Yukimasa Shiotsu

A pharmacodynamic study of the FLT3 inhibitor KW-2449 yields insight into the basis for clinical response

Internal tandem duplication mutations of FLT3 (FLT3/ITD mutations) are common in acute myeloid leukemia (AML) and confer a poor prognosis. This would suggest that FLT3 is an ideal therapeutic target, but FLT3 targeted therapy has produced only modest benefits in clinical trials. Due to technical obstacles, the assessment of target inhibition in patients treated with FLT3 inhibitors has been limited and generally only qualitative. KW-2449 is a novel multitargeted kinase inhibitor that induces cytotoxicity in Molm14 cells (which harbor an FLT3/ITD mutation). The cytotoxic effect occurs primarily at concentrations sufficient to inhibit FLT3 autophosphorylation to less than 20% of its baseline. We report here correlative data from a phase 1 trial of KW-2449, a trial in which typical transient reductions in the peripheral blast counts were observed. Using quantitative measurement of FLT3 inhibition over time in these patients, we confirmed that FLT3 was inhibited, but only transiently to less than 20% of baseline. Our results suggest that the failure to fully inhibit FLT3 in sustained fashion may be an underlying reason for the minimal success of FLT3 inhibitors to date, and stress the importance of confirming in vivo target inhibition when taking a targeted agent into the clinical setting.

A Nonfucosylated Anti-HER2 Antibody Augments Antibody-Dependent Cellular Cytotoxicity in Breast Cancer Patients

Purpose: Removal of fucose residues from the oligosaccharides of human antibody is a powerful approach to enhance antibody-dependent cellular cytotoxicity (ADCC), a potential important antitumor mechanism of therapeutic antibodies. To provide clinically relevant evidence of this mechanism, we investigated ADCC of a fucose-negative version of trastuzumab [anti–human epidermal growth factor receptor 2 (HER2) humanized antibody] using peripheral blood mononuclear cells (PBMC) from breast cancer patients as effector cells. Experimental Design: Thirty volunteers, including 20 breast cancer patients and 10 normal healthy control donors, were recruited randomly, and aliquots of peripheral blood were collected. ADCC of commercial trastuzumab (fucosylated) and its fucose-negative version were measured using PBMCs drawn from the volunteers as effector cells and two breast cancer cell lines with different HER2 expression levels as target cells. Relationships between cytotoxicity and characteristics of the patients, such as content of natural killer cells in PBMCs, type of therapy, FCGR3A genotypes, etc. were also analyzed. Results: ADCC was significantly enhanced with the fucose-negative antibody compared with the fucose-positive antibody using PBMCs from either normal donors or breast cancer patients. Enhancement of ADCC was observed irrespective of the various clinical backgrounds of the patients, even in the chemotherapy cohort that presented with a reduced number of natural killer cells and weaker ADCC. Conclusions: This preliminary study suggests that the use of fucose-negative antibodies may improve the therapeutic effects of anti-HER2 therapy for patients independent of clinical backgrounds.

Development of radicicol analogues.

Radicicol, a macrocyclic antibiotic produced by fungi, was originally isolated many years ago, and was described as tyrosine kinase inhibitor. We also rediscovered radicicol as an inhibitor of signal transduction of oncogene products, such as K-ras and v-Src, using yeast and mammalian cell-based assays. In a study of mechanisms of action, it was revealed that radicicol depletes the Hsp90 client signaling molecules in cells, and thus inhibit the signal transduction pathway. In addition, direct binding of radicicol to the N-terminal ATP/ADP binding site of Hsp90 was shown, and thus radicicol has been recognized as a structurally unique antibiotic that binds and inhibits the molecular chaperone Hsp90. Although radicicol itself has little or no activity in animals because of instability in animals, its oxime derivatives showed potent antitumor activities against human tumor xenograft models. Hsp90 client proteins were depleted and apoptosis was induced in the tumor specimen treated with radicicol oxime derivatives. Taken together, these results suggest that the antitumor activity of radicicol oxime derivatives is mediated by binding to Hsp90 and destabilization of Hsp90 client proteins in the tumor. Among Hsp90 clients, we focused on ErbB2 and Bcr-Abl as examples of important targets of Hsp90 inhibitors. Radicicol oxime showed potent antitumor activity against ER negative/ErbB2 overexpressing breast cancer and Bcr-Abl expressing CML. Putative mechanisms of action and future directions of radicicol oxime against these kinds of tumor are discussed.

KW-2449, a novel multikinase inhibitor, suppresses the growth of leukemia cells with FLT3 mutations or T315I-mutated BCR/ABL translocation.

KW-2449, a multikinase inhibitor of FLT3, ABL, ABL-T315I, and Aurora kinase, is under investigation to treat leukemia patients. In this study, we examined its possible modes of action for antileukemic effects on FLT3-activated, FLT3 wild-type, or imatinib-resistant leukemia cells. KW-2449 showed the potent growth inhibitory effects on leukemia cells with FLT3 mutations by inhibition of the FLT3 kinase, resulting in the down-regulation of phosphorylated-FLT3/STAT5, G(1) arrest, and apoptosis. Oral administration of KW-2449 showed dose-dependent and significant tumor growth inhibition in FLT3-mutated xenograft model with minimum bone marrow suppression. In FLT3 wild-type human leukemia, it induced the reduction of phosphorylated histone H3, G(2)/M arrest, and apoptosis. In imatinib-resistant leukemia, KW-2449 contributed to release of the resistance by the simultaneous down-regulation of BCR/ABL and Aurora kinases. Furthermore, the antiproliferative activity of KW-2449 was confirmed in primary samples from AML and imatinib-resistant patients. The inhibitory activity of KW-2449 is not affected by the presence of human plasma protein, such as alpha1-acid glycoprotein. These results indicate KW-2449 has potent growth inhibitory activity against various types of leukemia by several mechanisms of action. Our studies indicate KW-2449 has significant activity and warrants clinical study in leukemia patients with FLT3 mutations as well as imatinib-resistant mutations.

Steroid sulfatase and estrogen sulfotransferase in normal human tissue and breast carcinoma.

Steroid sulfatase (STS) hydrolyzes inactive estrone sulfate (E1-S) to estrone (E1), while estrogen sulfotransferase (EST; SULT 1E1 or STE gene) sulfonates estrogens to estrogen sulfates. They are considered to play important roles in the regulation of local estrogenic actions in various human tissues, however, their biological significance remains largely unknown. Therefore, we examined the expression of STS and EST in non-pathologic human tissues and breast carcinomas. STS expression was very weak except for the placenta, while EST expression was markedly detected in various tissues examined. In breast carcinoma tissues, STS and EST immunoreactivity was detected in carcinoma cells in 74 and 44% of cases, respectively, and was significantly associated with their mRNA levels and enzymatic activities. STS immunoreactivity was significantly correlated with the tumor size, and an increased risk of recurrence. EST immunoreactivity was inversely correlated with the tumor size or lymph node status. Moreover, EST immunoreactivity was significantly associated with a decreased risk of recurrence or improved prognosis. Our results suggest that EST is involved in protecting various peripheral tissues from excessive estrogenic effects. In the breast carcinoma, STS and EST are suggested to play important roles in the regulation of in situ estrogen production in the breast carcinomas.

Role of steroid sulfatase in local formation of estrogen in post-menopausal breast cancer patients

More than two-thirds of breast cancers occur in post-menopausal women, and depend on the estrogens for their proliferation and survival. For the treatment of estrogen-dependent breast cancers, two major treatment options are now available. One is selective estrogen receptor modulator (SERM) such as Tamoxifen and another is aromatase inhibitor such as Anastrozole, Letrozole and Exemestane, which reduce local in situ formation of estrogens. Although these therapies are clinically active for advanced and early breast cancers, de novo and/or acquired resistance to SERM and/or aromatase inhibitors are also clinical problem. Recent studies suggest that local formation of estrogens in the breast tumors is more important than circulating estrogen in plasma for the growth and survival of estrogen-dependent breast cancer in post-menopausal women. The rationale for the importance of local formation of estrogens is based on the following evidences. Estradiol (E2) levels in breast tumors are equivalent to those of pre-menopausal patients, although plasma E2 levels are 50-fold lower after menopause. E2 concentrations in breast tumors of post-menopausal women are 10-40 times higher than serum level. Biosynthesis of estrogens in breast tumors tissues occurs via two major different routes, one is aromatase pathway and another is steroid-sulfatase (STS) pathway. Whereas many studies has been reported about aromatase inhibitor and its clinical trial results in breast cancer patients, limited information are available regarding to other estrogen regulating enzymes including STS, its role in breast tumors and STS inhibitors. STS is the enzyme that hydrolyses estrone 3-sulfate (E1S) and dehydroepiandrosterone-sulfate (DHEA-S) to their active un-sulfoconjugated forms, thereby stimulating the growth and survival of estrogen-dependent breast tumors. It has been well known that E1S level are much higher than E2 level both in plasma and tumor of post-menopausal patients. Recent reports show that more than 80% of breast tumors are stained with anti-STS antibody and the expression of STS is an independent prognostic factor in breast cancer. Taking these findings into consideration, local formation of estrogens could be partially synthesized from large amount of E1S by STS, which exist in breast cancer. On the other hand, aromatase localizes in stroma and adipocyte surrounding breast cancer. Furthermore, since estrogen formation from E1S and DHEA-S (STS pathway) cannot be blocked by aromatase inhibitors, STS is thought to be a new molecular target for the treatment of estrogen-dependent tumor post-SERM and/or aromatase inhibitors. In this symposium, these recent rationale for the importance of STS in post-menopausal breast cancer patients is reviewed as well as STS inhibitor.

New Molecular and Biological Mechanism of Antitumor Activities of KW-2478, a Novel Nonansamycin Heat Shock Protein 90 Inhibitor, in Multiple Myeloma Cells

Purpose: The heat shock protein 90 (Hsp90) plays an important role in chaperoning oncogenic client proteins in multiple myeloma (MM) cells, and several Hsp90 inhibitors have shown antitumor activities both in vitro and in vivo. However the precise mechanism of action of Hsp90 inhibitor in MM has not been fully elucidated. Experimental Design: We evaluated the antitumor activities of KW-2478, a nonansamycin Hsp90 inhibitor, in MM cells with various chromosomal translocations of immunoglobulin heavy chain (IgH) loci both in vitro and in vivo. Results: Our studies revealed that exposure of KW-2478 to MM cells resulted in growth inhibition and apoptosis, which were associated with degradation of well-known client proteins as well as a decrease in IgH translocation products (FGFR3, c-Maf, and cyclin D1), and FGFR3 was shown to be a new client protein of Hsp90 chaperon complex. In addition, KW-2478 depleted the Hsp90 client Cdk9, a transcriptional kinase, and the phosphorylated 4E-BP1, a translational inhibitor. Both inhibitory effects of KW-2478 on such transcriptional and translational pathways were shown to reduce c-Maf and cyclin D1 expression. In NCI-H929 s.c. inoculated model, KW-2478 showed a significant suppression of tumor growth and induced the degradation of client proteins in tumors. Furthermore, in a novel orthotopic MM model of i.v. inoculated OPM-2/green fluorescent protein, KW-2478 showed a significant reduction of both serum M protein and MM tumor burden in the bone marrow. Conclusions: These results suggest that targeting such diverse pathways by KW-2478 could be a promising strategy for the treatment of MM with various cytogenetic abnormalities. Clin Cancer Res; 16(10); 2792–802. ©2010 AACR.

Hsp90 Inhibitors as Anti-Cancer Agents, from Basic Discoveries to Clinical Development

Heat shock protein (Hsp) 90 is an ATP-dependent molecular chaperone which stabilizes various oncogenic kinases, including HER2, EGFR, BCR-ABL, B-Raf and EML4-ALK, which are essential for tumor growth. Several monoclonal antibodies and small molecule kinase inhibitors which target these kinases have been identified as potential new molecular target therapeutics. Previous reports have shown that many oncogenic proteins essential for cancer transformation are chaperoned by the Hsp90 complex, and some of these client proteins have been discovered by using Hsp90 inhibitors, such as geldanamycin (GA) and radicicol (RD).Thus far more than 200 client proteins have been identified. In past derivatives of these natural products have been evaluated in clinical trials, but none of the 1st generation of Hsp90 inhibitors has been approved yet because of their limitations in physico-chemical properties and/or safety profiles. However, recent reports have indicated that more than 10 new agents, 2nd generation of Hsp90 inhibitors with different chemotypes from GA and RD, have entered clinical trials and some of them showed clinical efficacy. In this review article, we describe the discoveries of major Hsp90 client proteins in the cancer field by RD derivatives, the history of KW-2478 discovery and development by Kyowa Hakko Kirin, and gave an update on the current status of new Hsp90 inhibitors in clinical trials.

Stereospecific antitumor activity of radicicol oxime derivatives.

Abstract.Purpose: Radicicol is a novel hsp90 antagonist, distinct from the chemically unrelated benzoquinone ansamycin compounds, geldanamycin and herbimycin. Both geldanamycin and radicicol bind in the aminoterminal nucleotide-binding pocket of hsp90, destabilizing the hsp90 client proteins, many of which are essential for tumor cell growth. We describe here antitumor activity of a novel oxime derivative of radicicol, KF58333. We also investigated the mechanism of antitumor activity of KF58333 in comparison with its oxime isomer KF58332. Methods: Antiproliferative activities were determined in a panel of breast cancer cell lines in vitro. We also examined inhibition of hsp90 function and apoptosis induction in erbB2-overexpressing human breast carcinoma KPL-4 cells in vitro. Direct binding activity to hsp90 was assessed by hsp90-binding assays using geldanamycin or radicicol beads. In animal studies, we investigated plasma concentrations of these compounds after i.v. injection in BALB/c mice and antitumor activity against KPL-4 cells transplanted into nude mice. Inhibition of hsp90 function and induction of apoptosis in vivo were investigated using tumor specimens from drug-treated animals. Results: KF58333 showed potent antiproliferative activity against all breast cancer cell lines tested in vitro, and was more potent than its stereoisomer KF58332. These results are consistent with the ability of KF58333 to deplete hsp90 client proteins and the induction of apoptosis in KPL-4 cells in vitro. Interestingly, KF58333, but not KF58332, showed significant in vivo antitumor activity accompanied by induction of apoptosis in KPL-4 human breast cancer xenografts. Although the plasma concentrations of these compounds were equivalent, KF58333, but not KF58332, depleted hsp90 client proteins such as erbB2, raf-1 and Akt in the tumor specimen recovered from nude mice. Conclusions: These results suggest that inhibition of hsp90 function, which causes depletion of hsp90 client proteins in tumor, contributes to the antitumor activity of KF58333, and that the stereochemistry of the oxime moiety is important for the biological activity of radicicol oxime derivatives.

A Novel FLT3 Inhibitor FI-700 Selectively Suppresses the Growth of Leukemia Cells with FLT3 Mutations

Purpose: The aim of this study was to evaluate the antileukemia activity of a novel FLT3 kinase inhibitor, FI-700. Experimental Design: The antileukemia activity of FI-700 was evaluated in human leukemia cell lines, mutant or wild-type (Wt)-FLT3–expressing mouse myeloid precursor cell line, 32D and primary acute myeloid leukemia cells, and in xenograft or syngeneic mouse leukemia models. Results: FI-700 showed a potent IC50 value against FLT3 kinase at 20 nmol/L in an in vitro kinase assay. FI-700 showed selective growth inhibition against mutant FLT3-expressing leukemia cell lines and primary acute myeloid leukemia cells, whereas it did not affect the FLT3 ligand (FL)–driven growth of Wt-FLT3–expressing cells. These antileukemia activities were induced by the significant dephosphorylations of mutant FLT3 and STAT5, which resulted in G1 arrest of the cell cycle. Oral administration of FI-700 induced the regression of tumors in a s.c. tumor xenograft model and increased the survival of mice in an i.v. transplanted model. Furthermore, FI-700 treatment eradicated FLT3/ITD-expressing leukemia cells, both in the peripheral blood and in the bone marrow. In this experiment, the depletion of FLT3/ITD-expressing cells by FI-700 was more significant than that of Ara-C, whereas bone marrow suppression by FI-700 was lower than that by Ara-C. Conclusions: FI-700 is a novel and potent FLT3 inhibitor with promising antileukemia activity.