Duoli Sun

Combination treatment with MEK and AKT inhibitors is more effective than each drug alone in human non- small cell lung cancer in vitro and in vivo

AZD6244 and MK2206 are targeted small-molecule drugs that inhibit MEK and AKT respectively. The efficacy of this combination in lung cancer is unknown. Our previous work showed the importance of activated AKT in mediating resistance of non-small cell lung cancer (NSCLC) to AZD6244. Thus we hypothesized that dual inhibition of both downstream MEK and AKT pathways would induce synergistic antitumor activity. In this study, we evaluated the efficacy of AZD6244 and MK2206 individually on a large panel of lung cancer cell lines. Then, we treated 28 human lung cancer cell lines with a combination of AZD6244 and MK2206 at clinically applicable drug molar ratios. The AZD6244-MK2206 combination therapy resulted in a synergistic effect on inhibition of lung cancer cell growth compared to the results of single drug treatment alone. MK2206 enhanced AZD6244-induced Bim overexpression and apoptosis in A549 and H157 cells. When we tested the combination of AZD6244 and MK2206 at ratios of 8∶1, 4∶1, 2∶1, and 1∶8, we found that the synergistic effect of the combination therapy was ratio-dependent. At ratios of 8∶1, 4∶1, and 2∶1, the drug combination consistently demonstrated synergy, whereas decreasing the ratio to 1∶8 resulted in a loss of synergy and produced an additive or antagonistic effect in most cell lines. Furthermore, the AZD6244-MK2206 combination therapy showed synergy in the suppression of A549 and H157 xenograft tumor growth and increased mean animal survival time. The AZD6244-MK2206 combination therapy resulted in effective inhibition of both p-ERK and p-AKT expression in tumor tissue. In addition, a significant increase of apoptosis was detected in tumor tissue from mice treated with AZD6244-MK2206 compared with that from the single agent treated mice. Our study suggests that the combination of AZD6244 and MK2206 has a significant synergistic effect on tumor growth in vitro and in vivo and leads to increased survival rates in mice bearing highly aggressive human lung tumors.

Designing and Developing S100P Inhibitor 5-Methyl Cromolyn for Pancreatic Cancer Therapy

We have previously shown that the antiallergic drug cromolyn blocks S100P interaction with its receptor receptor for advanced glycation end product (RAGE) and improves gemcitabine effectiveness in pancreatic ductal adenocarcinoma (PDAC). However, the concentration required to achieve its effectiveness was high (100 μmol/L). In this study, we designed and synthesized analogs of cromolyn and analyzed their effectiveness compared with the parent molecule. An ELISA was used to confirm the binding of S100P with RAGE and to test the effectiveness of the different analogs. Analog 5-methyl cromolyn (C5OH) blocked S100P binding as well as the increases in NF-κB activity, cell growth, and apoptosis normally caused by S100P. In vivo C5OH systemic delivery reduced NF-κB activity to a greater extent than cromolyn and at 10 times lesser dose (50 mg vs. 5 mg). Treatment of mice-bearing syngeneic PDAC tumors showed that C5OH treatment reduced both tumor growth and metastasis. C5OH treatment of nude mice bearing orthotopic highly aggressive pancreatic Mpanc96 cells increased the overall animal survival. Therefore, the cromolyn analog, C5OH, was found to be more efficient and potent than cromolyn as a therapeutic for PDAC. Mol Cancer Ther; 12(5); 654–62. ©2013 AACR.

SF2312 is a natural phosphonate inhibitor of enolase

Despite being critical for energy generation in most forms of life, few if any microbial antibiotics specifically inhibit glycolysis. To develop a specific inhibitor of the glycolytic enzyme Enolase 2 for the treatment of cancers with deletion of Enolase 1, we modeled the synthetic tool compound inhibitor, Phosphonoacetohydroxamate (PhAH) into the active site of human ENO2. A ring-stabilized analogue of PhAH, with the hydroxamic nitrogen linked to the alpha-carbon by an ethylene bridge, was predicted to increase binding affinity by stabilizing the inhibitor in a bound conformation. Unexpectedly, a structure based search revealed that our hypothesized back-bone-stabilized PhAH bears strong similarity to SF2312, a phosphonate antibiotic of unknown mode of action produced by the actinomycete Micromonospora, which is active under anaerobic conditions. Here, we present multiple lines of evidence, including a novel X-ray structure, that SF2312 is a highly potent, low nM inhibitor of Enolase.

Binding partners for curcumin in human schwannoma cells: biologic implications.

Curcumin (diferuloylmethane) is a potent anti-inflammatory and anti-tumorigenic agent that has shown preclinical activity in diverse cancers. Curcumin up-regulates heat shock protein 70 (hsp70) mRNA in several different cancer cell lines. Hsp70 contributes to an escape from the apoptotic effects of curcumin by several different mechanisms including prevention of the release of apoptosis inducing factor from the mitochondria and inhibition of caspases 3 and 9. Previously we showed that the combination of curcumin plus a heat shock protein inhibitor was synergistic in its down-regulation of the proliferation of a human schwannoma cell line (HEI-193) harboring an NF2 mutation, possibly because curcumin up-regulated hsp70, which also binds merlin, the NF2 gene product. In order to determine if curcumin also interacts directly with hsp70 and to discover other binding partners of curcumin, we synthesized biotinylated curcumin (bio-curcumin) and treated HEI-193 schwannoma cells. Cell lysates were prepared and incubated with avidin-coated beads. Peptides pulled down from this reaction were sequenced and it was determined that biotinylated curcumin bound hsp70, hsp90, 3-phosphoglycerate dehydrogenase, and a β-actin variant. These binding partners may serve to further elucidate the underlying mechanisms of curcumins actions.

Imatinib analogs as potential agents for PET imaging of Bcr-Abl and c-KIT expression at a kinase level.

We synthesized two series of imatinib mesylate (STI-571) analogs to develop a Bcr-Abl and c-KIT receptor-specific labeling agent for positron emission tomography (PET) imaging to measure Bcr-Abl and c-KIT expression levels in a mouse model. The methods of molecular modeling, synthesis of STI-571 and its analogs, in vitro kinase assays, and radiolabeling are described. Molecular modeling revealed that these analogs bind the same Bcr-Abl and c-KIT binding sites as those bound by STI-571. The analogs potently inhibit the tyrosine kinase activity of Bcr-Abl and c-KIT, similarly to STI-571. [(18)F]-labeled STI-571 was prepared with high specific activity (75 GBq/μmol) by nucleophilic displacement and an average radiochemical yield of 12%. [(131)I]-labeled STI-571 was prepared with high purity (>95%) and an average radiochemical yield of 23%. The uptake rates of [(18)F]-STI-571 in K562 cells expressing Abl and in U87WT cells overexpressing c-KIT were significantly higher than those in the U87 cell and could be inhibited by STI-71 (confirming the specificity of uptake). PET scans of K562 and U87WT tumor-bearing mice with [(18)F]-STI-571 as a contrast agent showed visible tumor uptake and tumor-to-non-target contrast.

Degrasyn-like symmetrical compounds: possible therapeutic agents for multiple myeloma (MM-I).

A series of degrasyn-like symmetrical compounds have been designed, synthesized, and screened against B cell malignancy (multiple myeloma, mantle cell lymphoma) cell lines. The lead compounds T5165804 and CP2005 showed higher nanomolar potency against these tumor cells in comparison to degrasyn and inhibited Usp9x activity in vitro and in intact cells. These observations suggest that this new class of compounds holds promise as cancer therapeutic agents.

Improved synthesis of 17β-hydroxy-16α-iodo-wortmannin, 17β-hydroxy-16α-iodoPX866, and the [131I] analogue as useful PET tracers for PI3-kinase

An improved method for the synthesis of 17β-hydroxy-16α-iodo-wortmannin along with the first synthesis of 17β-hydroxy-16α-iodoPX866 and [(131)I] radiolabeled 17β-hydroxy-16α-[(131)I]iodo-wortmannin, as potential PET tracers for PI3K was also described. The differences between wortmannin and its iodo analogue were compared by covalently docking each structure to L833 in PI3K.

Abstract A39: Pomhex, a cell-permeable high potency enolase inhibitor with utility for collateral lethality treatment of cancer

Glycolysis inhibition is an active area of investigation for the treatment of cancer. However, few compounds have progressed beyond the cell culture stage. We have recently demonstrated that genomic passenger deletion of the glycolytic enzyme Enolase 1 (ENO1) leaves gliomas harboring such deletions solely reliant on ENO2, rendering them exquisitely sensitive to enolase inhibitors Collateral Lethality. However, the tool compound that we employed for these in vitro studies, Phosphonoacetohydroxamate (PhAH), has very poor pharmacological properties and was ineffective in vivo. We recently reported that a structural analogue of PhAH, the natural phosphonate antibiotic SF2312, is a high potency inhibitor of Enolase. While more potent than PhAH, SF2312 remains poorly cell permeable. Here, we generated a Pivaloyloxymethyl (POM) ester pro-drug derivative of SF2312, termed POMSF, which increased the potency in cell based systems by ~50-fold. POMSF is selectively active against ENO1-deleted glioma cells in culture at ~19 nM, versus μM for SF2312. However, POMSF displayed poor aqueous stability. A derivative of POMSF, termed POMHEX, showed greater stability and its active form, HEX, showed 4-fold preference for ENO1 over ENO2. Labeled 13C-glucose tracing shows that POMHEX inhibits glycolysis at the Enolase step in all cell lines tested, but with ~100-fold greater potency in ENO1-deleted lines. POMHEX selectively killed ENO1-deleted glioma cells with an IC50 Citation Format: Yu-Hsi Lin, Nikunj Satani, Naima Hammoudi, Federica Pisaneschi, Paul Leonard, David Maxwell, Zhenghong Peng, Todd Link, Lee IV R. Gilbert, Ananth Bosajou, Duoli Sun, Joe Marszalek, Yuting Sun, John S. McMurray, Pijus K. Mandal, Maria E. Di Francesco, Barbara Czako, Alan Wang, William Bornmann, Ronald A. DePinho, Florian Muller. Pomhex, a cell-permeable high potency enolase inhibitor with utility for collateral lethality treatment of cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A39.

Abstract 1727: Development of novel TAK1 inhibitors for pancreatic cancer

Transforming growth factor- (TGF-) activation kinase (TAK1, MAP3K7) is a member of MAPKKK family [1]. It was well established that TAK1 plays a key role in both TNFα and IL-1 pathway. Knock down of TAK1 impaired the activation of IKK and JNK by TNFα and IL-1 [2, 3]. NF-B activation induced by TNFα and IL-1 are severely impaired in TAK1 knock out MEF cells [4]. TAK1 acts as the kinase of IKK and therefore regulates NF-κB activation. Our previous work has demonstrated that TAK1 is upregulated and plays an important role in chemotherapeutic drug resistance in pancreatic cancer cells. Block TAK1 activity can reverse pancreatic cancer cell resistance to chemotherapeutic drug and reduce tumor size in Orthotopic Xenograft models [5]. These findings underscore the significance of TAK1 in mediating drug resistance. Strategies to target TAK1 may play a critical role in the treatment of this deadly malignancy. Utilizing computational-based structural modeling and a recently published crystal structure of TAK1, compounds were designed to inhibit the ATP-binding pocket of the TAK1. Synthetic templates were proposed for building a urea based library of potential inhibitors. A multiple stage docking involving both pre- and post-filtering and progressively more stringent docking criteria led to approximately 1,000 structures. The resulting scores from two docking programs were fed into workflow software that combined the results and calculated ADME properties. To provide better consistency in scoring and less bias in setting the filtering cutoffs, we also included docking decoys and utilized a more statistically relevant Z-score. After application of a final binding mode filter, there were 22 structures that were selected and ranked. A seven-step synthesis was developed and a focused library of compounds was synthesized from those top-ranked compounds. Note: This abstract was not presented at the meeting. Citation Format: Paul J. Chiao, Zhuonan Zhuang, Qianghua Xia, Paul T. Schuber, Duoli Sun, Zhenghong Peng, David S. Maxwell, William G. Bornmann. Development of novel TAK1 inhibitors for pancreatic cancer. [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 1727. doi:10.1158/1538-7445.AM2014-1727

Abstract 2698: Degrasyn-like symmetrical compounds: Possible therapeutic agents for multiple myeloma (MM-I)

A series of degrasyn-like symmetrical compounds have been designed, synthesized, and screened against B cell malignancy (multiple myeloma, mantle cell lymphoma) cell lines. The lead compounds T5165804 and CP2005 showed higher nanomolar potency against these tumor cells in comparison to degrasyn and inhibited Usp9x activity in vitro and in intact cells. These observations suggest that this new class of compounds holds promise as cancer therapeutic agents. Citation Format: William G. Bornmann, Zhenghong Peng, David Maxwell, Duoli Sun, Basvoju A. Bhanu, Moshe Talpaz, Nicholas Donato, Alexander Levitzki. Degrasyn-like symmetrical compounds: Possible therapeutic agents for multiple myeloma (MM-I). [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 2698. doi:10.1158/1538-7445.AM2014-2698