Edina Komlodi-Pasztor

Mitosis is not a key target of microtubule agents in patient tumors

Mitosis-specific agents have, to date, not been clinically successful. By contrast, microtubule-targeting agents (MTAs) have a long record of success, usually attributed to the induction of mitotic arrest. Indeed, it was this success that led to the search for mitosis-specific inhibitors. We believe the clinical disappointment of mitosis-specific inhibitors stands as evidence that MTAs have been successful not only by interfering with mitosis but, more importantly, by disrupting essential interphase cellular mechanisms. In this Perspective we will review literature that supports a paradigm shift in how we think about one of our most widely used classes of chemotherapeutics—MTAs. We believe that the steady presence and constant physiological role of microtubules are responsible for the overall success of MTAs. While mitosis-specific inhibitors are effective on only a small fraction of the tumor mass (dividing cells), MTAs target tubulin, a protein that has crucial roles in both mitotic and non-mitotic cells.

Inhibitors Targeting Mitosis: Tales of How Great Drugs against a Promising Target Were Brought Down by a Flawed Rationale

Although they have been advocated with an understandable enthusiasm, mitosis-specific agents such as inhibitors of mitotic kinases and kinesin spindle protein have not been successful clinically. These drugs were developed as agents that would build on the success of microtubule-targeting agents while avoiding the neurotoxicity that encumbers drugs such as taxanes and vinca alkaloids. The rationale for using mitosis-specific agents was based on the thesis that the clinical efficacy of microtubule-targeting agents could be ascribed to the induction of mitotic arrest. However, the latter concept, which has long been accepted as dogma, is likely important only in cell culture and rapidly growing preclinical models, and irrelevant in patient tumors, where interference with intracellular trafficking on microtubules is likely the principal mechanism of action. Here we review the preclinical and clinical data for a diverse group of inhibitors that target mitosis and identify the reasons why these highly specific, myelosuppressive compounds have failed to deliver on their promise. Clin Cancer Res; 18(1); 51–63. ©2012 AACR.

Microtubule-targeting agents augment the toxicity of DNA-damaging agents by disrupting intracellular trafficking of DNA repair proteins

Significance Drugs targeting microtubules are among the most active anticancer agents. In vitro and in preclinical models, these agents are said to interfere with mitosis. However human tumors divide too slowly for this paradigm to apply, evidenced by the failure of over a dozen well-designed antimitotic agents targeting the aurora kinases and kinesin spindle protein that had minimal antitumor activity but caused severe bone marrow suppression. We have proposed that microtubule-targeting agents interfere with the trafficking of critical proteins in interphase microtubules. If true, then one must identify critical proteins whose traffic on microtubules is impacted. We identify nine DNA repair proteins that traffic on microtubules, explaining why combinations of a microtubule-targeting agent and a DNA-damaging agent are frequently used in cancer therapy. The paradigm that microtubule-targeting agents (MTAs) cause cell death via mitotic arrest applies to rapidly dividing cells but cannot explain MTA activity in slowly growing human cancers. Many preferred cancer regimens combine a MTA with a DNA-damaging agent (DDA). We hypothesized that MTAs synergize with DDAs by interfering with trafficking of DNA repair proteins on interphase microtubules. We investigated nine proteins involved in DNA repair: ATM, ATR, DNA-PK, Rad50, Mre11, p95/NBS1, p53, 53BP1, and p63. The proteins were sequestered in the cytoplasm by vincristine and paclitaxel but not by an aurora kinase inhibitor, colocalized with tubulin by confocal microscopy and coimmunoprecipitated with the microtubule motor dynein. Furthermore, adding MTAs to radiation, doxorubicin, or etoposide led to more sustained γ-H2AX levels. We conclude DNA damage-repair proteins traffic on microtubules and addition of MTAs sequesters them in the cytoplasm, explaining why MTA/DDA combinations are common anticancer regimens.

Schedule-dependent synergy of histone deacetylase inhibitors with DNA damaging agents in small cell lung cancer

Small cell lung cancer (SCLC) is an aggressive lung cancer subtype in need of better therapies. Histone deacetylase inhibitors (HDIs) promote increased lysine acetylation in nucleosomal histones and are thought to relax chromatin, thereby allowing increased access of transcription factors and DNA damaging agents alike to DNA. We studied whether two HDIs, belinostat and romidepsin, could be effectively combined with cisplatin or etoposide (VP-16) for SCLC cells. Analysis of cell survival and synergy was performed using CalcuSyn mathematical modeling to calculate a combination index. Immunostaining of γH2AX was performed to evaluate persistence of DNA damage following simultaneous or sequential exposure. Based on CalcuSyn modeling, HDIs synergized with DNA damaging agents only when added simultaneously. An additive-to-antagonistic effect was seen with HDI pretreatment for 24 h or with addition after cisplatin or etoposide. Furthermore, pretreatment with HDIs resulted in normalization of cell cycle and reduced PARP degradation as compared with simultaneous treatment. The increase in γH2AX phosphorylation confirmed that simultaneous but not sequential treatment enhanced double-stranded DNA breaks. These results suggest that DNA relaxation is not required for synergy of HDIs with DNA damaging agents, and that scheduling of drug administration will be critical for rational development of clinical protocols.

Retained platinum uptake and indifference to p53 status make novel transplatinum agents active in platinum-resistant cells compared to cisplatin and oxaliplatin.

Despite the clinical success of platinum-containing drugs in the treatment of solid tumors, acquired resistance remains a major obstacle. We previously identified a group of novel transplanaramine or transplatinum compounds based on distinct activity profiles in the NCI-60 panel. In the present study, parental KB-3.1 cells with wild-type p53 and its cisplatin- and oxaliplatin-resistant sublines harboring mutant p53 proteins were used to contrast several transplatinum compounds with cisplatin and oxaliplatin. The transplatinum compounds retained cytotoxic activity in the resistant cell lines. While intracellular accumulation and DNA platination of cisplatin and oxaliplatin was decreased in the resistant cells, the transplatinum compounds both accumulated intracellularly and platinated DNA at comparable levels in all cell lines. Cytoflow analysis confirmed that cisplatin and oxaliplatin alter the cell cycle distribution and result in apoptosis; however, at comparably toxic concentrations, the transplatinum compounds did not alter the cell cycle distribution. Analysis of the cytoplasmic fraction treated with acetone showed that cisplatin and oxaliplatin readily bound to macromolecules in the pellet, whereas a larger percentage of the transplatinum compounds remained in the supernatant. We concluded that, distinct from platinum compounds currently in use, transplatinum compounds accumulate intracellularly in resistant cells at levels comparable to those in drug-sensitive cells, do not affect the cell cycle and thus retain cytotoxicity independent of p53 status and likely have cytoplasmic targets that are important in their activity.

Tales of How Great Drugs Were Brought Down by a Flawed Rationale—Response

We argue that drugs that specifically target mitosis will be more effective if mitosis is common and less so if mitosis is rare. Hence, the occurrence of myelosuppression, following therapy with microtubule-targeting agents and with mitosis-specific agents, reflects the high mitotic activity in the

Authors' reply: (Not) too early to say, |[ldquo]|no targeting of mitosis!|[rdquo]|

We appreciate Dr Katsumi Kitagawa’s com‐ ments and interest in our article: Mitosis is not a key target of microtubule agents in patient tumors. Nat. Rev. Clin. Oncol. 8, 244–2501 (Too early to say, “no targeting of mitosis!” Nat. Rev. Clin. Oncol. doi:10.1038/ nrclinonc.2010.228‐c1)2. The main points of our article were these: first, although microtubule target‐ ing agents (MTAs) have been successful as cancer therapeutics, to date, the clinical results from mitosis‐specific agents have been disappointing despite clear evidence of their ability to affect mitosis in patients, as evidenced by their principal toxicity, neutro penia. Second, MTAs can target critical microtubule functions in cells while they are in mitosis as well as critical cellular functions in other phases of the cell cycle, where many cells spend more than 98% of their time. By comparison, mitosis‐specific agents can only target cells in mitosis, and this occurs infrequently in patient tumors. Finally, we concluded that MTAs are suc‐ cessful because they target both mitotic and non‐mitotic microtubule functions. In cell culture, xenografts and in humans, mitosis in normal or cancer cells is uni‐ formly fast, lasting on average about 1 h. By contrast, one can find marked differences in the rates of cell growth when comparing human tumors with xenografts or cells in culture, with tumors having rates that are relatively slower. In our article, we did not suggest that a tumor’s growth rate is defined by mitotic frequency, but it is true that cells in mitosis are uncommon in human tumors (Table 1). Slow‐growing tumors present fewer mitotic cells as targets for mitotic agents compared with rapidly doubling cells in culture and xenografts. We agree with Dr Kitagawa2 that the net increase in the size of a tumor in xenografts and in human cancers is the result of concomitant cell death and cell replication. While both of these events occur in patient tumors, losses from cell death do not contribute substantially to the ‘sum’ value.3–5 In addi‐ tion, although the growth rate of a tumor AUTHORS’ REPLY

Abstract LB-106: Microtubule-targeting agents (MTAs) disrupt intracellular trafficking of DNA repair proteins and augment the toxicity of DNA damaging agents (DDAs)

MTAs have long been thought to cause cell death primarily by inducing mitotic arrest, a paradigm that applies to rapidly dividing cells in preclinical models. However, mitotic arrest cannot explain the activity of MTAs in much more slowly growing human cancers. In the latter, we have proposed interference with trafficking on interphase microtubules (MTs) as the principal mechanism of cytotoxicity. Satisfying this paradigm requires identification of the proteins whose trafficking on MTs, when disrupted, leads to cytotoxicity. MTAs in combination with DNA-damaging agents (DDAs) have emerged as preferred regimens for the treatment of ovarian, lung, some head and neck cancers, and many lymphomas. We proposed that by interfering with the trafficking of DNA repair proteins, MTAs could synergize with DDAs, augmenting their toxicity and enhancing cell death. To explore this hypothesis, we systematically investigated the effects of either paclitaxel or vincristine on treatment-induced DNA damage and on the distribution and biology of nine different proteins involved in DNA repair: ATM, ATR, DNA-PK, Rad50, Mre11, p95/NBS1, TP53, 53BP1 and p63. In several cell models including A549 cells and four Burkitt9s lymphoma models (CA46, DG-75, Ramos and ST486) addition of vincristine increased cytoplasmic retention of the DNA repair proteins, thus excluding a greater fraction from the nucleus. The latter effect was observed only with MTAs and not with an inhibitor of Aurora kinase, despite similar cell cycle effects, confirming the cytoplasmic retention seen with MTAs is not due to alterations in the cell cycle. Increased cytoplasmic retention of DNA repair proteins following MT disruption suggests these proteins traffic on MTs and are vulnerable to MTAs, and this is supported by both confocal microscopy demonstrating co-localization of DNA repair proteins and α- or αβ-tubulin, as well as co-immunoprecipitation of these proteins with antibodies against the MT motor, dynein. In both A549 and MCF cells, the repair of DNA as measured by the level and persistence of γ-H2AX, was prolonged by the addition of paclitaxel to radiation. Moreover, when MCF7 or A549 cells were treated with either adriamycin or etoposide, γ-H2AX was induced to higher levels and for longer times when cells were treated with vincristine prior to and with the DDA and during DDA washout, when vincristine was present. In comparison, γ-H2AX decline was more rapid after DDA washout for cells treated only with the DDA. Together these data demonstrate that many DNA damage repair proteins travel on MTs and that the addition of MTAs promotes their sequestration in the cytoplasm. By interfering with the repair of DNA, cytoplasmic retention results in greater toxicity and likely explains why combinations of MTAs and DDAs have emerged as favored drug combinations for therapy of a diverse group of cancers. Citation Format: Marianne S. Poruchynsky, Edina Komlodi-Pasztor, Julia Wilkerson, Shana Trostel, Mauricio Burroto-Pichun, Tito Fojo. Microtubule-targeting agents (MTAs) disrupt intracellular trafficking of DNA repair proteins and augment the toxicity of DNA damaging agents (DDAs). [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 LB-106. doi:10.1158/1538-7445.AM2014-LB-106

Abstract 2554: Copper transporter 2 (CTR2) is able to transport cisplatin and/or oxaliplatin in drug-resistant cell lines

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The clinical use of platinum (Pt) agents is often compromised by the occurrence of drug resistance. Studies have shown reduced intracellular Pt accumulation in cells resistant to Pt drugs. Copper transporters have been reported to have a role in Pt influx and efflux. While the first member of this family, CTR1, has been shown to transport Pt, the role of the structurally similar CTR2 in Pt transport is unclear. The aim of this study was to assess the role of CTR2 in Pt based drug-transport using KB-3.1 cervical carcinoma cells and its cisplatin (KB-CP20) or oxaliplatin (KB-OX60) resistant subclones. In previous studies we reported that compared to parental cells the resistant cell lines have lower levels of intracellular Pt following cisplatin (CIS) or oxaliplatin (OX) exposure. We transfected parental KB-3.1 cells and its drug resistant sublines with CTR1, CTR2 or both transporters, and incubated cells with either CIS or OX. Intracellular Pt accumulation was not increased in any of the transfected parental KB-3.1 cells. In contrast, KB-CP20 cells transfected with CTR1, CTR2 or both trasnporters accumulated more CIS but not more OX compared to non-transfected KB-CP20 cells; while transfected KB-OX60 cells accumulated higher levels of both CIS and OX compared to non-transfected KB-OX 60 cells. In both resistant cell lines, the increased intracellular Pt observed after transtfection of the transporters was accompanied by increased DNA platination. Given the clinical utility of OX in colon carcinoma we next determined intracellular CIS and OX levels, the expression of proteins involved in copper transport (ATP7A, ATP7B, CTR1 and CTR2), and the IC50 for CIS and OX of six colon carcinoma cell lines. We found an inverse correlation between intracellular Pt levels and the IC50 of both drugs, and correlations between: (1) the IC50 of CIS and expression levels of CTR1, (2) intracellular CIS levels and expression levels of ATP7A, (3) expression levels of ATP7B and CTR2, and (4) OX accumulation and expression levels of ATP7A. In summary, we have shown CTR2 is able to transport Pt-containing drugs and increases intracellular Pt accumulation that is accompanied by greater DNA-platination. In unselected colon cancer cells, we have shown that the IC50 of a Pt based drug is determined by the intracellular accumulation of that drug, and to varying extent by the expression of copper transporters. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2554.