Diletta Laccabue

Current Status and Perspectives in the Development of Camptothecins

Camptothecins are cytotoxic agents with a wide spectrum of antitumor activity. The unique mechanism of action, the impressive preclinical efficacy and the clinical success of irinotecan and topotecan have stimulated intensive efforts to identify novel analogues. The development of novel camptothecins was recently rationalized on the basis of the detailed knowledge of mechanism of drug-target interaction and was aimed to overcome the major limitations of these drugs (i.e. lactone ring instability and reversibility of topoisomerase I-DNA cleavage complexes). The development of novel series of analogues (7-substituted camptothecins, silatecans and homocamptothecins) resulted in identification of promising compounds, which are currently in clinical development. Considering the lack of precise correlations between preclinical activity and clinical efficacy of camptothecins, the potential advantages of novel analogs in clinical therapy remains to be documented. However, a rational basis for drug selection and development is now provided by the recognition of major limitations of these agents and by a detailed knowledge of multiple interactions between drug, cellular target and serum albumin. Inhibition of the nuclear enzyme DNA topoisomerase I has proven to be a promising strategy in the design of antitumor agents, in spite of a limited cellular basis of selectivity in cytotoxic action of camptothecins (i.e., overexpression of the target enzyme in tumor cells, and increased sensitivity of proliferating cells). The interest in topoisomerase I as a therapeutic target promoted various efforts to identify other chemotypes effective as topoisomerase inhibitors and chemical/modelling efforts to rationally design specific analogs among known inhibitors. Additional approaches, including drug delivery/formulation, optimization of dose/schedule and route of administration, are expected to improve the therapy with camptothecins and other inhibitors.

Inhibition of c-Met and prevention of spontaneous metastatic spreading by the 2-indolinone RPI-1

Hepatocyte growth factor (HGF) and its tyrosine kinase receptor Met play a pivotal role in the tumor metastatic phenotype and represent attractive therapeutic targets. We investigated the biochemical and biological effects of the tyrosine kinase inhibitor RPI-1 on the human lung cancer cell lines H460 and N592, which express constitutively active Met. RPI-1-treated cells showed down-regulation of Met activation and expression, inhibition of HGF/Met-dependent downstream signaling involving AKT, signal transducers and activators of transcription 3 and paxillin, as well as a reduced expression of the proangiogenic factors vascular endothelial growth factor and basic fibroblast growth factor. Cell growth in soft agar of H460 cells was strongly reduced in the presence of the drug. Furthermore, RPI-1 inhibited both spontaneous and HGF-induced motility/invasiveness of both H460 and human endothelial cells. Targeting of Met signaling by alternative methods (Met small interfering RNA and anti-phosphorylated Met antibody intracellular transfer) produced comparable biochemical and biological effects. Using the spontaneously metastasizing lung carcinoma xenograft H460, daily oral treatment with well-tolerated doses of RPI-1 produced a significant reduction of spontaneous lung metastases (−75%; P < 0.001, compared with control mice). In addition, a significant inhibition of angiogenesis in primary s.c. tumors of treated mice was observed, possibly contributing to limit the development of metastases. The results provide preclinical evidence in support of Met targeting pharmacologic approach as a new option for the control of tumor metastatic dissemination. [Mol Cancer Ther 2006;5(9):2388–97]

A novel taxane active against an orthotopically growing human glioma xenograft.

The development of effective chemotherapy for central nervous system tumors is hampered by the blood‐brain barrier and by limited drug diffusion in the brain tissue. BAY 59‐8862 is a new taxane analog that was selected and developed for its activity against tumors with a P‐glycoprotein‐mediated, multidrug‐resistant phenotype. Because P‐glycoprotein is implicated in limiting the access of drugs to central nervous system tumor targets, the objective of this study was to evaluate the ability of intravenously administered BAY 59‐8862 to affect the growth of central nervous system tumors.

RET/PTC oncoproteins: molecular targets of new drugs.

Ret oncoproteins expressed in thyroid carcinomas represent possible targets for therapeutic intervention. Oncogenic activation of the receptor tyrosine kinase encoding RET gene occurs typically by gene rearrangement in papillary thyroid carcinomas (PTC) and by missense mutation in medullary thyroid carcinomas (MTC). These genetic alterations lead to the expression of deregulated products characterized by ligand-independent activation of the intrinsic tyrosine kinase of Ret. Such features suggest the possibility of using specific tyrosine kinase inhibitors to block the Ret oncoproteins signaling. The present report summarizes the cellular effects of the arylidene 2-indolinone Ret inhibitor RPI-1 (formerly Cpd1) on the human PTC cell line TPC-1 which spontaneously harbors the RET/PTC1 oncogene. The results provide evidence that RPI-1 is able to inhibit cell growth and to interfere with Ret/ptc1-driven signaling. These findings support a role for Ret oncoproteins as therapeutic targets and the pharmacological interest of RPI-1 as a candidate drug for preclinical evaluation on thyroid tumors expressing RET oncogenes.

Concomitant downregulation of proliferation/survival pathways dependent on FGF-R3, JAK2 and BCMA in human multiple myeloma cells by multi-kinase targeting

The identification of proliferation/survival pathways constitutively activated by genetic alterations in multiple myeloma (MM), or sustained by the bone marrow (BM) microenvironment, provides novel opportunities for the development of targeted therapies. The deregulated function of protein tyrosine kinases plays a critical role in driving MM malignant phenotype. We investigated the effects of the multi-target tyrosine kinase inhibitor RPI-1 in a panel of human MM cell lines, including t(4;14) positive cell lines expressing the TK receptor FGF-R3. Cells harboring FGF-R3 activating mutations (KMS11 and OPM2) displayed the highest sensitivity to RPI-1 antiproliferative effect. The stimulating effect of the aFGF ligand was abrogated in cells harboring a non-constitutively active receptor. Drug treatment inhibited activation and expression of the FGF-R3(Y373C) mutant as well as aFGF-dependent signaling involving AKT and ERKs. Inhibition of JAK2, an additional RPI-1 target, resulted in STAT3 inactivation. Blockade of these proliferation/survival pathways was associated with caspase-dependent apoptosis. Moreover, drug treatment abrogated proliferative and pro-invasive stimuli provided by conditioned medium from mesenchymal stromal cells. Gene expression profile of KMS11 cells showed 22 upregulated and 52 downregulated genes upon RPI-1 treatment, with an early modulation of genes implicated in MM pathobiology such as SAT-1, MYC, MIP-1alpha/beta, FGF-R3, and the growth factor receptor B-cell maturation antigen (BCMA). Thus, concomitant blockade of FGF-R3 and JAK2 results in inhibition of several MM-promoting pathways, including BCMA-regulated signaling, and downregulation of disease-associated proteins. These data may have therapeutic implications in the design of treatment strategies resulting in the concomitant inhibition of FGF-R3 and JAK2 signaling pathways in t(4;14) MM.

Abstract B224: Multi‐tyrosine kinase targeting resulting in concomitant downregulation of proliferation/survival pathways dependent on FGF‐R3, Jak2 and BCMA in human multiple myeloma cells

Proliferation/survival pathways constitutively activated by genetic alterations in multiple myeloma (MM), or sustained by the bone marrow (BM) microenvironment, provide opportunities for the development of novel targeted therapies. Deregulated protein tyrosine kinases (TKs) involved in these pathways may play a critical role in driving the MM malignant phenotype thus representing attractive therapeutic targets. We have investigated the effects of a multi‐target tyrosine kinase inhibitor (1,3‐dihydro‐5,6‐dimethoxy‐3‐[(4‐hydrophenyl)methylene]1‐indol‐2‐one) (named RPI‐1 in our laboratory) in a panel of human MM cell lines, including t(4;14) positive cell lines expressing the TK receptor FGF‐R3. Cells harboring FGF‐R3 activating mutations (KMS11 and OPM2) displayed the highest sensitivity to the drug antiproliferative effect. However, proliferation induced by the FGF‐R3 ligand, aFGF, was abrogated even in cells harboring a non‐constitutively active receptor. Drug treatment inhibited activation and expression of the FGF‐R3Y373C mutant as well as aFGF‐dependent signaling involving AKT and ERKs. Jak2 has been identified as an additional target of RPI‐1 in MM cells. Inhibition of Jak2 TK resulted in STAT3 inactivation. Blockade of these proliferation/survival pathways was associated with activation of caspase‐dependent apoptosis. Moreover, the TK inhibitor abrogated proliferative and pro‐invasive stimuli provided by conditioned medium from mesenchymal stromal cells. Gene expression analysis of KMS11 cells showed 22 upregulated and 52 downregulated genes upon RPI‐1 treatment, with an early modulation of genes implicated in MM pathobiology such as SAT‐1, MYC, MIP‐1α/β, FGF‐R3, and the growth factor receptor B‐cell maturation antigen (BCMA). Thus, concomitant blockade of FGF‐R3 and Jak2 results in inhibition of several MM‐promoting pathways, including BCMA‐regulated signaling, and downregulation of disease‐associated proteins. Overall, these data support the therapeutic interest for FGF‐R3/JAK2 multi‐target approaches in the treatment of t(4;14) MM. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B224.