For instance, doxorubicin, commonly used anticancer drug, was conjugated with RGD-peptides for integrin targeted therapy. A doxorubicin-RGD4C conjugate (doxo-RGD4C) is one of good examples. Doxo-RGD4C demonstrated equal efficacy as free doxorubicin in vitro and, more importantly, doxo-RGD4C showed improved inhibition of tumor growth and spreading of pulmonary metastases than free doxorubicin in mouse MDA-MB-435 breast cancer model, in which integrin αvβ3 is expressed by the endothelium in the angiogenic blood vessels and by the tumor cells themselves 53. In addition, doxo-RGD4C was also found to be less toxic to liver and heart. In another similar study, doxo-RGD4C was tested in mouse αvβ3-negative MH134 hepatoma tumor model. As compared to doxorubicin alone, the doxo-RGD4C conjugate showed less treatment efficacy in vitro; however, the doxo-RGC4C conjugate demonstrated better anti-tumor activity in vivo. Because direct anti-tumor cell effects of the targeted doxorubicin are not expected in this integrin αvβ3-negative tumor model, this study suggests the anti-angiogenetic effect on the endothelial cells induced by doxo-RGD4C conjugate may lead to tumor recession 54.
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Tissue Factor (TF) is a cell membrane receptor protein that is the initiator of the extrinsic pathway of the blood coagulation cascade and normally released from damaged tissues 82. It is expected that this potent thrombogenic protein in its truncated form (tTF) can be targeted to the tumor, occlude the tumor's blood supply and, thus cause rapid tumor destruction. To test this hypothesis, three fusion proteins, chTNT-3/tTF, chTV-1/tTF, and RGD/tTF, which target DNA exposed in degenerative areas of tumors, fibronectin on the tumor vascular basement membrane, and integrin αvβ3 on the luminal side of tumor vessels, respectively, were developed and tested for their antitumor effects 82. In vitro, all fusion proteins retained similar thrombotic activity. In MAD109 mouse lung tumor model, RGD-tTF was found to be localized mainly in capillaries and small vessels of the tumor. In vivo, daily injections of RGD-tTF resulted in thrombosis of about 40% of the tumor blood vessels, but no significant inhibition of tumor growth was observed. In contrast, the other two fusion proteins showed thrombosis in up to 80% of the scored blood vessels, leading to massive tumor necrosis and more than 50% reduction in tumor volume versus the RGD-tTF group. Similar results were obtained in the C26 colon carcinoma model. The data implied that these thrombogenic agents had to occlude medium and large vessels within the tumor in order to attain a significant antitumor effect. Interestingly, most impressive tumor suppression was observed for the combination therapy of all three-fusion proteins, suggesting the delivery of tTF to all available targets produced an additive thrombotic effect.
Interleukin 12 (IL-12) plays an important role in the activities of natural killer (NK) cells and T lymphocytes. It mediates enhancement of the cytotoxic activity of NK cells and CD8+ cytotoxic T lymphocytes. However, systemic administration of IL-12 was associated with dose-limiting toxicity, thus preventing IL-12 from attaining its full therapeutic potential 83. A fusion protein was synthesized by conjugating mouse IL-12 with RGD4C 84. The results showed that RGD4C-IL-12 retained the immunostimulatory activity of IL-12. In corneal angiogenesis assay, RGD4C-IL-12 demonstrated excellent inhibition of bFGF-induced vessel growth, whereas native mIL-12 only partially inhibited neovascularization. In addition, in a neuroblastoma model (NXS2 model), RGD4C-IL-12 showed an improved antitumor effect, whereas native IL-12 was not effective. Moreover, RGD4C-mediated targeting prevented IL-12 induced hepatic necrosis, which was observed after continuous subcutaneous infusion for two weeks via surgically implanted osmotic pumps. While RGD-IL-12 was tested in knockout mice lacking the IL-12 receptor, RGD4C-IL-12 showed the neovascularization inhibition for up to 25%, whereas mIL-12 was completely ineffective. The enhanced antiangiogenic effect of mrIL-12vp may involve several mechanisms, including increased IL-12 concentrations delivered directly to angiogenic endothelial cells, activation of immune cells within the angiogenic site, and contribution of RGD4C in suppressing endothelial cell survival pathways.
The RGD-containing peptide-decorated nanoparticulate delivery system has been extensively investigated. For example, RGD-peptides were coupled to the distal end of poly(ethylene glycol)-coated liposomes (LCL) to obtain a stable long-circulating drug delivery system functioning as a platform for multivalent interaction with integrin αvβ3 91. The results showed that RGD-peptide-modified LCL exhibited increased binding to endothelial cells in vitro. Moreover, intravital microscopy demonstrated a specific interaction of these liposomes with tumor vasculature, a characteristic not observed for LCL. In in vivo study, RGD-LCL encapsulating doxorubicin inhibited tumor growth in a doxorubicin-insensitive murine C26 colon carcinoma model, whereas doxorubicin in LCL failed to decelerate tumor growth. Overall, RGD-LCL containing doxorubicin showed superior efficacy over non-targeted LCL in inhibiting C26 doxorubicin-insensitive tumor outgrowth. Likely, these RGD-LCL-doxorubicin antitumor effects are brought about through direct effects on tumor endothelial cells 91. Recently, cRGDfK peptide was coupled to PEGylated liposome encapsulating anticancer drug (doxorubicin) to target integrin αvβ3-expressing tumor vasculature 92. The results showed that delivery of targeted nanoparticles inhibited the growth of metastases while eliminating the toxicity and weight loss associated with systemic administration of doxorubicin. The delivery resulted in a 15-fold improvement in tumor and anti-metastatic activity when compared with administration of the free drug. The preferential activity of these nanoparticles on metastases implies that growing metastatic tumors may have a greater dependence on angiogenic vessels and thus could be more susceptible to integrin αvβ3-targeted therapy 93. In another example, Xiong et al. achieved high tumor accumulation and intercellular delivery of doxorubicin after conjugating synthetic RGD mimic compound with the drug-loading liposome in syngeneic B16 melanoma mouse model. Administration of RGD mimic-modified nanoparticle resulted in retarded tumor growth and prolonged lifespan compared with the non-modified one 94-95. Similar RGD liposome modification strategies have also been used to deliver other anticancer drugs, such as 5-fluorouracil (5-FU) and paclitaxel, to malignant tumor-bearing animals. The significant anti-primary tumor and antimetastatic activities can also be achieved 96, 52, 97. The liposomal delivery of a new snake venom disintegrin, contortrostatin (CN) has been reported in an orthotopic human breast tumor xenograft model 98. This disintegrin modulates its interaction with integrins on tumor cells and angiogenic vascular endothelial cells.
The JAK2 inhibitor, ruxolitinib, has been shown to reduce tumor burden in xenograft mouse models harboring BCR-JAK2 [t(9;22)(p24;q11.2)] [88], and has demonstrated promising results in the treatment of CRLF2-rearranged, JAK2-mutated leukemic cells in vitro [87]. Additionally, the PI3K inhibitor, rapamycin, has been shown to control leukemic burden [88]. Clinical trial NCT01251965, utilizing ruxolitinib in refractory or relapsed ALL or AML (acute myelogenous leukemia), is currently ongoing.
More recently, another potential molecular target in JAK2-mutated B-ALL was revealed. In a mouse model, overexpression of mutant JAK2 led to downstream upregulation of prosurvival Bcl-2 gene family members, and combined use of the Bcl-2/Bcl-xL inhibitor ABT-737 with JAK2 inhibitors prolonged disease regression time [89].
Epigenetic studies have also shown insights into our understanding of MLL- rearranged B-ALL. MicroRNAs (miRNAs), short noncoding RNAs involved in the regulation of signaling pathways of cell differentiation, proliferation, and apoptosis, have been shown to promote leukemogenesis through aberrant epigenetic activity [98]. The presence of these epigenetic aberrancies suggests that histone deacetylase (HDAC), DNA methyltransferase (DNMT), and/or histone methyltransferase (HMT) inhibitors may play a therapeutic role in MLL-rearranged B-ALL [99, 100]. Notably, selective DOT1L HMT inhibitors, such as EPZ-004777, have been shown to selectively destroy MLL-rearranged cells in mouse models [101, 102]. Clinical trial NCT01684150 is currently evaluating the use of the DOT1L HMT inhibitor, EPZ-5676, in adults with MLL-rearrangements. 2ff7e9595c
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