Dimethylaminoparthenolide Research Articles

Analysis of the Anti-Leukemia Mechanism of Parthenolide.

Abstract 2734Poster Board II-710We have previously demonstrated that parthenolide (PTL), a naturally occurring small molecule found in feverfew Chrysanthemum parthenium, induces apoptosis in primary acute myeloid leukemia (AML) cells, including the stem and progenitor cell compartment. Based on these preclinical findings, a PTL derivative (dimethylamino parthenolide) is currently being evaluated in a phase I clinical trial. However, despite the promising activity of PTL, its underlying mechanism of action remains poorly understood. Thus, we have undertaken biochemical studies to better characterize how PTL mediates leukemia-specific cell death. Chemically, the key structural feature of PTL is its alpha-metheylene-gamma-lactone moiety, which via Michael reaction is predicted to mediate potent free thiol scavenging, an activity readily observed in PTL-treated cells. Reported consequences of PTL chemical reactivity in a variety of cell types, represent a broad range of activities that include inhibition of NFkB, activation of p53, ubiquitination of MDM2, inhibition of DNMT1 and inhibition of HDAC1. To better define the specific activities responsible for parthenolide-mediated leukemia cell death, we have employed two general approaches. First, we generated a biotinylated analog of parthenolide (PTL-biotin), which was shown to retain the anti-leukemia activity of the parent compound. PTL-biotin was then used in biochemical pull-down assays to purify parthenolide target proteins, followed by liquid phase chromatography-mass spectrometry (LC-MS) for protein identification. To further verify molecular interactions, native non-biotinylated parthenolide was used to compete binding between candidate targets and PTL-biotin. These studies identified HSP70 as a direct target of PTL. Notably, cysteine-17 of HSP70 is exposed to the ADP/ATP binding site crevice and a molecular docking study indicates that the covalent attachment of PTL to this residue should disrupt the ATP hydrolysis function of the protein. These findings imply that inhibition of HSP70 may contribute to the cell death mechanism underlying PTL anti-leukemia based activity. As a second approach to characterizing PTL, we have performed comparative studies using the closely related compound costunolide (CSN). Since previous structure-activity studies with PTL analogs revealed that opening of the epoxide ring at C4-C5 of the molecule completely destroys the anti-tumor activity, we sought to utilize a compound lacking this feature. CSN lacks the epoxide group, but is otherwise identical to PTL, and retains the key alpha-metheylene-gamma-lactone moiety. Interestingly, at concentrations where PTL is highly cytotoxic, CSN does not induce leukemia-specific cell death (less than 10% death for primary AML cells at 7.5 microM). Analysis of CSN activity demonstrated that despite the lack of AML cell death, CSN still induced loss of free thiols and increased reactive oxygen species in a fashion comparable to PTL (as measured by mBBR and CM-H2DCFDA based flow cytometry). However, CSN is markedly less effective as an inhibitor of NFkB activity (measured by phosphorylation level of NFkB p65). Taken together these findings indicate that oxidative stress alone is not sufficient for PTL-mediated cell death, and further extend previous molecular genetic data demonstrating that NFkB inhibition is an important component of the overall cell death mechanism. The data also show that the alpha-metheylene-gamma-lactone moiety alone is not sufficient to mediate all aspects of PTL activity, and that at least some activity/specificity is created by juxtaposition of the epoxide group. Based on these studies, as well as previous data, we propose that inhibition of NFkB and HSP70 are components of the parthenolide-mediated cell death mechanism, and that oxidative stress is a necessary but not sufficient aspect of its leukemia-specific activity. Disclosures:No relevant conflicts of interest to declare.

Dimethylaminoparthenolide (DMAPT) in Multiple Myeloma

Background: The nuclear factor kappa B (NFkB) signaling pathway is constitutively activated and central to the pathogenesis of multiple myeloma (MM). We previously published the antitumor activity of the NFkB inhibitor parthenolide in MM. Dimethylaminoparthenolide (DMAPT) is a water-soluble analog of parthenolide. The improved bioavailability of DMAPT makes its appealing for clinical development.Objectives: To validate the effects of DMAPT both in vitro and in vivo MM models and to develop a rational strategy for a combination of DMAPT with other anti-MM agents.Methods: The in vitro cytotoxicity of DMAPT was tested in human MM cell lines and primary samples both alone and in combination with bortezomib and lenalidomide. The in vivo activity of DMAPT was tested in a model of NOD/Scid mice intravenously transplanted with GFP-tagged NCI-H929 MM cells.Results: Based on MTT assays on MM cell lines and primary MM cells, the IC50 for proliferation inhibition of DMAPT was similar to that of parthenolide (1–3 μM). When used in combination with bortezomib, we observed synergy (combinatorial index < 1.0) at higher doses of DMPT and bortezomib (DMAPT 2.5–5 μM and bortezomib 5–12.5 nM), while an antagonistic effect was observed at lower doses. Similarly, when combined with lenalidomide, synergy was observed at DMAPT doses 1.6–6.4 μM and lenalidomide doses 0.5–2 μM, while an antagonistic effect was observed at lower doses.The in vivo cytotoxicity of DMAPT was explored in a model of NOD/Scid mice intravenously transplanted with GFP-tagged NCI-H929 cells. At 3 weeks post-transplantation, all animals had measurable serum human kappa light chain levels, as measured by ELISA assays. Animals were randomized to receive either 50 mg/kg/day DMAPT (n=12), 100 mg/kg/day DMAPT (n=12) or vehicle (n=10) via oral gavage for a duration of 6 weeks or until criteria for euthanasia were met. Weight loss of over 15% and hind leg paralysis were criteria for euthanasia. Toxicities in both treatment groups were minimal. Weight loss correlated with serum human kappa light chain levels and, hence, represented disease progression. At the end of treatment, there were 2 animals alive in the control group, while 5 were alive in each of the treated groups. Using Kaplan-Meier curves and log-rank analysis, the mean overall survival (OS) was 56.3 days in the control cohort versus 61.0 days and 62.4 days in groups treated with 50 and 100 mg/kg/day DMAPT, respectively. There was no statistically significant difference in the mean OS of mice treated with the two doses of DMAPT (p=0.07).Prior to treatment initiation, the serum levels of human kappa light chains were similar in all groups (ANOVA test, p= 0.38). At 2 weeks and 4 weeks post treatment (Week 5 and week 7, respectively; See table), the levels of human kappa light chains were significantly lower in the DMAPT treated groups (ANOVA test p = 0.017 and 0.0061, respectively).Week 3Week 5Week 7Control10.46 ± 5.79531.31 ± 2386272.1 ± 3211DMAPT 50 mg/kg/day6.94 ± 4.48264.03 ± 1982928.8 ± 2131DMAPT 100 mg/kg/day7.99 ± 7.21334.15 ± 1983374.9 ± 1835ANOVA (P value)0.380.0170.0061Conclusions: DMAPT demonstrated excellent efficacy in preclinical models of MM and supports a rationale for its clinical development in MM patients with relapsed MM either alone or in combination with bortezomib or lenalidomide.

Effect of Celecoxib and the Novel Anti-Cancer Agent, Dimethylamino-Parthenolide, in a Developmental Model of Pancreatic Cancer

Cancer of the exocrine pancreas is the fourth leading cause of cancer-related deaths in the United States. The efficacy of a novel bioavailable anticancer agent, dimethylamino-parthenolide (DMAPT), and the cyclooxygenase 2 inhibitor, celecoxib, was evaluated in a carcinogen-induced developmental model of pancreatic cancer. Syrian golden hamsters were injected with N-nitrosobis(2-oxopropyl)amine, once weekly for 6 weeks. Upon the first injection, hamsters were randomized as follows: placebo, low-/high-dose DMAPT (20 and 40 mg/kg per day), low-/high-dose celecoxib (10and 50 mg/kg per day), or combination DMAPT/celecoxib (low/low, high/high). The 32-week trial showed that 40 mg/kg DMAPT alone significantly decreased the size of gross pancreatic cancers relative to placebo. No significant difference in gross tumor number was observed between the treatment groups and placebo with the exception of 50 mg/kg celecoxib with a higher tumor incidence; this group also exhibited lower lymphotactin levels suggestive of decreased immune surveillance. Tumor invasion into adjacent organs and metastasis were not observed in the DMAPT/celecoxib treatment groups. Drug targets including prostaglandin E2, prostaglandin E2 metabolite and activated nuclear factor kappaB were significantly decreased. Dimethylamino-parthenolide and celecoxib have the potential to be novel chemotherapeutic agents for pancreatic cancer; however, further optimization or the use of other modalities may be required for chemoprevention.

An orally bioavailable parthenolide analog selectively eradicates acute myelogenous leukemia stem and progenitor cells

Leukemia stem cells (LSCs) are thought to play a central role in the pathogenesis of acute leukemia and likely contribute to both disease initiation and relapse. Therefore, identification of agents that target LSCs is an important consideration for the development of new therapies. To this end, we have previously demonstrated that the naturally occurring compound parthenolide (PTL) can induce death of human LSCs in vitro while sparing normal hematopoietic cells. However, PTL has relatively poor pharmacologic properties that limit its potential clinical use. Consequently, we generated a family of PTL analogs designed to improve solubility and bioavailability. These studies identified an analog, dimethylamino-parthenolide (DMAPT), which induces rapid death of primary human LSCs from both myeloid and lymphoid leukemias, and is also highly cytotoxic to bulk leukemic cell populations. Molecular studies indicate the prevalent activities of DMAPT include induction of oxidative stress responses, inhibition of NF-kappaB, and activation of p53. The compound has approximately 70% oral bioavailability, and pharmacologic studies using both mouse xenograft models and spontaneous acute canine leukemias demonstrate in vivo bioactivity as determined by functional assays and multiple biomarkers. Therefore, based on the collective preclinical data, we propose that the novel compound DMAPT has the potential to target human LSCs in vivo.