recombinant fusion protein that blocks the CD47-SIRPa axis by binding to human CD47 and enhancing phagocytosis of malignant cells. Blockade of this inhibitory axis using TTI-621 has emerged as a promising therapeutic strategy to promote tumor cell eradication.Experimental Design: The ability of TTI-621 to promote macrophage-mediated phagocytosis of human tumor cells was assessed using both confocal microscopy and flow cytometry. In vivo antitumor efficacy was evaluated in xenograft and syngeneic models and the role of the Fc region in antitumor activity was evaluated using SIRPaFc constructs with different Fc tails.Results: TTI-621 enhanced macrophage-mediated phagocytosis of both hematologic and solid tumor cells, while sparing normal cells. In vivo, TTI-621 effectively controlled the growth of aggressive AML and B lymphoma xenografts and was efficacious in a syngeneic B lymphoma model. The IgG1 Fc tail of TTI-621 plays a critical role in its antitumor activity, presumably by engaging activating Fcg receptors on macrophages. Finally, TTI-621 exhibits minimal binding to human erythrocytes, thereby differentiating it from CD47 blocking antibodies.Conclusions: These data indicate that TTI-621 is active across a broad range of human tumors. These results further establish CD47 as a critical regulator of innate immune surveillance and form the basis for clinical development of TTI-621 in multiple oncology indications.

Overexpression of vsr in Escherichia coli stimulates transition and frameshift mutations. The pattern of mutations suggests that mutagenesis is due to saturation or inactivation of dam-directed mismatch repair.Escherichia coli has a DNA repair system dedicated to converting T/G mismatches into C ⅐ G base pairs in C(T/G)WGG and related sequences (8,12). Very short patch (VSP) repair is mediated by a sequence-and mismatch-specific endonuclease (10), the product of the vsr gene (20). The second C in CCW GG sequences in methylated in E. coli by the Dcm methylase. Thus, the main function of VSP repair is probably to correct T/G mismatches which result from deamination of 5-methylcytosine to thymine, thereby minimizing CCWGG-to-CTWGG mutations (11).C(T/G)WGG heteroduplexes can also arise from errors in DNA replication. Such errors are normally corrected by the dam-directed repair system in favor of the base on the template strand (16). Thus, a T/G mismatch arising from misreplication of CTWGG should be repaired to T ⅐ A. However, there is good evidence that VSP repair outcompetes damdirected repair for C(T/G)WGG substrates, converting the mismatch to C ⅐ G (21). Dominance of VSP repair over damdirected repair may account for the overrepresentation of CC WGG in the E. coli genome and the underrepresentation of CTWGG (1,14).The purpose of our research was to determine whether overexpression of vsr would stimulate CTAGG-to-CCAGG mutations ( Fig. 1). Overexpression of vsr was accomplished by subcloning it from pDVW (17) into pKK233-2 (Pharmacia), under the control of the strong, constitutive trc promoter (pKK-V). vsr-lacZ fusion plasmids were used to estimate levels of vsr expression from the synthetic trc promoter (pKK-VZ) and from the wild-type promoter 5Ј of the overlapping dcm gene (7) (pVZ). All four plasmids are shown in Fig. 2. CC221 cells containing pKK-VZ produce about 6,000 U of ␤-galactosidase, while cells containing pVZ produce about 300 U, indicating that pKK-V overproduces Vsr.CC221 [ara ⌬(gpt-lac)5 thi ⌬(supD-dcm-fla) zee3129::Tn10], a derivative of CSH142 (15), contains a large chromosomal deletion which removes dcm, vsr, and at least 20 kb of surrounding sequence. The deletion was introduced by P1 transduction from RP4182 (courtesy of A. S. Bhagwat) using a tetracycline resistance marker from CAG12099 (19). The deletion was verified by Southern hybridization using a dcm-vsr probe. Mutagenesis assays were done by introducing pKK-V and pKK233-2 into a set of CC221 derivatives containing FЈ lacZ lacY ϩ A ϩ proA ϩ B ϩ episomes. Each episome has a point mutation in lacZ which makes the cell Lac Ϫ ; reversion to Lac ϩ requires a specific base substitution or frameshift mutation. FЈ CC101 to CC111 have been described previously (4, 6). FЈ CC113 was constructed for this study.FЈ CC113 contains an amber mutation at codon 999 in lacZ, introduced by site-directed mutagenesis as described previously (5). The substitution of an amber codon (TAG) for the wild-type tryptophan codon (TGG) eliminates ␤-galactosidase activ...

ML-133 is a novel small molecule with potent antiproliferative activity, as shown in cancer cell lines and in a human colon tumor xenograft model. ML-133 reduces the concentration of intracellular labile zinc in HT-29 colon cancer cells, leading to induction of the Krüppel-like factor 4 transcription factor. Krüppel-like factor 4 displaces the positive regulator SP1 from the cyclin D1 promoter, thereby negatively regulating the expression of cyclin D1 and promoting the G 1 -S phase arrest of cell proliferation. The antiproliferative and antitumor activity of ML-133 described in the present study suggests modulation of intracellular zinc homeostasis as a potential strategy for the treatment of several cancer types, and ML-133 represents a promising new class of antitumor agents that deserves further development. [Mol Cancer Ther 2009;8(9):2586-96]

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Although clotrimazole (CLT), an antifungal drug, inhibits tumor cell proliferation and angiogenesis, its clinical application is hampered by significant hepatotoxicity due to the presence of an imidazole moiety. In our attempts to develop CLT analogs that are devoid of imidazole and are as efficacious as CLT, one pharmacophore designated NC381 was generated and shown to inhibit tumor cell growth via a mechanism similar to that of CLT. In vitro, treatment of NCI-H460 nonsmall cell lung cancer (NSCLC) cells with NC381 inhibited growth in a time-dependent manner. Flow cytometric analysis demonstrated that the decrease in cell growth was associated with inhibition of cell cycle progression at the G 1 -S phase transition, resulting in G 0 -G 1 arrest. There was a concomitant inhibition of cyclin D1 expression and subsequent reduction in the formation of the cyclin D1-CDK4 complex. Consistent with a decrease in the cyclin D1-CDK4 complex, NC381 treatment resulted in significant inhibition of pRb phosphorylation. There also were changes in the activity of cell cycle-related proteins, including p16Ink4 and p27 Kip1 . Together, these results are consistent with a model in which NC381 arrests cell cycle progression via inhibition of the pathway that promotes exit from the G 1 phase of the cell cycle. Furthermore, the clinical applicability of NC381 was evaluated in an in vivo murine xenograft model of human NSCLC (NCI-H460). NC381 treatment resulted in significant inhibition of tumor growth. Given the poor prognosis and the limited treatment options available, the present results underscore the potential of NC381 in the treatment of human NSCLC.