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To date, over 100 small molecule oncology drugs have been approved by the US Food and Drug Administration. Due to the inherent heterogeneity of tumors, these small molecules are often administered in combination to prevent emergence of resistant cell subpopulations. Therefore, new combination strategies to overcome drug resistance in patients with advanced cancer are needed. In this study, we performed a systematic evaluation of the therapeutic activity of over 5,000 pairs of FDA-approved cancer drugs against a panel of 60 well-characterized human tumor cell lines (NCI-60) to uncover combinations with greater than additive growth-inhibitory activity. Screening results were compiled into a database, termed the NCI-ALMANAC (A Large Matrix of Anti Neoplastic Agent Combinations), publically available at https://dtp.cancer.gov/ncialmanac. Subsequent in vivo experiments in mouse xenograft models of human cancer confirmed combinations with greater than single-agent efficacy. Concomitant detection of mechanistic biomarkers for these combinations in vivo supported the initiation of two phase I clinical trials at the NCI to evaluate clofarabine with bortezomib and nilotinib with paclitaxel in patients with advanced cancer. Consequently, the hypothesis-generating NCI-ALMANAC web-based resource has demonstrated value in identifying promising combinations of approved drugs with potent anticancer activity for further mechanistic study and translation to clinical trials.

Comparison of biochemical, molecular biological, and chemosensitivity data obtained from screening a large number of cell lines (e.g., the NCI tumor cell line panel) may facilitate investigation of factors influencing drug antitumor activity. The knowledge gained may be of value in the development of new anticancer agents or in the selection of patients to receive specific therapies.

The heat shock protein Hsp90 is a potential target for drug discovery of novel anticancer agents. By affecting this protein, several cell signaling pathways may be simultaneously modulated. The geldanamycin analog 17AAG has been shown to inhibit Hsp90 and associated proteins. Its clinical use, however, is hampered by poor solubility and thus, difficulties in formulation. Therefore, a water-soluble derivative was desirable and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17DMAG) is such a derivative. Studies were carried out in order to evaluate the activity and molecular mechanism(s) of 17DMAG in comparison with those of 17-allylamino-demethoxygeldanamycin (17AAG). 17DMAG was found to be more potent than 17AAG in a panel of 64 different patient-derived tumor explants studied in vitro in the clonogenic assay. The tumor types that responded best included mammary cancers (six of eight), head and neck cancers (two of two), sarcomas (four of four), pancreas carcinoma (two of three), colon tumors (four of eight for 17AAG and six of eight for 17DMAG), and melanoma (two of seven). Bioinformatic comparisons suggested that, while 17AAG and 17DMAG are likely to share the same mode(s) of action, there was very little similarity with standard anticancer agents. Using three permanent human melanoma cell lines with differing sensitivities to 17AAG and 17DMAG (MEXF 276L, MEXF 462NL and MEXF 514L), we found that Hsp90 protein was reduced following treatment at a concentration associated with total growth inhibition. The latter occurred in MEXF 276L cells only, which are most sensitive to both compounds. The depletion of Hsp90 was more pronounced in cells exposed to 17DMAG than in those treated with 17AAG. The reduction in Hsp90 was associated with the expression of erbB2 and erbB3 in MEXF 276L, while erbB2 and erbB3 were absent in the more resistant MEXF 462NL and MEXF 514L cells. Levels of known Hsp90 client proteins such as phosphorylated AKT followed by AKT, cyclin D1 preceding cdk4, and craf-1 declined as a result of drug treatment in all three melanoma cell lines. However, the duration of drug exposure needed to achieve these effects was variable. All cell lines showed increased expression of Hsp70 and activated cleavage of PARP. No change in PI3K expression was observed and all melanoma cells were found to harbor the activating V599E BRAF kinase mutation. The results of our in vitro studies are consistent with both 17AAG and 17DMAG acting via the same molecular mechanism, i.e. by modulating Hsp90 function. Since 17DMAG can be formulated in physiological aqueous solutions, the data reported here strongly support the development of 17DMAG as a more pharmaceutically practicable congener of 17AAG.

Tubulysin A (tubA) is a natural product isolated from a strain of myxobacteria that has been shown to depolymerize microtubules and induce mitotic arrest. The potential of tubA as an anticancer and antiangiogenic agent is explored in the present study. tubA shows potent antiproliferative activity in a panel of human cancer cell lines irrespective of their multidrug resistance properties. It induces apoptosis in cancer cells but not in normal cells and shows significant potential antiangiogenic properties in several in vitro assays. It is efficacious in initial animal studies using a hollow fibre assay with 12 different human tumour cell lines. This study suggests that both in vitro and preclinical profiles of tubA may translate into clinically useful anticancer properties.

Mouse epidermal keratinocytes (MK cells) were grown as replicating subcultures at clonal density, in a serum-free, low calcium basal medium supplemented with seven different growth factors (Bertolero et al., Exp. Cell. Res. 155:64-80, 1984). This serum-free system was used to investigate the activity of fetal bovine serum (FBS) and of serum-derived factors on the growth and differentiation of MK cells. Unfractionated, whole FBS inhibited growth and induced terminal differentiation of normal MK cells. The growth inhibitory activity was considerably reduced by passing whole FBS over a resin (Chelex) to remove Ca2+ and other di- and trivalent cations. It is not known whether this treatment removed other factors. Addition of individual serum components either stimulated or inhibited cell-growth and differentiation. Fetuin, a major alpha-globulin of FBS, and high density lipoprotein strongly inhibited the colony forming efficiency (CFE) of MK cells, whereas bovine serum albumin increased the CFE 4.5-fold and stimulated the growth rate as well. The addition of impure commercial preparations of platelet-derived growth factor inhibited the CFE and induced the morphological features of squamous terminally-differentiating keratinocytes. As reported in other systems, transforming growth factor beta (TGF-beta) inhibited the growth of secondary keratinocytes in a dose-dependent manner. Thus, at least three factors present in FBS inhibited growth whereas others were stimulatory. These observations explain the difficulties in obtaining replicating subcultures of mouse keratinocytes in serum-supplemented media and emphasize the importance of a serum-free system for studies on growth control and carcinogenesis in keratinocytes.

Various concentrations of oxygen were used to determine the optimum culture medium Po, for survival and proliferation of attached human and mouse fibroblasts grown from different inoculum sizes. When T-15 flasks were seeded with =Z 2 X lo4 cells (E 1.3 x lo3 celldcrn'), the highest plating efficiencies and cell yields were obtained with a culture medium Po, of 40-60 mm Hg.At higher inoculum sizes (lo5 cells per T-15) used routinely for mass cultures, no difference in cell yield or glycolytic activity was observed between cultures gassed with atmospheric, i.e., 18% 0, (growth medium Po, 125-135 mm Hg) and those gassed with 1% O2 (growth medium Po, zz 40-60 mm Hg). The enhanced clonal growth observed at the latter Po, results from an increased proliferation rate rather than more efficient attachment and survival of inoculated cells. Glucose uptake and lactic acid accumulation were increased in sparse cultures sparged with 1% 0,. A slight extension of lifespan was observed in WI-38 cells serially subcultured with a gas phase of 1% 0,.Numerous reports of the deleterious effects of excessive or insufficient concentrations of oxygen on cell growth appear in the literature (McLimans e t al., '68, '72; Balin et al., '76a). As an essential respiratory gas, oxygen dissolved in the culture medium must be sufficient to sustain a normal respiration rate but must not be at a partial pressure such that the cell is damaged (Swartz, '73). Generally, organ cultures require a gas phase containing up to 95% oxygen, and most cell cultures, whether of one or several layers, are routinely grown with a gas phase containing a n atmospheric oxygen concentration, 16 to 20% depending on altitude. An even lower concentration of l-3% oxygen is required for both enhanced clonal growth (Richter et al., '72; Taylor et al., '74) and chromosome stability (Parshad and Sanford, '71; Parshad e t al., '77). To explore further this relationship between cell numbers and oxygen requirements, several concentrations of gaseous oxygen and inoculum sizes were used to assess the Po, of the culture medium that results in optimal proliferation of attached cells. In contrast to the report of Balin et al. ('76a) MATERIALS AND METHODS Cells, culture media, and test gasesHuman Phase I1 WI-38 cells (CCL-75) were obtained from the American Type Culture Collection (ATCC), Rockville, Maryland. A culture of human WI-26 cells was a gift from Monroe Vincent, Microbiological Associates (MA), Bethesda, Maryland. Stock cultures of WI-26 and WI-38 were grown in minimum essential medium (Eagle, '59) (MEM) supplemented with 1 mM sodium pyruvate, 2 mM glutamine, and the "nonessential amino acids" a t a final concentration of 0.1 mM; all MEM and additives were obtained from MA. Lowpassage adult human lines BiRil (CRL 1104) and WilMac (CRL 124) were obtained from ATCC and were grown in Dulbecco-Vogt's modified MEM (DV) obtained from Schwaid Mann, Orangeburg, New York. Cells of line NCTC 9032 are fibroblast-like and derived from an explant culture of normal adult human s...