Richard D. May

Differential toxicity of camptothecin, topotecan and 9-aminocamptothecin to human, canine, and murine myeloid progenitors (CFU-GM) in vitro

Abstract Purpose: 20(S)-Camptothecin (CAM), topotecan (TPT, active ingredient in Hycamtin) and 9-amino-20(S)-camptothecin (9AC) are topoisomerase I inhibitors that cause similar dose-limiting toxicities to rapidly renewing tissues, such as hematopoietic tissues, in humans, mice, and dogs. However, dose-limiting toxicity occurs at tenfold lower doses in humans than in mice. The purpose of the current study was to determine whether hematopoietic progenitors of the myeloid lineage from humans, mice, and dogs exhibit the differential sensitivity to these compounds that is evident in vivo. Methods: Drug-induced inhibition of in vitro colony formation by a myeloid progenitor in human, murine, and canine marrow colony-forming unit-granulocyte/macrophage (CFU-GM) provided the basis for interspecies comparisons at concentrations which inhibited colony formation by 50% (IC50) and 90% (IC90). Results: Murine IC90 values were 2.6-, 2.3-, 10-, 21-, 5.9-, and 11-fold higher than human values for CAM lactone (NSC-94600) and sodium salt (NSC-100880), TPT (NSC-609699), and racemic (NSC-629971), semisynthetic and synthetic preparations (NSC-603071) of 9AC, respectively. In contrast, canine IC90 values were the same as, or lower than, the human IC90 values for all six compounds. Conclusions: The greater susceptibility of humans and dogs to the myelotoxicity of camptothecins, compared to mice, was evident in vitro at the cellular level. Differential sensitivity between murine and human myeloid progenitors explains why the curative doses of TPT and 9AC in mice with human tumor xenografts are not achievable in patients. Realizing the curative potential of these compounds in humans will require the development of therapies to increase drug tolerance of human CFU-GM at least to a level equal to that of murine CFU-GM. Because these interspecies differences are complicated by species-specific effects of plasma proteins on drug stability, not all in vitro assay conditions will yield results which can contribute to the development of such therapies.

Altered immunomodulating and toxicological properties of degraded Quillaja saponaria Molina saponins

Quillaja saponins are readily hydrolyzed under physiological conditions, yielding deacylated forms that are significantly less toxic than their precursors. Yet, deacylated saponins are unable to stimulate a strong primary immune response. Although deacylated saponins elicit a strong total IgG response, their capacity to stimulate a Thl type IgG isotype profile (i.e. high levels of IgG2a and IgG2b) has been significantly diminished. Instead, an IgG profile closer to that of a Th2 immune response is stimulated (i.e. high IgG1 levels). Deacylated saponins have also lost their capacity to elicit an effective T cell immunity, as shown by their stimulation of a marginal lymphoproliferative response and their inability to elicit the production of cytotoxic lymphocytes (CTL). Modification of the immune-modulating properties brought by the degradation of quillaja saponins during vaccine storage may change the intended immune response from a Th1 to a Th2 type. This alteration would have negligible effects on vaccines depending on Th2 immunity mediated by neutralizing antibodies. However, the performance of vaccines directed against intracellular pathogens as well as therapeutic cancer vaccines may be seriously affected by the loss of their capacity to stimulate both a Th1 immune response and the production of CTL.

Fractionation, structural studies, and immunological characterization of the semi-synthetic Quillaja saponins derivative GPI-0100

Unfractionated GPI-0100 (UFGPI-0100) containing semi-synthetic derivatives of deacylated Quillaja saponins (DS saponins) modified at the glucuronic acid residue was resolved by reverse phase low-pressure liquid chromatography (RP-LPLC) into two fractions, RP18-1 and RP18-2, with different compositions and adjuvanticity. The fraction RP18-1 contained DS saponin adducts of N-dicyclohexylurea, and stimulated Th2 immunity with production of IgG1, while the RP18-2 fraction contained the dodecylamide derivatives of DS saponins and stimulated Th1 immunity with production of IgG2a, IFN-gamma, IL-2, and CTL. The strong immune stimulatory properties of RP18-2, relative to RP18-1, and the formation of RP18-1/RP18-2 mixed micelles may account for the effective stimulation of Th1 immunity by UFGPI-0100. UFGPI-0100 was free of acylated quillaja saponin components, including the more stable QS-7.

Structure/Function Relationships of Immunostimulating Saponins

Abstract Saponins are widely distributed plant glycosides comprising either steroidal or triterpene aglycones linked to carbohydrate chains. The triterpene saponins from Quillaja saponaria Molina, Gypsophila sp. , and Saponaria officinalis , have unique immunostimulating and immunomodulating properties. Analysis of their structure/function relationships shows common structural characteristics (i.e. a triterpene aglycone containing an aldehyde group at C-4, and oligosaccharide chains attached to positions 3 and 28) that appear to account for these saponins’ effects on the immune system. The crucial role of the saponins triterpene imine-forming carbonyl group in immune stimulating and modulating activities is supported by the following: i) saponins lacking such an aldehyde group are devoid of these activities, and ii) modification of the quillaja saponins aldehyde group results in the loss of such activities. The oligosaccharide chains of these saponins appear to mediate targeting of saponins to antigen presenting cells (APCs), thus enhancing their immunological properties. The saponins from Quillaja saponaria Molina also contain two acyl moieties: a normonoterpene carboxylic acid and a normonoterpene carboxylic acid glycoside, which are linked linearly to a fucosyl residue attached at position C-28. These acyl groups appear to be responsible for the stimulation of cytotoxic T cells (CTLs) against exogenous antigens. Removal of the acyl moieties by mild basic hydrolysis yields deacylated quillaja saponins that are structurally and functionally comparable to the non-acylated Gypsophila sp. and Saponaria officinalis saponins; all these non-acylated saponins stimulate a poor primary immune response. Replacement of the quillaja saponins acyl moieties with other hydrophobic groups alters their immune stimulating and modulating properties.

Reduced proliferation of non-megakaryocytic acute myelogenous leukemia and other leukemia and lymphoma cell lines in response to eltrombopag

Leukemia cell lines were treated with eltrombopag or thrombopoietin and their proliferative response was determined. Eltrombopag did not increase proliferation of cell lines that did not express high levels of megakaryocyte markers. Instead, treatment with eltrombopag alone inhibited proliferation of many cell lines (IC(50) range=0.56-21 microg/mL). The addition of other cytokines, such as G-CSF, Epo or Tpo, did not affect the decrease in proliferation. The decrease in proliferation appears to be through a TpoR-independent, nonapoptotic mechanism. These findings suggest that eltrombopag does not enhance, but rather inhibits, proliferation of leukemia cell lines in vitro.

Abstract 3730: A quick and cost effective 12-cell line panel assay to predict drug activity in human tumor xenograft models

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The procedure to identify and develop an anti-cancer drug first involves testing drug candidates in cell lines followed by human tumor xenograft models, usually selected based upon the histotype of the cell lines in which the drug showed optimal activity. Many drugs fail at this stage, as activity in cell lines does not often correlate with activity in xenograft models. This is not surprising, as we have previously shown that gene expression in xenograft models does not necessarily correlate with the cell line from which it was derived. In an attempt to improve the success rate of drugs tested in xenograft models, we have developed a fast and cost effective 12-panel human tumor cell line assay that represents the genetic diversity of all our xenograft models and several different cancer histotypes. Affymetrix genomic analysis was performed on 100 human tumor xenograft and cell line models. The genomic profiles obtained underwent Unsupervised Hierarchical Cluster Analysis to group models with similar genetic profiles. This analysis resulted in 12 distinct clusters; a representative cell line was chosen from each cluster. Stocks of each representative cell line were frozen and tested to ensure exponential growth immediately upon thawing, resulting in no waiting time for drug testing. It follows that if a candidate drug shows activity in one or more of these representative cell lines, other cell lines and/or xenograft models in the same cluster can also be tested. As the cell lines and xenograft models within the same cluster will have a similar genetic profile, the chances of success should thus be increased. To test the effectiveness of this approach, we used our database to further develop an internal compound. SRI-20900 had been tested previously in the CCRF-CEM and CAKI-1 xenograft models. The compound showed no activity in CCRF-CEM cells, but excellent activity in CAKI-1 cells. These models were in completely different clusters. So, based on these data, we tested the compound in the SKOV-3 and IGROV-1 xenograft models, as these clustered closely to the CAKI-1 model. The compound showed excellent activity in both SKOV-3 and IGROV-1 models. Although these data provide proof of principle, further work needs to be done by testing targeted compounds in the 12-cell line panel, followed by testing in xenograft models within the same cluster as the cell lines that show optimal activity. In addition, it would follow that a xenograft model within the same cluster as an inactive cell line should also be tested. We hope to start these studies early in 2014. Citation Format: Michael J. Roberts, Tommie A. Gamble, Richard D. May, Murray Stackhouse, Kristy L. Berry, Andrew D. Penman, Robert J. Rooney, Yulia Maxuitenko, Michael S. Koratich. A quick and cost effective 12-cell line panel assay to predict drug activity in human tumor xenograft models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3730. doi:10.1158/1538-7445.AM2014-3730

Abstract 3943: Affymetrix whole genome microarray analysis of 51 human tumor cell lines representative of 16 different tissues of origin: Unsupervised hierarchical clustering

The total mRNA for each sample of 51 human tumor cell lines representative of 16 different tissues of origin was split into 3 replicates, and analyzed against the entire human genome using standard Affx WT procedures. Approximately 50% of the samples (25 out of the 51 cell lines tested) exhibited low-level clustering related to their tissue of origin. The remaining 50% (26 out of the 51 cell lines tested) did not cluster with other samples of the same tissue of origin. These data reveal the importance of testing potential anticancer agents in multiple models representative of several different tumors of origin, as there is a 50% chance that the model chosen is not actually representative of the intended tissue of origin. This analysis also showed that the pancreatic cancer cell line CFPAC-1 did not cluster with any other cell line tested, revealing the unique genetic profile of this cell line. Interestingly, the reported lung cancer cell lines NCI-H69 and NCI-H82 clustered more closely with leukemic lines than with lung or any other solid tumor. This is particularly interesting as these lines are known to grow/behave more like a suspension culture than a monolayer. The NCI recently published its genetic analysis of their 60-panel, and they revealed that the MDA-MB-435 cell line, traditionally thought to be a breast cancer cell line, more closely resembled a melanoma line; hence, it was re-classified as a melanoma (likely a metastasized melanoma that was taken from the breast site). Our analysis reveals that another traditional breast cancer cell line, UISO-BCA-1, also clusters more closely with the melanomas (including the MDA-MB-435 cell line), suggesting that this cell line also may have been misclassified. Based on these data, we suggest that any potential anticancer agent showing activity in a particular cell line should be tested in other cell lines that cluster with the active line, and not merely in other lines supposedly representative of the same tissue of origin. Furthermore, in early stage testing, it would be more prudent to test several cell lines from different clusters, rather than several cell lines from different tissues of origin. It follows that by testing orphan drugs against several cell lines from each cluster, it would be possible to significantly narrow (and possibly identify), the likely drug target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3943. doi:10.1158/1538-7445.AM2011-3943

Abstract 4418: Affymetrix whole genome microarray analysis of 24 human tumor xenograft models and the cell lines from which they were developed: Clustering of targets vs tumor tissue of origin

The NCI recently performed microarray expression analysis on its 60-panel of human tumor cell lines. This revealed important information, with some cell lines shown to be from a different tissue of origin than originally believed. Traditionally, potential anti-cancer agents have been evaluated in these in vitro models, and then moved into the corresponding in vivo xenograft model(s) based on the in vitro results. It has been shown that drugs which are effective in vitro are not necessarily effective in vivo and vice versa. Systematic microarray analysis of traditional xenograft models in conjunction with their in vitro counterparts has not been performed. The development of a human tumor xenograft in a mouse might be expected to lead to changes in gene expression, and this could account, in some instances, for the disconnect in results observed between in vitro and in vivo models. Our aim was to perform a genetic analysis against the entire human genome using 24 cell lines from 11 differing tissues of origin that were implanted into immune-deficient mice to establish a xenograft model for each. Once the tumors reached approximately 1 cm3 in size, the tumors were removed, cut into approx. 2-3 mm3 fragments, and an in vivo tumor passage was established. Microarray expression in fragments of those xenografted tumors was compared to microarray expression in the cell line from which they were developed. The total mRNA for each sample was split into 3 replicates, and analyzed against the entire human genome using standard Affx WT procedures. The results showed that over 60% (15 of 24) of the xenograft samples clustered with the cell line from which it was developed, whereas approximately 40% (9 of 24) did not, revealing that major changes in gene expression had occurred in 40% of these xenograft samples. Furthermore, when analyzed alone, these particular 24 cell line samples clustered according to their tissue of origin, whereas the tumor fragment samples did not appear to cluster. On the basis of these data we are currently performing the same analysis on an additional 25 tumor fragments and their corresponding matched cell lines to allow for a more accurate, in-depth cluster analysis. These data strongly suggest that although precedent exists to select in vitro models on the basis of their tissue of origin, no such precedent exists for in vivo models. In vivo models should be more carefully selected to ensure that the model chosen is still representative of the tissue to be tested. It follows that a drug candidate effective in a particular in vitro model might be expected to show activity in other in vitro lines from the same tissue of origin. However, a drug candidate effective in a particular in vivo model representative of a tissue of origin should not be expected to show efficacy in other in vivo models representing the same tissue type. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4418. doi:10.1158/1538-7445.AM2011-4418