Dimethylaminoparthenolide Research Articles

Targeting Vasohibins to Promote Axon Regeneration.

Treatments accelerating axon regeneration in the nervous system are still clinically unavailable. However, parthenolide promotes adult sensory neurons' axon growth in culture by inhibiting microtubule detyrosination. Here, we show that overexpression of vasohibins increases microtubule detyrosination in growth cones and compromises growth in culture and in vivo. Moreover, overexpression of these proteins increases the required parthenolide concentrations to promote axon regeneration. At the same time, the partial knockdown of endogenous vasohibins or their enhancer SVBP in neurons facilitates axon growth, verifying them as pharmacological targets for promoting axon growth. In vivo, repeated intravenous application of parthenolide or its prodrug di-methyl-amino-parthenolide (DMAPT) markedly facilitates the regeneration of sensory, motor, and sympathetic axons in injured murine and rat nerves, leading to acceleration of functional recovery. Moreover, orally applied DMAPT was similarly effective in promoting nerve regeneration. Thus, pharmacological inhibition of vasohibins facilitates axon regeneration in different species and nerves, making parthenolide and DMAPT the first promising drugs for curing nerve injury.

Inhibition of microtubule detyrosination by parthenolide facilitates functional CNS axon regeneration

Injured axons in the central nervous system (CNS) usually fail to regenerate, causing permanent disabilities. However, the knockdown of Pten knockout or treatment of neurons with hyper-IL-6 (hIL-6) transforms neurons into a regenerative state, allowing them to regenerate axons in the injured optic nerve and spinal cord. Transneuronal delivery of hIL-6 to the injured brain stem neurons enables functional recovery after severe spinal cord injury. Here we demonstrate that the beneficial hIL-6 and Pten knockout effects on axon growth are limited by the induction of tubulin detyrosination in axonal growth cones. Hence, cotreatment with parthenolide, a compound blocking microtubule detyrosination, synergistically accelerates neurite growth of cultured murine CNS neurons and primary RGCs isolated from adult human eyes. Systemic application of the prodrug dimethylamino-parthenolide (DMAPT) facilitates axon regeneration in the injured optic nerve and spinal cord. Moreover, combinatorial treatment further improves hIL-6-induced axon regeneration and locomotor recovery after severe SCI. Thus, DMAPT facilitates functional CNS regeneration and reduces the limiting effects of pro-regenerative treatments, making it a promising drug candidate for treating CNS injuries.

Inhibition of microtubule detyrosination by parthenolide facilitates functional CNS axon regeneration.

Injured axons in the central nervous system (CNS) usually fail to regenerate, causing permanent disabilities. However, the knockdown of Pten knockout or treatment of neurons with hyper-IL-6 (hIL-6) transforms neurons into a regenerative state, allowing them to regenerate axons in the injured optic nerve and spinal cord. Transneuronal delivery of hIL-6 to the injured brain stem neurons enables functional recovery after severe spinal cord injury. Here we demonstrate that the beneficial hIL-6 and Pten knockout effects on axon growth are limited by the induction of tubulin detyrosination in axonal growth cones. Hence, cotreatment with parthenolide, a compound blocking microtubule detyrosination, synergistically accelerates neurite growth of cultured murine CNS neurons and primary RGCs isolated from adult human eyes. Systemic application of the prodrug dimethylamino-parthenolide (DMAPT) facilitates axon regeneration in the injured optic nerve and spinal cord. Moreover, combinatorial treatment further improves hIL-6-induced axon regeneration and locomotor recovery after severe SCI. Thus, DMAPT facilitates functional CNS regeneration and reduces the limiting effects of pro-regenerative treatments, making it a promising drug candidate for treating CNS injuries.

Pharmacological NF-κB inhibition decreases cisplatin chemoresistance in muscle-invasive bladder cancer and reduces cisplatin-induced toxicities.

Most patients with muscle-invasive bladder cancer (MIBC) are not cured with platinum chemotherapy. Up-regulation of nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) is a major mechanism underlying chemoresistance, suggesting that its pharmacological inhibition may increase platinum efficacy. NF-κB signaling was investigated in two patient cohorts. The Cancer Genome Atlas (TCGA) was used to correlate NF-κB signaling and patient survival. The efficacy of cisplatin plus the NF-κB inhibitor dimethylaminoparthenolide (DMAPT) versus cisplatin or DMAPT alone was tested in vitro. Xenografted and immunocompetent MIBC mouse models were studied in vivo. Platinum-naive claudin-low MIBC showed constitutive NF-κB signaling and this was associated with reduced disease-specific survival in TCGA patients. Chemotherapy up-regulated NF-κB signaling and chemoresistance-associated genes, including SPHK1, PLAUR, and SERPINE1. In mice, DMAPT significantly improved the efficacy of cisplatin in both models. The combination preserved body weight, renal function, and morphology, reduced musclefatigue and IL-6 serum levels, and did not aggravate immuno-hematological toxicity compared with cisplatin alone. These data provide a rationale for combining NF-κB inhibition with platinum-based chemotherapy and conducting a clinical trial in MIBC patients.

Effects of Gemcitabine/Nab-Paclitaxel and DMAPT in PDAC Cachexia in vivo and in vitro

Cachexia is the involuntary wasting of skeletal muscle and adipose tissue, affecting over 80% of patients with pancreatic ductal adenocarcinoma (PDAC). Gemcitabine and nab-paclitaxel (GemNP) combination are commonly given as first-line treatments for PDAC. Data from our lab showed GemNP reduced tumor growth and prevented muscle and fat wasting. Dimethylaminoparthenolide (DMAPT), a small molecule inhibitor, prevented muscle wasting and prolonged survival in a genetic murine model of breast cancer. It has yet to determine if GemNP in combination with DMAPT can improve indices of cachexia and overall survival. Therefore, I hypothesize that GemNP in combination with DMAPT will improve indices of cachexia and overall survival in a murine model of pancreatic cancer. Male 10-week-old C57BL/6J mice underwent orthotopic injection of 5x104 KPC cells or SHAM surgery. Mice were randomly assigned to 1 of 4 groups (N=10/group): SHAM+Vehicle, KPC+Vehicle, KPC+GemNP, KPC+GemNP+DMAPT. Gemcitabine (120mg/kg) and Nab-paclitaxel (10mg/kg) were injected intraperitoneally starting on day 4 and continued every 6 days. DMAPT (100mg/kg) was administered by gavage Monday-Friday. To determine the role of GemNP and DMAPT in vitro, KPC cells were treated with GemNP and/or DMAPT and cell viability evaluated. Additionally, we treated myotubes with GemNP with/without DMAPT to assess myotube diameter in an established KPC conditioned media. In KPC mice, GemNP (24.4days) increased survival compared to Vehicle (16.8days, p=.0007). However, the combination GemNP+DMAPT did not extend survival over GemNP alone (25.4days, p=.693). Tumor mass was similar between all groups (p=0.411). Interestingly, at the time of sacrifice, all KPC treated mice independent of treatment had similar reduction in adipose tissue and muscle mass compared to SHAM. In conclusion, the addition of DMAPT to GemNP did not extend survival over GemNP alone in an aggressive pancreatic tumor cell line. Future studies should determine if less aggressive tumor cell lines might benefit from GemNP and DMAPT.

Parthenolide and Its Soluble Analogues: Multitasking Compounds with Antitumor Properties.

Due to its chemical properties and multiple molecular effects on different tumor cell types, the sesquiterpene lactone parthenolide (PN) can be considered an effective drug with significant potential in cancer therapy. PN has been shown to induce either classic apoptosis or alternative caspase-independent forms of cell death in many tumor models. The therapeutical potential of PN has been increased by chemical design and synthesis of more soluble analogues including dimethylaminoparthenolide (DMAPT). This review focuses on the molecular mechanisms of both PN and analogues action in tumor models, highlighting their effects on gene expression, signal transduction and execution of different types of cell death. Recent findings indicate that these compounds not only inhibit prosurvival transcriptional factors such as NF-κB and STATs but can also determine the activation of specific death pathways, increasing intracellular reactive oxygen species (ROS) production and modifications of Bcl-2 family members. An intriguing property of these compounds is its specific targeting of cancer stem cells. The unusual actions of PN and its analogues make these agents good candidates for molecular targeted cancer therapy.

Dimethylaminoparthenolide (DMAPT) as an alternative approach for treatment of Familial Mediterranean Fever (FMF).

Objective(s): Familial Mediterranean Fever (FMF) is a hereditary auto-inflammatory disorder that is caused by mutations in the Mediterranean fever (MEFV) gene and is associated with an increase in pro-inflammatory cytokines, such as interleukin-1β (IL-1β) and interleukin-18 (IL-18), leading to excess inflammation. Colchicine is a common drug widely used for treatment of FMF attacks, but about 5–15% of the patients show resistance to the regular colchicine treatment. In this study, we used dimethylamino-parthenolide (DMAPT), as a small molecule inhibitor of Nuclear factor-κB (NF-κB), NLR family Pyrin domain containing 3 (NLRP3), and cysteine-aspartic acid protease 1(Caspase-1) on FMF-derived peripheral blood mononuclear cells (PBMCs).Materials and Methods:The effects of DMAPT and colchicine on metabolic activity and apoptosis of FMF-derived PBMCs were evaluated by MTT and Annexin V/PI assays, respectively. Also, the expression levels of NF-κB, NLRP3, MEFV, CASP1, and IL-1β mRNA were investigated using a TaqMan real-time PCR, and the protein levels of IL-1β, IL-18, and IL-37 were assessed via an enzyme-linked immunosorbent assay (ELISA) in LPS/ ATP-stimulated PBMCs.Results:DMAPT decreased the expression levels of NFκB (0.38±0.096, P<0.0001), NLRP3 (0.39±0.12, P<0.001), MEFV (0.384±0.145, P<0.001), CASP1 (0.48±0.13, P=0.0023), and IL-1β (0.09±0.09, P<0.0001) and reduced the secretion levels of IL-1β (8.92±5.3 vs. 149.85±20.92, P<0.0001), IL-18 (135±32.1 vs. 192±22.18, P=0.01), and IL-37 (27.5±6.3 vs. 78.19±14.3, P<0.0001) as compared to untreated cells.Conclusion:Given the obtained results in comparison with previous research, the future clinical development of DMAPT could result in the expansion of new anti-inflammatory therapeutics for FMF disorder.

NF-κB Blockade with Oral Administration of Dimethylaminoparthenolide (DMAPT), Delays Prostate Cancer Resistance to Androgen Receptor (AR) Inhibition and Inhibits AR Variants.

NF-κB activation has been linked to prostate cancer progression and is commonly observed in castrate-resistant disease. It has been suggested that NF-κB-driven resistance to androgen-deprivation therapy (ADT) in prostate cancer cells may be mediated by aberrant androgen receptor (AR) activation and AR splice variant production. Preventing resistance to ADT may therefore be achieved by using NF-κB inhibitors. However, low oral bioavailability and high toxicity of NF-κB inhibitors is a major challenge for clinical translation. Dimethylaminoparthenolide (DMAPT) is an oral NF-κB inhibitor in clinical development and has already shown favorable pharmacokinetic and pharmacodyanamic data in patients with heme malignancies, including decrease of NF-κB in circulating leuchemic blasts. Here, we report that activation of NF-κB/p65 by castration in mouse and human prostate cancer models resulted in a significant increase in AR variant-7 (AR-V7) expression and modest upregulation of AR. In vivo castration of VCaP-CR tumors resulted in significant upregulation of phosphorylated-p65 and AR-V7, which was attenuated by combination with DMAPT and DMAPT increased the efficacy of AR inhibition. We further demonstrate that the effects of DMAPT-sensitizing prostate cancer cells to castration were dependent on the ability of DMAPT to inhibit phosphorylated-p65 function. IMPLICATIONS: Our study shows that DMAPT, an oral NF-κB inhibitor in clinical development, inhibits phosphorylated-p65 upregulation of AR-V7 and delays prostate cancer castration resistance. This provides rationale for the development of DMAPT as a novel therapeutic strategy to increase durable response in patients receiving AR-targeted therapy.

DMAPT‑D6 induces death‑receptor‑mediated apoptosis to inhibit glioblastoma cell oncogenesis via induction of DNA damage through accumulation of intracellular ROS.

Glioblastoma (GBM) is an aggressive malignancy with a high rate of tumor recurrence after treatment with conventional therapies. Parthenolide (PTL), a sesquiterpene lactone extracted from the herb Tanacetum parthenium or feverfew, possesses anticancer properties against a wide variety of solid tumors. In the present study, a series of PTL derivatives were synthesized and screened. An inhibitor, dimethylaminoparthenolide (DMAPT)‑D6, a derivative of the PTL prodrug DMAPT in which the hydrogen of the dimethylamino group is substituted for the isotope deuterium, induced significant cytotoxicity in GBM cells invitro and induced cell cycle arrest at the S‑phase in a dose‑dependent manner. Furthermore, mechanistic investigation indicated that through increasing the levels of intracellular accumulation of reactive oxygen species (ROS), DMAPT‑D6 triggered DNA damage and finally death receptor‑mediated extrinsic apoptosis in GBM cells, suggesting that DNA damage induced by DMAPT‑D6 initiated caspase‑dependent apoptosis to remove damaged GBM cells. Taken together, these data suggested that ROS accumulation following treatment with DMAPT‑D6 results in DNA damage, and thus, death‑receptor‑mediated apoptosis, highlighting the potential of DMAPT‑D6 as a novel therapeutic agent for the treatment of GBM.

Role of Inhibition of DNA Double-Strand Break Repair and Autophagy in Pancreatic Cancer Cytotoxicity and Radiation-Induced Cell Killing

Low survival rates of pancreatic ductal adenocarcinoma (PDAC) are attributed to its detection at advanced stage and high chemotherapy and radiation resistance. Previous work in the lab has shown that resistance to radiation therapy can be decreased through simultaneous inhibition of the anti-apoptotic NF-kB cell survival signaling pathway by dimethyl-amino-parthenolide (DMAPT) and inhibition of Warburg cancer metabolism by dichloroacetate (DCA). We sought to determine the mechanism by which dual treatment with DMAPT and DCA increases cytotoxicity and radiation-induced cell killing in pancreatic cancer. Established pancreatic cancer cell lines, MIA-PaCa-2, Panc-1, and AsPc-1 cells were plated in T-25 flasks. For single fraction experiments, cells were treated with no drugs or both DMAPT and DCA at 24 and 48 hours after plating and irradiated with 0-6Gy with 160 kVp X-rays. To assess split dose repair, experimental groups were 5Gy, 2 x 2.5Gy, 5Gy with DMAPT and DCA, and 2x2.5Gy with DMAPT and DCA. Colonies were counted after 10 days of growth, and plating efficiency and survival fractions were calculated. Western blots of autophagy specific ATGs, LC-3a/b, and P62- SQSTM1 proteins were also determined. To assess radiation DNA damage response (DDR), cells were grown on chambered slides, treated with no drugs or dual DMAPT and DCA, irradiated at 2Gy, and fixed at 0hr, 1hr, and 24hrs after irradiation. After labeling with Gamma-H2Ax antibody, 53BP1 antibody, and DAPI, cells were imaged on a Leica Sp8 confocal microscope. Foci quantification was performed using ImageJ software. Forty-eight hours of DMAPT and DCA dual treatment significantly reduced plating efficiency of MIA-PaCa-2 cells from 0.406±0.092 (40.6%) to 0.253±.0.10 (25.3%), of Panc-1 cells from 0.528±0.11 (52.8%) to 0.0755±0.044 (7.55%), and of AsPc-1 cells from 0.294±0.16 (29.4%) to 0.0300±0.031 (3.0%). Cell death involves alterations of autophagy regulatory proteins. Treatment of cells with two 2.5Gy fractions given four hours apart with DMAPT and DCA dual treatment significantly reduced cell survival fraction compared to split-dose fractionated cells without drug treatment in MIA-PaCA-2 (0.224±0.098 to 0.0716±0.064), Panc-1(0.260±0.040 to 0.0130±0.012), and AsPc-1 cells (0.121±0.011 to 0.0370±0.026). Furthermore, for all three cell lines, persistence of gamma-H2Ax foci was seen at 24 hours post 2Gy irradiation in DMAPT and DCA dual treated cells compared to 2 Gy irradiated only cells, where foci levels returned to baseline levels. Dual DMAPT and DCA treatment of pancreatic cancer decreases plating efficiency, inhibits autophagy, inhibits split dose repair, and results in persistence of gamma-H2Ax foci at 24 hours post irradiation. We conclude that dual DMAPT and DCA treatment of pancreatic cancer increases radiation-induced cell killing through inhibition of DNA double-strand break repair.

Aging‐associated skeletal muscle defects in HER2/Neu transgenic mammary tumour model

AbstractBackgroundLoss of skeletal muscle volume and functional limitations are poor prognostic markers in breast cancer patients. Several molecular defects in skeletal muscle including reduced myoblast determination protein 1 (MyoD) levels and increased protein turn over due to enhanced proteosomal activity have been suggested as causes of skeletal muscle loss in cancer patients. However, it is unknown whether molecular defects in skeletal muscle are dependent on tumour aetiology.MethodsWe characterized functional and molecular defects of skeletal muscle in mouse mammary tumour virus (MMTV)‐Neu (Neu+) mice (n = 6–12), an animal model that represents HER2 + human breast cancer, and compared the results with well‐characterized luminal B breast cancer model MMTV‐PyMT (PyMT+). Functional studies such as grip strength, rotarod performance, and ex vivo muscle contraction were performed to measure the effects of cancer on skeletal muscle. Expression of muscle‐enriched genes and microRNAs as well as circulating cytokines/chemokines were measured. Because nuclear factor‐kappaB (NF‐κB) pathway plays a significant role in skeletal muscle defects, the ability of NF‐κB inhibitor dimethylaminoparthenolide (DMAPT) to reverse skeletal muscle defects was examined.ResultsNeu+ mice showed skeletal muscle defects similar to accelerated aging. Compared with age and sex‐matched wild type mice, Neu+ tumour‐bearing mice had lower grip strength (202 ± 6.9 vs. 179 ± 6.8 g grip force, P = 0.0069) and impaired rotarod performance (108 ± 12.1 vs. 30 ± 3.9 s, P &lt; 0.0001), which was consistent with reduced muscle contractibility (P &lt; 0.0001). Skeletal muscle of Neu+ mice (n = 6) contained lower levels of CD82 + (16.2 ± 2.9 vs. 9.0 ± 1.6) and CD54 + (3.8 ± 0.5 vs. 2.4 ± 0.4) muscle stem and progenitor cells (P &lt; 0.05), suggesting impaired capacity of muscle regeneration, which was accompanied by decreased MyoD, p53, and miR‐486 expression in muscles (P &lt; 0.05). Unlike PyMT+ mice, which showed skeletal muscle mitochondrial defects including reduced mitochondria levels and peroxisome proliferator‐activated receptor gamma co‐activator 1 (beta), Neu + mice displayed accelerated aging‐associated changes including muscle fibre shrinkage and increased extracellular matrix deposition. Circulating ‘aging factor’ and cachexia and fibromyalgia‐associated chemokine C‐C motif chemokine ligand 11 (Ccl11) was elevated in Neu+ mice (1439.56 ± 514 vs. 1950 ± 345 pg/mL, P &lt; 0.05). Treatment of Neu+ mice with DMAPT significantly restored grip strength (205 ± 6 g force), rotarod performance (74 ± 8.5 s), reversed molecular alterations associated with skeletal muscle aging, reduced circulating Ccl11 (1083.26 ± 478 pg/mL), and improved animal survival.ConclusionsThese results suggest that breast cancer subtype has a specific impact on the type of molecular and structural changes in skeletal muscle, which needs to be taken into consideration while designing therapies to reduce breast cancer‐induced skeletal muscle loss and functional limitations.

Abstract B038: Dimethylaminoparthenolide-mediated inhibition of NF-κB decreases resistance to ADT by targeting AR variants in lethal prostate cancer

Abstract Background: Inflammation, mediated by NF-κB, is linked to prostate cancer (PCa) progression and is commonly observed in castrate-resistant disease. We have previously shown that low expression of tristetraprolin (TTP), which modulates the NF-κB pathway and inflammation, is prognostic for lethal PCa after surgery. It has been suggested that NF-κB-driven resistance in PCa cells may be mediated by aberrant androgen receptor (AR) activation and AR splice variant production. Reversal of resistance to androgen deprivation therapy (ADT) may therefore be achieved by utilizing NF-κB inhibitors, such as dimethylaminoparthenolide (DMAPT). Materials and Methods: To examine clinical correlations between TTP/NF-κB and AR-V7, gene-expression profiling data and clinical data was obtained from several publicly available PCa cohorts (TCGA, SU2C, Taylor et. al.). Quantitative immunofluorescence for TTP and PTEN was performed on tissue microarrays from a clinical cohort of patients treated by radical prostatectomy with known time to biochemical recurrence (BCR). In vitro, mouse (MYC-CaP) and human (VCaP-CR, LNCaP-95) PCa cell lines were treated with TNF-α (20 ng/mL) to stimulate NF-κB expression, DMAPT (5-10 μM) to inhibit NF-κB and Enzalutamide (10 μM) in various combinations. Cell lines stably expressing the IκBα super repressor (MUT IκBα) were generated for comparison to DMAPT-mediated inhibition of NF-κB. Expression of AR and AR variants was measured by western blot and qPCR. For in vivo experiments, SCID mice bearing VCaP-CR xenograft tumors were treated with surgical castration, DMAPT (100 mg/kg, oral gavage, QD), surgical castration plus DMAPT or water vehicle control (n = 7-8 per group). Xenograft tumors were measured thrice weekly until they reached an ethical endpoint or after 80 days of treatment. Time-to-progression of tumors was assessed by Kaplan-Meier analysis and molecular differences within tumors analyzed by qPCR and immunohistochemistry (IHC). Results: In multiple clinical PCa cohorts, reduced TTP expression correlated with an increased AR-V7 signature. In a cohort of patients with localized PCa, decreased TTP expression reduced time to BCR and co-loss of TTP and PTEN (a known PCa tumor suppressor) further decreased time to BCR. Activation of NF-κB in PCa cell lines resulted in a significant increase in AR variant expression and minor upregulation of AR. Chemical (DMAPT) and genetic (MUT IκBα) inhibition of NF-κB slowed tumor proliferation and decreased AR variant expression. Treatment of PCa cell lines with Enzalutamide resulted in AR variant upregulation, which was blocked by DMAPT. Castration of mice bearing VCaP-CR tumors significantly increased AR and AR-V7 tumor expression. DMAPT treatment reduced NF-κB and AR-V7 expression in xenograft tumors, but did not significantly alter tumor growth compared to vehicle treatment. The combination of castration and DMAPT slowed median time-to-progression of xenograft tumors compared to vehicle-treated tumors (79 and 36 days respectively, p &amp;lt; 0.001). Combination therapy decreased Ki-67 (IHC), AR-V7 and AR (IHC and qPCR) expression in xenograft tumors. Conclusions: Current treatments for PCa mainly target AR; however, despite initial response, these treatments commonly fail. Our data highlight the relationship between NF-κB and AR variant expression in aggressive PCa and support the rationale for clinical use of DMAPT to treat castrate-resistant tumors in combination with AR antagonists. Citation Format: Katherine L Morel, Anis A Hamid, Leigh Ellis, Christopher J Sweeney. Dimethylaminoparthenolide-mediated inhibition of NF-κB decreases resistance to ADT by targeting AR variants in lethal prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B038. doi:10.1158/1535-7163.TARG-19-B038

Dimethylaminoparthenolide reduces the incidence of dysplasia and ameliorates a wasting syndrome in HPV16‐transgenic mice

The nuclear factor kappa light chain enhancer of activated B cells (NF-κB) has been implicated in the progression of cancers induced by high-risk human papillomaviruses (HPV). In cancer patients, NF-κB is also thought to drive a chronic systemic inflammatory status, leading to cachexia. This study addressed the ability of dimethylaminoparthenolide (DMAPT), a water-soluble NF-κB inhibitor, to block the development of HPV-induced lesions and wasting syndrome in HPV16-transgenic mice. Mice received DMAPT orally (100 mg/kg/day), once a day, for 6 consecutive weeks. Body weight was monitored weekly along with food and water intake. After 6 weeks the animals were submitted to a grip strength test and sacrificed for specimen collection. Skin samples were analyzed histologically and for expression of NF-κB-regulated genes Bcl2 and Bcl2l1. Gastrocnemius muscles were weighted and analyzed for expression of NF-κB subunits p50, p52, p65, and Rel-B. DMAPT reduced the incidence of epidermal dysplasia (18.2% versus 33.3% in HPV16+/- untreated mice). This was associated with reduced expression of Bcl2 and Bcl2l1 (p = .0003 and p = .0014, respectively) and reduced neutrophilic infiltration (p = .0339). Treated mice also showed partially preserved bodyweight and strength, which were independent of the expression levels of NF-κB subunits in skeletal muscle.These results suggest that NF-κB inhibition may be a valid strategy against HPV-induced lesions in vivo and warrant further preclinical tests particularly in the set of combination therapies. In addition, the data may support the use of DMAPT to prevent wasting syndrome.

DMAPT is an Effective Radioprotector from Long-Term Radiation-Induced Damage to Normal Mouse Tissues In Vivo.

While radiotherapy is widely used in cancer treatment, the benefits can be limited by radiation-induced damage to neighboring healthy tissues. We previously demonstrated in mice that the anti-inflammatory compound dimethylaminoparthenolide (DMAPT) selectively induces radiosensitivity in prostate tumor tissue from transgenic adenocarcinoma of mouse prostate (TRAMP) mice, while simultaneously protecting healthy tissues from 6 Gy whole-body radiation-induced apoptosis. Here, we examined the radioprotective effect of DMAPT on fibrosis in normal tissues after a partial-body fractionated radiation protocol that more closely mimics the image-guided fractionated radiotherapy protocols used clinically. Male C57BL/6J mice, 16 weeks old, received 20 Gy fractionated doses of X rays (2 Gy daily fractions, five days/week for two weeks) or sham irradiation to the lower abdomen, with or without a prior 20 mGy dose to mimic an image dose. In addition, mice received thrice weekly DMAPT (100 mg/kg by oral gavage) or vehicle control from 15 weeks of age until time of analysis at 6 weeks postirradiation. In the absence of exposure to radiation, there were no significant differences observed in the tissues of DMAPT and vehicle-treated mice (P > 0.05). DMAPT treatment significantly reduced radiation-induced testis weight loss by 60.9% (P < 0.0001), protected against a decrease in the seminiferous tubule diameter by 42.1% (P < 0.0001) and largely preserved testis morphology. Inclusion of the image dose had no significant effect on testis mass, seminiferous tubule diameter or testis morphology. DMAPT reduced radiation-induced fibrosis in the corpus cavernous region of the penis (98.1% reduction, P = 0.009) and in the muscle layer around the bladder (80.1% reduction, P = 0.0001). There was also a trend towards reduced collagen infiltration into the submucosal and muscle layers in the rectum. These results suggest that DMAPT could be useful in providing protection from the radiation-induced side effects of impotence and infertility, urinary incontinence and fecal urgency resulting from prostate cancer radiotherapy. DMAPT is a very well-tolerated drug and can conveniently be delivered orally without strict time windows relative to radiation exposure. Protection of normal tissues by DMAPT could potentially be useful in radiotherapy of other cancer types as well.

The NF-KB Inhibitor DMAPT in Combination with Ruxolitinib Displays Efficacy in Jak2V617F Knock-in Mouse Model of Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPN) are a group of hematologic malignancies characterized by excessive proliferation of myeloid/erythroid lineage cells. The JAK2V617F mutation has been found in a majority of patients with MPN including polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF). Ruxolitinib, a JAK1/JAK2 inhibitor, has been approved for treatment of MF and PV. Although ruxolitinib treatment provides some benefits, it does not produce disease remission or reverse bone marrow (BM) fibrosis in patients with MPN/MF. This underscores the need to identify new therapeutic targets and develop novel therapies for treatment of MPN.In order to identify new therapeutic targets in MPN, we performed microarray gene expression analysis on sorted hematopoietic stem cells (HSC) from wild type and JAK2V617F mice. We found significant enrichment of the NF-κB signaling pathway in JAK2V617F HSC compared with wild type HSC. We also observed increased phosphorylation/activation of NF-κB (p65 RelA) in the BM of JAK2V617F mice. Hyper activation of NF-κB also has been observed in patients with MPN. Lentiviral shRNA-mediated knockdown of RelA significantly reduced the proliferation of murine Ba/F3-EpoR cells expressing JAK2V617F. RelA knockdown also significantly inhibited human JAK2V617F-positive HEL, SET-2 and UKE-1 leukemia cells. Furthermore, forced expression of a dominant negative IκBα mutant markedly reduced proliferation in Ba/F3-EpoR-JAK2V617F cells. These data suggest a role for the NF-κB signaling pathway in MPN pathogenesis.In this study, we investigated the efficacy of the NF-κB inhibitor, dimethylaminoparthenolide (DMAPT), in hematopoietic cells expressing JAK2V617F and in JAK2V617F knock-in mouse model of MPN. DMAPT is a water soluble, orally bioavailable NF-κB inhibitor that has shown promise against several solid tumors in pre-clinical models. Treatment of DMAPT (1-2.5 uM) significantly reduced the proliferation of Ba/F3-EpoR-JAK2V617Fcells. DMAPT (2.5-7.5 uM) treatment also significantly inhibited the human JAK2V617F-positive HEL, SET-2 and UKE-1 leukemia cells. Furthermore, DMAPT treatment significantly inhibited the hematopoietic progenitor colony outgrowth in JAK2V617F mice BM and MPN patient peripheral blood CD34+ cells.We previously generated conditional Jak2V617F knock-in mice. Whereas expression of heterozygous Jak2V617F induces a PV-like disease, homozygous Jak2V617F expression promotes rapid progression to myelofibrosis. We have utilized the homozygous Jak2V617F mice to test the in vivo efficacy of DMAPT alone and in combination with ruxolitinib against MPN. We observed that combined treatment of DMAPT and ruxolitinib significantly reduced the WBC and neutrophil counts and spleen size in Jak2V617F mice compared with vehicle or single drug treatment. Combined treatment of DMAPT and ruxolitinib also significantly reduced hematopoietic stem/progenitors and myeloid precursors in the BM and spleens of Jak2V617F mice. Furthermore, combined treatment of DMAPT and ruxolitinib significantly decreased the Jak2V617F mutant-expressing myeloid precursors in the BM and spleens of transplanted mice. Histopathologic analysis revealed significant reduction of fibrosis in the bone marrow of Jak2V617F mice treated with the combination of DMAPT and ruxolitinib. Overall, our results suggest that simultaneous inhibition of JAK2 and NF-κB pathways might be useful for treatment of MPN/MF. DisclosuresCrooks:University of Arkansas for Medical Sciences: Patents & Royalties: Inventor on the DMAPT patents.

Actinomycin-D and dimethylamino-parthenolide synergism in treating human pancreatic cancer cells.

Preclinical Research & Development Pancreatic cancer is the third leading cause of death in the US with a poor 5-year survival rate of 8.5%. A novel anti-cancer drug, dimethylamino parthenolide (DMAPT), is the water-soluble analog of the natural sesquiterpene lactone, parthenolide. The putative modes of action of DMAPT are inhibition of the Nuclear chain factor kappa-light-chain enhancer of activated B cells (NFκB) pathway and depletion of glutathione levels; the latter causing cancer cells to be more susceptible to oxidative stress-induced cell death. Actinomycin-D (ActD) is a polypeptide antibiotic that binds to DNA, and inhibits RNA and protein synthesis by inhibiting RNA polymerase II. A phase 2 clinical trial indicated that ActD could be a potent drug against pancreatic cancer; however, it was not a favored drug due to toxicity issues. New drug entities and methods of drug delivery, used alone or in combination, are needed to treat pancreatic cancer more effectively. Thus, it was postulated that combining DMAPT and ActD would result in synergistic inhibition of Panc-1 pancreatic cancer cell growth because DMAPT's inhibition of NFκB would enhance induction of apoptosis by ActD, via phosphorylation of c-Jun, by minimizing NFκB inhibition of c-Jun phosphorylation. Combining these two drugs induced a higher level of cell death than each drug alone. A fixed drug ratio of DMAPT: ActD (1,200:1) was used. Data from metabolic (MTT) and colony formation assays were analyzed for synergism with CompuSyn software, which utilizes the Chou-Talalay equation. The analyses indicated synergism and moderate synergism at combination concentrations of DMAPT/ActD of 12/0.01 and 18/0.015 μM, respectively.

Parthenolide and DMAPT induce cell death in primitive CML cells through reactive oxygen species.

Tyrosine kinase inhibitors (TKI) have become a first‐line treatment for chronic myeloid leuakemia (CML). TKIs efficiently target bulk CML cells; however, they are unable to eliminate the leukaemic stem cell (LSC) population that causes resistance and relapse in CML patients. In this study, we assessed the effects of parthenolide (PTL) and dimethyl amino parthenolide (DMAPT), two potent inhibitors of LSCs in acute myeloid leukaemia (AML), on CML bulk and CML primitive (CD34+lin−) cells. We found that both agents induced cell death in CML, while having little effect on the equivalent normal hematopoietic cells. PTL and DMAPT caused an increase in reactive oxygen species (ROS) levels and inhibited NF‐κB activation. PTL and DMAPT inhibited cell proliferation and induced cell cycle arrest in G0 and G2 phases. Furthermore, we found cell cycle inhibition to correlate with down‐regulation of cyclin D1 and cyclin A. In summary, our study shows that PTL and DMAPT have a strong inhibitory effect on CML cells. Given that cell cycle arrest was not dependent on ROS induction, we speculate that this effect could be a direct consequence of NF‐κB inhibition and if this mechanism was to be evaded, PTL and DMAPT induced cell death would be potentiated.

Chronic low dose ethanol induces an aggressive metastatic phenotype in TRAMP mice, which is counteracted by parthenolide.

Despite advances in prostate cancer therapy, dissemination and growth of metastases results in shortened survival. Here we examined the potential anti-cancer effect of the NF-κB inhibitor parthenolide (PTL) and its water soluble analogue dimethylaminoparthenolide (DMAPT) on tumour progression and metastasis in the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model of prostate cancer. Six-week-old male TRAMP mice received PTL (40mg/kg in 10% ethanol/saline), DMAPT (100mg/kg in sterile water), or vehicle controls by oral gavage thrice weekly until palpable tumour formation. DMAPT treatment slowed normal tumour development in TRAMP mice, extending the time-to-palpable prostate tumour by 20%. PTL did not slow overall tumour development, while the ethanol/saline vehicle used to administer PTL unexpectedly induced an aggressive metastatic tumour phenotype. Chronic ethanol/saline vehicle upregulated expression of NF-κB, MMP2, integrin β1, collagen IV, and laminin, and induced vascular basement membrane degradation in primary prostate tumours, as well as increased metastatic spread to the lung and liver. All of these changes were largely prevented by co-administration with PTL. DMAPT (in water) reduced metastasis to below that of water-control. These data suggest that DMAPT has the potential to be used as a cancer preventive and anti-metastatic therapy for prostate cancer. Although low levels of ethanol consumption have not been shown to strongly correlate with prostate cancer epidemiology, these results would support a potential effect of chronic low dose ethanol on metastasis and the TRAMP model provides a useful system in which to further explore the mechanisms involved.

NF-\u03baB inhibition by dimethylaminoparthenolide radiosensitizes non-small-cell lung carcinoma by blocking DNA double-strand break repair

Despite optimal chemotherapy, radiotherapy (RT), and/or surgery, non-small-cell lung carcinoma (NSCLC) remains the leading cause of cancer-related death in the US and worldwide. Thoracic RT, a mainstay in the treatment of locally advanced NSCLC, is often restricted in efficacy by a therapeutic index limited by sensitivity of tissues surrounding the malignancy. Therefore, radiosensitizers that can improve the therapeutic index are a vital unmet need. Inhibition of the NF-κB pathway is a proposed mechanism of radiosensitization. Here we demonstrate that inhibition of the canonical NF-κB pathway by dimethylaminoparthenolide (DMAPT) radiosensitizes NSCLC by blocking DNA double-strand break (DSB) repair. NF-κB inhibition results in significant impairment of both homologous recombination (HR) and non-homologous end joining (NHEJ), as well as reductions in ionizing radiation (IR)-induced DNA repair biomarkers. NF-κB inhibition by DMAPT shows preclinical potential for further investigation as a NSCLC radiosensitizer.

Pharmacological Dual Inhibition of Tumor and Tumor-Induced Functional Limitations in a Transgenic Model of Breast Cancer.

Breast cancer progression is associated with systemic effects, including functional limitations and sarcopenia without the appearance of overt cachexia. Autocrine/paracrine actions of cytokines/chemokines produced by cancer cells mediate cancer progression and functional limitations. The cytokine-inducible transcription factor NF-κB could be central to this process, as it displays oncogenic functions and is integral to the Pax7:MyoD:Pgc-1β:miR-486 myogenesis axis. We tested this possibility using the MMTV-PyMT transgenic mammary tumor model and the NF-κB inhibitor dimethylaminoparthenolide (DMAPT). We observed deteriorating physical and functional conditions in PyMT+ mice with disease progression. Compared with wild-type mice, tumor-bearing PyMT+ mice showed decreased fat mass, impaired rotarod performance, and reduced grip strength as well as increased extracellular matrix (ECM) deposition in muscle. Contrary to acute cachexia models described in the literature, mammary tumor progression was associated with reduction in skeletal muscle stem/satellite-specific transcription factor Pax7. Additionally, we observed tumor-induced reduction in Pgc-1β in muscle, which controls mitochondrial biogenesis. DMAPT treatment starting at 6 to 8 weeks age prior to mammary tumor occurrence delayed mammary tumor onset and tumor growth rates without affecting metastasis. DMAPT overcame cancer-induced functional limitations and improved survival, which was accompanied with restoration of Pax7, Pgc-1β, and mitochondria levels and reduced ECM levels in skeletal muscles. In addition, DMAPT restored circulating levels of 6 out of 13 cancer-associated cytokines/chemokines changes to levels seen in healthy animals. These results reveal a pharmacological approach for overcoming cancer-induced functional limitations, and the above-noted cancer/drug-induced changes in muscle gene expression could be utilized as biomarkers of functional limitations. Mol Cancer Ther; 16(12); 2747-58. ©2017 AACR.