A phase I pharmacokinetic and safety analysis of epothilone folate (BMS-753493), a folate receptor targeted chemotherapeutic agent in humans with advanced solid tumors.

Background The folate receptor alpha is selectively over-expressed in a number of human cancers. BMS-753493 is a folate conjugate of the epothilone analog BMS-748285 that was designed to selectively target folate receptor expressing cancer cells. Methods BMS-753493 was investigated in two parallel multi-institutional first-in-human phase I/IIa studies in patients with advanced solid tumors. In Study 1, patients were treated on a schedule of once daily dosing of BMS-753493 administered on Days 1, 4, 8 and 11 every 21 days with a starting dose of 5 mg daily and in Study 2, patients were treated once daily on Days 1-4 every 21 days, with a starting dose of 2.5 mg daily. Results A total of 65 patients were treated across the two studies. The maximum tolerated dose (MTD) was 26 mg in Study 1 and 15 mg in Study 2. Fatigue, transaminitis, gastrointestinal toxicity, and mucositis were dose-limiting toxicities. One patient in Study 2 developed Stevens-Johnson syndrome attributed to BMS-753493. Plasma exposures of both the conjugated and free epothilone increased in a dose related fashion in both studies and the half-life of the conjugated epothilone was 0.2-0.6 h across dose levels. No objective tumor responses were seen in either study. Conclusions BMS-753493 was generally tolerable and toxicities known to be associated with epothilone class of anticancer agents were common, although peripheral neuropathy and neutropenia appear to have been less frequent and less severe as compared to epothilones. Antitumor activity was not demonstrated and further development of BMS-753493 has been discontinued.

Reduced cardiotoxicity and comparable efficacy in a phase III trial of pegylated liposomal doxorubicin HCl (CAELYX/Doxil) versus conventional doxorubicin for first-line treatment of metastatic breast cancer.

BACKGROUND: This study was designed to demonstrate that efficacy [progression-free survival (PFS)] of CAELYX [pegylated liposomal doxorubicin HCl (PLD)] is non-inferior to doxorubicin with significantly less cardiotoxicity in first-line treatment of women with metastatic breast cancer (MBC). PATIENTS AND METHODS: Women (n=509) with MBC and normal cardiac function were randomized to receive either PLD 50 mg/m2 (every 4 weeks) or doxorubicin 60 mg/m2 (every 3 weeks). Cardiac event rates were based on reductions in left ventricular ejection fraction as a function of cumulative anthracycline dose. RESULTS: PLD and doxorubicin were comparable with respect to PFS [6.9 versus 7.8 months, respectively; hazard ratio (HR)=1.00; 95% confidence interval (CI) 0.82-1.22]. Subgroup results were consistent. Overall risk of cardiotoxicity was significantly higher with doxorubicin than PLD (HR=3.16; 95%CI 1.58-6.31; P<0.001). Overall survival was similar (21 and 22 months for PLD and doxorubicin, respectively; HR=0.94; 95%CI 0.74-1.19). Alopecia (overall, 66% versus 20%; pronounced, 54% versus 7%), nausea (53% versus 37%), vomiting (31% versus 19%) and neutropenia (10% versus 4%) were more often associated with doxorubicin than PLD. Palmar-plantar erythrodysesthesia (48% versus 2%), stomatitis (22% versus 15%) and mucositis (23% versus 13%) were more often associated with PLD than doxorubicin. CONCLUSIONS: In first-line therapy for MBC, PLD provides comparable efficacy to doxorubicin, with significantly reduced cardiotoxicity, myelosuppression, vomiting and alopecia.

Gene-target recognition among members of the myc superfamily and implications for oncogenesis.

Myc and Mad family proteins regulate multiple biological processes through their capacity to influence gene expression directly. Here we show that the basic regions of Myc and Mad proteins are not functionally equivalent in oncogenesis, have separable E-box-binding activities and engage both common and distinct gene targets. Our data support the view that the opposing biological actions of Myc and Mxi1 extend beyond reciprocal regulation of common gene targets. Identification of differentially regulated gene targets provides a framework for understanding the mechanism through which the Myc superfamily governs the growth, proliferation and survival of normal and neoplastic cells.

Histone deacetylase associated with mSin3A mediates repression by the acute promyelocytic leukemia-associated PLZF protein.

The PLZF gene was identified first by its fusion with the retinoic acid receptor alpha gene in the t(11;17) translocation associated with a retinoic acid resistant form of acute promyelocytic leukemia (APL). It encodes a krüppel-like zinc finger protein with a POZ domain shared with a subset of regulatory proteins including the BCL6 leukemogenic protein. PLZF, like BCL6, strongly represses transcription initiated from different promoters. Here we show that PLZF associates in vitro and in vivo with the Mad co-repressor mSin3A and the histone deacetylase HDAC1. Two domains in PLZF and the PAH1 structure of mSin3A mediate these interactions. Trichostatin A, a specific inhibitor of histone deacetylases, significantly reduces PLZF repression. These data strongly suggest that, like nuclear receptors and Mad, PLZF represses transcription by recruiting a histone deacetylase through the SMRT-mSin3-HDAC co-repressor complex. We also show that BCL6 associates with HDAC1 indicating that this type of regulation might be common to POZ/Zinc finger proteins involved in human leukemias. This work supports a role for deregulated histone deacetylation in the development of both lymphoid and myeloid neoplasia in human and suggests that targeted histone deacetylase inhibitors may be useful for treatment of certain types of malignancies.

The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53.

The INK4a gene encodes two distinct growth inhibitors--the cyclin-dependent kinase inhibitor p16Ink4a, which is a component of the Rb pathway, and the tumor suppressor p19Arf, which has been functionally linked to p53. Here we show that p19Arf potently suppresses oncogenic transformation in primary cells and that this function is abrogated when p53 is neutralized by viral oncoproteins and dominant-negative mutants but not by the p53 antagonist MDM2. This finding, coupled with the observations that p19Arf and MDM2 physically interact and that p19Rrf blocks MDM2-induced p53 degradation and transactivational silencing, suggests that p19Arf functions mechanistically to prevent MDM2's neutralization of p53. Together, our findings ascribe INK4a's potent tumor suppressor activity to the cooperative actions of its two protein products and their relation to the two central growth control pathways, Rb and p53.

Role for N-CoR and histone deacetylase in Sin3-mediated transcriptional repression.

Normal mammalian growth and development are highly dependent on the regulation of the expression and activity of the Myc family of transcription factors. Mxi1-mediated inhibition of Myc activities requires interaction with mammalian Sin3A or Sin3B proteins, which have been purported to act as scaffolds for additional co-repressor factors. The identification of two such Sin3-associated factors, the nuclear receptor co-repressor (N-CoR) and histone deacetylase (HD1), provides a basis for Mxi1/Sin3-induced transcriptional repression and tumour suppression.

Mouse Sin3A interacts with and can functionally substitute for the amino-terminal repression of the Myc antagonist Mxi1.

Mxi1 is a basic region helix-loop-helix leucine zipper (bHLH/LZ) protein that, in association with Max, antagonizes Myc oncogenic activities. A possible mechanistic basis for Mxi1-mediated repression was provided by the recent demonstration that the repressive potential of Mxi1 correlates with its ability to physically associate with mSin3B, one of two mammalian homologues of the yeast transcriptional repressor SIN3. Here, we sought to characterize more fully the physical properties of the second homologue, mSin3A and to determine whether the recruitment of mSin3A by Mxi1 is indeed required for anti-Myc activity. Transient transfection of mammalian cells showed that the mSin3A protein can associate with the strong repressive isoform of Mxi1 (Mxi1-SR) and that, like other Myc superfamily members, both mSin3A and Mxi1-SR localize to the nucleus. From a developmental standpoint, a comparative analysis of Myc, Mxi1-SR and Sin3A expression during postnatal mouse development and in differentiating mouse erythroleukemia (MEL) cells revealed that dramatic and reciprocal changes in Myc and Mxi1-SR mRNA levels are accompanied by minimal stage-specific changes in mSin3A gene expression. This constant expression profile, coupled with the observation that over-expression of mSin3A does not augment the anti-Myc activity of Mxi1-SR in the rat embryo fibroblast (REF) transformation assay, suggests that mSin3A is not a limiting factor in the regulation of Myc superfamily function. Finally, a mSin3A-Mxi1 fusion protein, in which the amino terminal mSin3-interacting domain of Mxi1-SR was replaced with the full-length mSin3A, exhibited a level of repression activity equivalent to, or greater than, the level of repression obtained with Mxi1-SR. Taken together, these observations directly demonstrate that the amino-terminal repression domain of Mxi1-SR functions solely to recruit mSin3A and possibly other proteins like mSin3A and this association is necessary for the anti-Myc activity of Mxi1-SR.

Dual myristylation and palmitylation of Src family member p59fyn affects subcellular localization.

The Src family consists of nine related tyrosine protein kinases with a common domain structure, including a myristylated N-terminal glycine residue. In this report, we identify cysteine residues within the N-terminal region of the Src family member Fyn which serve as sites for palmitylation. To facilitate detection of protein fatty acylation, p59fyn was overexpressed in COS cells and incubated with radioiodinated fatty acid analogs of myristate (IC13) or palmitate (IC16). Incorporation of both fatty acids into p59fyn was readily observed. Acylation with the palmitate analog was prevented when Gly-2 was mutated to alanine, implying that N-myristylation is required for palmitylation, and when either Cys-3 or Cys-6 was mutated to serine. Palmitylation was shown to alter the distribution of p59fyn between membrane-bound and soluble fractions. In contrast, no incorporation of the palmitate analog into pp60v-src, which lacks N-terminal cysteine residues, was observed. Mutation of Ser-3 of Src to cysteine, but not Ser-6, resulted in incorporation of the palmitate analog. These results serve to delineate sequence elements important for dual acylation of proteins, and further illustrate the utility of radioiodinated fatty acid analogs for studies of protein fatty acid acylation.