Caroline Haglund

Screening of an annotated compound library for drug activity in a resistant myeloma cell line

Purpose: Resistance to anticancer drugs is a major problem in chemotherapy. In order to identify drugs with selective cytotoxic activity in drug-resistant cancer cells, the annotated compound library LOPAC1280, containing compounds from 56 pharmacological classes, was screened in the myeloma cell line RPMI 8226 and its doxorubicin-resistant subline 8226/Dox40. Methods: Cell survival was measured by the Fluorometric Microculture Cytotoxicity Assay. Results: Selective cytotoxic activity in 8226/Dox40 was obtained for 33 compounds, with the most pronounced difference observed for the glucocorticoids. A microarray analysis of the cells showed a difference in mRNA-expression for the glucocorticoid receptor suggesting potential mechanisms for the difference in glucocorticoid sensitivity. In the presence of the glucocorticoid-receptor antagonist RU486, the sensitivity to the glucocorticoids was reduced and a similar effect level in RPMI 8226 and 8226/Dox40 was achieved. Conclusion: In conclusion, screening of mechanistically annotated compounds on drug-resistant cancer cells can identify compounds with selective activity and provide a basis for the development of novel treatments of drug-resistant malignancies.

Piperlongumine induces inhibition of the ubiquitin-proteasome system in cancer cells.

Piperlongumine, a natural product from the plant Piperlongum, has demonstrated selective cytotoxicity to tumor cells and to show anti-tumor activity in animal models [1]. Cytotoxicity of piperlongumine has been attributed to increase in reactive oxygen species (ROS) in cancer cells. We here report that piperlongumine is an inhibitor of the ubiquitin-proteasome system (UPS). Exposure of tumor cells to piperlongumine resulted in accumulation of a reporter substrate known to be rapidly degraded by the proteasome, and of accumulation of ubiquitin conjugated proteins. However, no inhibition of 20S proteolytic activity or 19S deubiquitinating activity was observed at concentrations inducing cytotoxicity. Consistent with previous reports, piperlongumine induced strong ROS activation which correlated closely with UPS inhibition and cytotoxicity. Proteasomal blocking could not be mimicked by agents that induce oxidative stress. Our results suggest that the anti-cancer activity of piperlongumine involves inhibition of the UPS at a pre-proteasomal step, prior to deubiquitination of malfolded protein substrates at the proteasome, and that the previously reported induction of ROS is a consequence of this inhibition.

Identification of Agents that Induce Apoptosis of Multicellular Tumour Spheroids: Enrichment for Mitotic Inhibitors with Hydrophobic Properties

Cell‐based anticancer drug screening generally utilizes rapidly proliferating tumour cells grown as monolayer cultures. Hit compounds from such screens are not necessarily effective on hypoxic and slowly proliferating cells in 3‐D tumour tissue. The aim of this study was to examine the potential usefulness of 3‐D cultured tumour cells for anticancer drug screening. We used colon carcinoma multicellular spheroids containing hypoxic and quiescent cells in core areas for this purpose. Three libraries (∼11 000 compounds) were screened using antiproliferative activity and/or apoptosis as end‐points. Screening of monolayer and spheroid cultures was found to identify different sets of hit compounds. Spheroid screening enriched for hydrophobic compounds: median XLogP values of 4.3 and 4.4 were observed for the hits in two independent screening campaigns. Mechanistic analysis revealed that the majority of spheroid screening hits were microtubuli inhibitors. One of these inhibitors was examined in detail and found to be effective against non‐dividing cells in the hypoxic centres of spheroids. Spheroid screening represents a conceptually new strategy for anticancer drug discovery. Our findings have implications for drug library design and hit selection in projects aimed to develop drugs for the treatment of solid tumours.

Clonal Variation in Drug and Radiation Response among Glioma-Initiating Cells Is Linked to Proneural-Mesenchymal Transition

Intratumoral heterogeneity is a hallmark of glioblastoma multiforme and thought to negatively affect treatment efficacy. Here, we establish libraries of glioma-initiating cell (GIC) clones from patient samples and find extensive molecular and phenotypic variability among clones, including a range of responses to radiation and drugs. This widespread variability was observed as a continuum of multitherapy resistance phenotypes linked to a proneural-mesenchymal shift in the transcriptome. Multitherapy resistance was associated with a semi-stable cell state that was characterized by an altered DNA methylation pattern at promoter regions of mesenchymal master regulators and enhancers. The gradient of cell states within the GIC compartment constitutes a distinct form of heterogeneity. Our findings may open an avenue toward the development of new therapeutic rationales designed to reverse resistant cell states.

In vitro evaluation of clinical activity and toxicity of anticancer drugs using tumor cells from patients and cells representing normal tissues

PurposeThe aim of this study was to evaluate a phenotypic cell panel with tumor cells from various patients and normal cells for preclinical profiles of antitumor efficacy and toxicity of anticancer drugs.MethodsThe antitumor activity of fourteen anticancer drugs was tested in over one hundred tumor samples from patients with solid or hematological malignancies. Drug activity against four normal cell types was used for the assessment of normal tissue toxicity. In vitro activity of the drugs was compared with indications approved by the Food and Drug Administration and established adverse event profiles.ResultsIn general, in vitro drug activity in tumor cells from patients reflected known clinical activity of the drugs investigated. For example, the clinical activity of imatinib in chronic myeloid leukemia was clearly detected in the tumor panel. Further, and in accordance with clinical use, cisplatin and bortezomib showed high activity in ovarian cancer and myeloma samples, respectively. The normal cell models roughly reflected known clinical toxicity profiles and were able to detect differences in therapeutic index, e.g., between targeted drugs and classical cytotoxic agents. For example, the high tolerability of imatinib and the well-known renal toxicity of cisplatin were demonstrated.ConclusionsIn preclinical drug development, primary tumor cells from patients can be used for the prediction of cancer diagnosis–specific activity and may aid in the selection of diagnoses for clinical trials. By using tumor and toxicity panels together, information about therapeutic index may be derived, which may be useful when choosing among drug candidates with similar tumor effects.

The FMCA-GM assays, high throughput non-clonogenic alternatives to CFU-GM in preclinical hematotoxicity testing

One of the most common dose limiting adverse effects in cancer treatment is myelotoxicity. The aim of this study was to develop an in vitro method for measuring potential myelotoxic properties of a drug candidate in a high throughput setting. Human CD34(+) progenitor cells from umbilical cord blood were plated in 384-well microplates with drugs in liquid culture, supplemented with specific cytokines for the granulocytopoietic-macrophage lineage. After 7 or 14 days of proliferation and differentiation the cells were analyzed using the automated non-clonogenic fluorometric microculture cytotoxicity assay (FMCA). Two types of assays setups were evaluated, the FMCA-GM7 where cells were exposed to drugs directly after thawing and cytotoxicity measured on day 7 in contrast to the FMCA-GM14 where the cells were cultured 7 days prior to plating and drug exposure, with viability analysis on day 14 of differentiation. Drug sensitivity was similar in both assays and method validation was performed using 24 drugs with known myelotoxic profile (acyclovir, bortezomib, busulfan, carboplatin, chloramphenicol, chlorpromazine, cisplatin, cytarabine, clozapine, doxorubicin, erlotinib, etoposide, 5-fluorouracil, fludarabine, gefitinib, gemcitabine, hydroxyurea, imatinib, lomustine, melphalan, sorafenib, sunitinib, taxol and 6-thioguanine). The 50% inhibitory concentrations (IC(50)) from the FMCA-GM7 and the FMCA-GM14 correlated highly (r = 0.83) and (r = 0.82), respectively, with IC(50) from the established clonogenic assay (CFU-GM), obtained from the literature. The current data suggests that the FMCA-GM could offer a simple and robust alternative to the CFU-GM assay in preclinical hematotoxicity studies.

The novel alkylating prodrug J1: diagnosis directed activity profile ex vivo and combination analyses in vitro

SummaryObjective: The dipeptide J1 acts as a prodrug of melphalan with a significant increased potency in vitro resulting from activation by cellular aminopeptidases. The current study was performed to evaluate the ex vivo profile of J1 using 176 primary tumor cell cultures from patients. In addition, the activity of J1 in combination with eight standard drugs, representing different mechanistic classes, was studied in nine different human tumor cell lines of different histopathological origin. Methods: Ex vivo evaluation of tumor type selectivity, was performed using the established fluorometric microculture cytotoxicity assay (FMCA). Combinations between J1 and eight standard chemotherapeutic drugs were analyzed using the median-effect method. Results: The prodrug J1 expressed approximately 50- to 100-fold higher potency but similar activity profile as that of its metabolite, melphalan. The difference was greater in some diagnoses (e.g. breast cancer, NHL and AML), and exceptionally high in some breast cancer samples with aggressive phenotypes. Combination analysis of J1 and standard chemotherapeutics yielded several potentially additive and synergistic interactions, most striking for etoposide with significant synergism in all studied cell lines. Conclusions: In conclusion, the ex vivo profile suggests that further evaluation of J1 as the alkylating agent in for example aggressive breast cancer might be of particular interest, preferentially in combination with DNA-topoisomerase II inhibitors like etoposide.

Gambogic acid is cytotoxic to cancer cells through inhibition of the ubiquitin-proteasome system

SummaryGambogic acid (GA), displays cytotoxicity towards a wide variety of tumor cells and has been shown to affect many important cell-signaling pathways. In the present work, we investigated the mechanism of action of GA by analysis of drug-induced changes in gene expression profiles and identified GA and the derivative dihydro GA as possible inhibitors of the ubiquitin-proteasome system (UPS). Both GA and dihydro GA inhibited proteasome function in cells resulting in the accumulation of polyubiquitin complexes. In vitro experiments showed that both GA and dihydro GA inhibited 20S chymotrypsin activity and the inhibitory effects of GA and dihydro GA on proteasome function corresponded with apoptosis induction and cell death. In conclusion, our results show that GA and dihydro GA exert their cytotoxic activity through inhibition of the UPS, specifically by acting as inhibitors of the chymotrypsin activity of the 20S proteasome.

Identification of an inhibitor of the ubiquitin–proteasome system that induces accumulation of polyubiquitinated proteins in the absence of blocking of proteasome function

The ubiquitin–proteasome system (UPS) represents one of the most promising therapeutic targets in oncology to emerge in recent years. Here we used a combination of cytotoxic and image-based screening assays to identify a novel UPS inhibitor, designated HRF-3. HRF-3 evokes a gene expression profile similar to that of other characterized UPS inhibitors, suggesting a common mechanism of action. Consistent with UPS inhibition, HRF-3 induced strong accumulation of polyubiquitinated proteins in cells. Surprisingly, HRF-3 induced only weak accumulation of two proteasome targeted reporter proteins, UbG76V-YFP and ZsGreen-ODC. Consistent with this observation, HRF-3 did not inhibit proteasome proteolytic activity in an in vitro assay. Similar to a number of other UPS inhibitors, HRF-3 increased the expression of the redox-inducible protein Hmox-1. In distinction to the 20S inhibitor bortezomib, but similarly to two different p97/VCP inhibitors, HRF-3 did not elicit strong induction of the chaperone Hsp70B′. Finally, we show that HRF-3 is cytotoxic to a variety of cancer cell lines and ex vivo patient tumour cells, with the strongest activity observed in cells of leukemic/myeloma origin. Taken together our data show that HRF-3 induces polyubiquitin accumulation in the absence of efficient proteasomal blocking, and suggest that induction of oxidative stress is a common denominator of UPS inhibitors.

In vitro and in vivo anti-leukemic activity of the peptidase-potentiated alkylator melflufen in acute myeloid leukemia

The novel aminopeptidase potentiated alkylating agent melflufen, was evaluated for activity in acute myeloid leukemia in a range of in vitro models, as well as in a patient derived xenograft study. All tested AML cell lines were highly sensitive to melflufen while melphalan was considerably less potent. In the HL-60 cell line model, synergy was observed for the combination of melflufen and cytarabine, an interaction that appeared sequence dependent with increased synergy when melflufen was added before cytarabine. Also, in primary cultures of AML cells from patients melflufen was highly active, while normal PBMC cultures appeared less sensitive, indicating a 7-fold in vitro therapeutic index. Melphalan, on the other hand, was only 2-fold more potent in the AML patient samples compared with PBMCs. Melflufen was equally active against non-malignant, immature CD34+ progenitor cells and a more differentiated CD34+ derived cell population (GM14), whereas the stem cell like cells were less sensitive to melphalan. Finally, melflufen treatment showed significant anti-leukemia activity and increased survival in a patient derived xenograft of AML in mice. In conclusion, melflufen demonstrates high and significant preclinical activity in AML and further clinical evaluation seem warranted in this disease.