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STAT

An MUSC blog
Keyword: cancer

"Juicing" Th17 cells with FDA-approved small molecule beta-catenin and p110 delta inhibitors during in vitro expansion for adoptive T cell therapy (ACT) profoundly improves their therapeutic properties, report investigators at the Medical University of South Carolina (MUSC) in an article published online ahead of print on April 20, 2017 by JCI Insight.

MUSC cancer immunologist Dr. Chrystal M. PaulosACT involves harvesting T cells, rapidly amplifying and/or modifying them in the laboratory to boost their cancer-fighting ability, and then reinfusing them back to the patient to boost anticancer immunity. One challenge for ACT has been that the rapid expansion of T cells in the laboratory can cause them to age and wear out, decreasing their longevity after reinfusion.

"Juicing" Th17 cells with the FDA-approved small molecules enhanced their potency, function and stem-like (less differentiated) quality, suggesting that they would persist better after reinfusion into patients, and also reduced regulatory T cells in the tumor microenvironment, which can blunt the immune response. These findings highlight novel investigative avenues for next-generation immunotherapies, including vaccines, checkpoint modulators, and ACT.

"This is exciting because we might be able to overcome some of the delays and disadvantages of rapid expansion in the laboratory," explains senior author Chrystal M. Paulos, Ph.D., associate professor of immunology and Endowed Peng Chair of Dermatology at MUSC and a member of the MUSC Hollings Cancer Center. "We might be able to use fewer cells (for ACT) because we can pharmaceutically 'juice' these T cells to make them more fit in the oppressive tumor microenvironment."

Building upon their previous findings that ICOS costimulation is critical for generating human Th17 cells and for enhancing their antitumor activity, an MUSC research team led by Paulos and including postdoctoral fellow Kinga Majchrzak report for the first time that repurposing FDA-approved small molecule drugs that inhibit two ICOS-induced pathways greatly enhances the antitumor potency of T cells.

Several biologic properties of the Wnt/ beta-catenin and P13K delta pathways led the team to suspect that they supported the antitumor activities of Th17 cells. For example, these pathways are active in both regulating T cell cytokine production during the immune response and in promoting self-renewal of hematopoietic stem cells (HSCs) and sustaining HSCs in an undifferentiated state. So, they designed a series of experiments to determine whether these two pathways were also active in enhancing Th17 antitumor memory and effectiveness.

To test this idea, they pharmaceutically inhibited PI3K delta and beta-catenin in Th17 cells (using idelalisib [CAL-101] to block the PI3K delta pathway and indomethacin [Indo] to inhibit beta-catenin)-anticipating that this would weaken Th17 cells' antitumor activity. To their surprise, the exact opposite occurred. ICOS-stimulated Th17 cells that were treated in vitro with CAL-101 plus Indo elicited a more potent antitumor response against melanoma in mice.

"My post-doc student came to me and said, 'I think I made a mistake because the data are going in the opposite direction to what we originally predicted!" says Paulos. "So, she repeated the experiment several times but we kept getting the same result. The data showed that using drugs to inhibit these pathways actually made the Th17 cells even better at killing tumors."

The team found that Th17 cells treated with CAL-101 express less FoxP3, suggesting that the drug suppresses Treg conversion while sustaining central memory-like Th17 cells. This finding is highly important because the phenotypic plasticity of Th17 cells in vivo allows their conversion to Tregs or Th1 cells with weak antitumor properties. These data suggest that treatment with CAL-101 can halt the development of these poorly therapeutic phenotypes and, thus, enhance the T cells' antitumor activity.

While the findings were initially counterintuitive and perplexing from a mechanistic perspective, in retrospect Paulos sees that they make sense. "Essentially, the T cells are younger," explains Paulos. "We know that T cells used for ACT age and wear out over time. Somehow these drugs sustain their youth and function. They're able to keep all the properties of their youth-they expand better and they're more functional and handle the oppressive tumor microenvironment better."

The discovery that existing FDA-approved drugs that block p110 delta and beta-catenin can make T cells more efficient tumor killers in vivo is an exciting prospect for Paulos' team. "From a clinical standpoint, this finding indicates that the therapeutic effectiveness of ACT could be improved by simple treatments with readily available drugs. It opens a lot of new investigative avenues for next-generation immunotherapy trials," she says.

"This research offers tremendous promise for the treatment of patients with serious forms of skin cancer," says Dirk M. Elston, M.D., chair of the Department of Dermatology and Dermatologic Surgery at MUSC.

Paulos has a patent on ICOS signaling in adoptive T cell transfer therapy (US 9133436), and Paulos, Majchrzak, and J.S. Bowers have a patent on pharmaceutical drug combinations or genetic strategies that instill durable antitumor T cell memory and activity (patent application P1685).

T cell attacking a tumor

T cell attacking a tumor

 

 

Stylized image of a T cell attacking a tumor. Illustration by Emma Vought.


 

 

 

 

 

 

 

 

 

 

 

 

Release Summary: The protein moesin could be a target for cancer immunotherapy, report Medical University of South Carolina (MUSC) investigators in an article in the Journal of Clinical Investigation. Their data suggest that moesin promotes conversion of naive T cells into regulatory T cells that suppress the immune response against cancer. Inhibiting moesin could help restore the anti-tumor T cell response and also improve the survival of cancer-killing CD8+ cells after adoptive T cell transfer.

In an article published online ahead of print on March 13, 2017 by the Journal of Clinical Investigation, Medical University of South Carolina (MUSC) investigators report preclinical research showing that moesin, a membrane-domain organizing protein, controls regulatory T cell (Treg) function as well as the abundance and stability of transforming growth factor-beta (TGF-beta) receptors on the surface of cells, providing a potential therapeutic target for cancer immunotherapy.

Their findings show that TGF-beta acts at the protein level to generate Tregs in the tumor microenvironment. Although the human immune system is capable of eradicating cancer, Tregs dampen the immune response and protect cancer cells against tumor-killing (i.e., cytotoxic) T cells. The MUSC study is the first to show that eliminating moesin reduces TGF-beta receptor expression and subsequent Treg generation to restore anti-tumor immunity.

T cells, a subtype of white blood cells, can effectively attack and kill tumor cells when activated by the protein TGF-beta. However, the immune system has a sophisticated network of checks and balances to ensure that the body does not produce so many of these cytotoxic T cells that it harms its own cells and tissues. When the immune reaction is complete, TGF-beta signals naive T cells to become Tregs that suppress and degrade the activated, inflammatory T cells, ensuring that they do not overproduce the immune factors that can lead to autoimmune disease.

Cancer cells have learned to hijack this system of checks and balances to hide from the tumor-killing T cells. Many cancers produce TGF-beta that binds the receptors on the tumor-killing helper T cells so they can’t be recruited to fight the tumor. The T cells convert instead to Tregs, which suppress the immune response against the cancer.

Inhibiting moesin could help prevent conversion of naive T cells into Tregs, thereby restoring the anti-tumor immune response. 

"Because moesin supports greater Treg production, we could design moesin inhibitors to halt or slow active TGF-beta signaling and slow down Treg conversion so that anti-tumor T cells can have a chance to see the cancer and eradicate it,” explains Zihai Li, M.D., Ph.D., chair of the Department of Microbiology and Immunology at MUSC and senior author on the paper.

Earlier studies by Philip Howe, Ph.D., chair of MUSC's Department of Biochemistry and Molecular Biology and a co-author on the paper, demonstrated that many TGF-beta-mediated epithelial mesenchymal transition genes, including moesin, were repressed by an RNA-binding protein in healthy epithelial cells and that moesin expression could be restored through TGF-beta stimulation.

This ability of TGF-beta to dramatically increase moesin expression led the team to investigate moesin’s role in Treg generation. Jointly with other colleagues at MUSC, the team compared the abilities of helper T cells with and without moesin to become Tregs. They found that moesin promotes Treg generation by interacting with a TGF-beta receptor to make it more available, thereby enhancing TGF-beta signaling. Conversely, TGF-beta signaling was reduced in the absence of moesin, impairing the development and function of Tregs.

Perhaps the most compelling results were provided by studies involving adoptive T cell therapy in a mouse model of melanoma. In adoptive T cell therapy, tumor-killing T cells are “harvested” from a human or animal with cancer and amplified or otherwise “supercharged” before being reinfused into the donor. Although these reinfused cells can be very effective at killing tumors, they do not always survive long-term, setting the stage for recurrence. 

The MUSC research team showed that these reinfused anti-cancer CD8+ T cells not only underwent rapid activation and expansion in mice lacking moesin, but that they also survived longer, reducing the likelihood of recurrence. Indeed, after adoptive T cell transfer, all of the mice having moesin relapsed while most of the mice lacking moesin were cured.

"When the mice lacking moesin had no recurrence, this was really exciting. We were not only deleting moesin but, when we gave T cells to the active tumors, those T cells could control the cancer for a very long time,” explains Ephraim Ansa-Addo, Ph.D., a postdoctoral fellow in the Department of Microbiology and Immunology and lead author on the paper.

These findings suggest that moesin could be a therapeutic target in developing new treatments for cancer and Treg-related immune disorders. Chemical modulators of moesin could control the function of T cells by inhibiting moesin in cancers or inducing it to treat autoimmune diseases. Moesin modulators could also be combined with current immunotherapy regimens.

“These findings are very interesting for the field and provide a lot of directions for further research into alternative therapies," says Li.

Screen Shot of New Medical Video Center

The MUSC Health Medical Video Center is now available online at MUSCHealth.org/medical-video. It profiles cutting-edge surgical procedures and innovative treatments available at MUSC Health and is intended for a health care audience. Its initial areas of focus are cardiology, oncology, neuroscience, and pediatrics. The site contains educational (and explicit) surgical video and photography.

Confocal-microscopy-showing-colocalization-of-mitochondria-and-autophagosomes

 

 

Left: Confocal microscopy detecting mitochondria (Tom20, red) colocalization with autophagosomes (LC3B, green), a process that happens during mitophagy in cancer cells treated with FLT3 inhibitor

 

 

 

Researchers at the Medical University of South Carolina Hollings Cancer Center have discovered a mechanism that confers resistance to drugs used to treat certain types of acute myeloid leukemia (AML). Targeting this pathway with a novel lipid-based therapeutic showed efficacy in a preclinical model of AML. These findings were reported in an article published online on August 18, 2016 by Blood.

“There are not many successful therapeutics at the moment for the treatment of patients with AML due to the problem of drug resistance,” said Besim Ogretmen, Ph.D., SmartStateTM Endowed Chair in Lipidomics and Drug Discovery at the Medical University of South Carolina (MUSC) Hollings Cancer Center and the senior author on the article.  

In the Blood article, Ogretmen and his colleagues, including clinicians at the MD Anderson Cancer Center who provided patient samples, report that ceramide-dependent mitophagy plays a key role in chemotherapeutic-mediated AML cell death.

“Ceramide, a pro-cell death lipid, kills cancer cells by causing them to eat their own mitochondria,” said Ogretmen. “This is called mitophagy.”

Patient cells with the FLT3 mutation inhibit ceramide synthesis and thereby become resistant to cell death. To combat this resistance, a number of FLT3 inhibitors have been developed and trialed in patients with AML.

“Unfortunately, regardless of the inhibitor, the problem of resistance to FLT3 targeted therapy has persisted,” said Mohammed Dany, MD/PhD student and the first author on the article.

By adding a synthetic ceramide analogue, LCL-461, the researchers were able to reactivate mitophagy and kill drug-resistant AML cells in a dish. Mice with drug-resistant human AML tumor xenografts—that is, mice into which drug-resistant tumor cells from AML patients had been grafted–were also treated with LCL-461. The treatment eliminated AML cells from the mice's bone marrow.

LCL-461 has clinical appeal because it is able to specifically target cancer cells. A positively charged molecule, LCL-461 is attracted to the mitochondria of cancer cells, which become negatively charged through the “Warburg effect.” This limits off-target effects that can occur with less specific inhibitors of FLT3 signaling. Previous studies in Ogretmen’s laboratory have tested the safety of LCL-461, finding that it had no major side effects at therapeutically active doses.

These results suggest the promise of LCL-461 as a potential therapeutic for patients with FLT3 mutated AML. LCL-461 was developed at MUSC in the Lipidomics Core. The MUSC Foundation for Research Development, MUSC’s technology transfer enterprise, has patented it and licensed it to Charleston-based startup SphingoGene, Inc.

Ogretmen and his colleagues are next seeking to perform large animal studies with LCL-461 to achieve Investigational New Drug (IND) approval, poising LCL-461 for translation.

“We are very excited about this. Head and neck cancers also respond to this drug very well,” said Ogretmen. “What we are trying to do is really cure cancer one disease at a time, and we are digging and digging to understand the mechanisms of how these cancer cells escape therapeutic interventions so that we can find mechanism-based therapeutics to have more tools for treatment.”

Immunofluorescence analysis to detect the expression and localization of Vps34 and Beclin-1 in cathepsin B overexpressing mouse mammary epithelial (CTSB/OE cells) treated -/+ TGF-beta for 7 days.

 

 

 

 

 

 

 

 

 

Summary: In an article published online in Nature Cell Biology on July 11, 2016, investigators at the Medical University of South Carolina report preclinical findings suggesting that disabled 2 (Dab2) serves as a molecular switch that regulates whether a tumor cell undergoes autophagy or apoptosis. Maintaining Dab2 by inhibiting cathepsin B could prevent tumor cell survival by blocking autophagy and promoting cell death. These insights provide important information for maximizing the efficacy of existing chemotherapeutic agents.

The results of preclinical studies by investigators at the Medical University of South Carolina (MUSC) reported in an article published online on July 11, 2016 in Nature Cell Biology (doi: 10.1038/ncb3388) demonstrate that disabled 2 (Dab2) serves as a molecular switch that regulates whether a tumor cell undergoes autophagy or apoptosis.

While expression of Dab2—an endocytic adaptor and tumor suppressor—is known to occur during transforming growth factor-beta (TGF-beta)–mediated epithelial-mesenchymal transition (EMT), the mechanisms by which it regulates apoptosis were, until now, poorly understood.

Exploring the pathways by which Dab2 is degraded and the effects of maintaining Dab2 levels reveals its pivotal role in preventing tumor cell survival by blocking autophagy and promoting cell death. These insights provide important information for maximizing the efficacy of existing chemotherapeutic agents.

TGF-beta induces EMT—a process by which cells transform from a polarized epithelial phenotype to a fibroblastic or mesenchymal one. Dab2 is expressed during TGF-beta–mediated EMT. While EMT is essential for normal cellular growth and homeostasis, it is abnormally activated in tumor cells and contributes to their chemo-resistance and metastasis.

TGF-beta has also been reported to regulate autophagy, which, in established tumors, ensures tumor cell survival through times of stress, as for example during chemotherapy. In other words, autophagy supports the chemo-resistance, growth, and metastasis of tumor cells.

Researchers focused on the Dab2 protein after noticing that, in cells treated with TGF-beta, Dab2 levels rose over the initial 24-48 hours as they went through EMT but then fell with continued TGF-beta treatment. By day 7, the tumor cells had transitioned to a morphological state suggestive of either autophagy or apoptosis. Furthermore, the mesenchymal markers N-cadherin and vimentin, which like Dab2 initially rose during EMT, began to decline with longer exposure to TGF-beta.

"This was an unexpected finding that we followed,” explains senior author Philip Howe, Ph.D., Professor and Chair of Biochemistry and Molecular Biology and Hans and Helen Koebig Chair in Clinical Oncology at MUSC. “We knew that if you give cells TGF-beta they go through EMT, and we knew you needed Dab2 for TGF-beta–mediated EMT. But, when we kept adding TGF-beta for more sustained periods (after EMT took place), cells took on a different morphology and we noticed a loss of Dab2. We investigated this loss of Dab2 and discovered that it was being cleaved and that the cells were undergoing autophagy. Upon sustained TGF-beta treatment, the cells had lost their mesenchymal phenotype they'd gained in EMT and entered into an autophagic state.”

The team began to explore how prolonged TGF-beta treatment led to loss of Dab2 and the mesenchymal phenotype.

First, they found that longer TGF-beta exposure significantly increased cathepsin B (CTSB) expression and promoted its co-localization with Dab2. The team then not only demonstrated that CTSB is responsible for cleaving Dab2 but also that it recognizes the cleavage site by the flanking amino acids Val499 and Gly500. Thus, while an unaltered Dab2 sequence (Leu-Val-Gly-Leu) was degraded by CTSB, it did not cleave a mutant Dab2 sequence (Leu-Val-Leu).

Second, findings showed that, after 7 days, continuous TGF-beta treatment induced autophagy and down-regulated markers of apoptosis. This was particularly notable because these conditions promote tumor cell chemo-resistance and metastasis.

Third, they found that CTSB inhibition or expression of a mutant Dab2 without the CTSB cleavage site (i.e., the Leu-Val-Leu mutant) led to time-dependent increases in pro-apoptotic markers. When TGF-beta was withdrawn, cells in which Dab2 had been preserved underwent cell death. This series of experiments show not only how Dab2 is modulated by CTSB but also that it serves as a switch for regulating TGF-b–induced autophagy and apoptosis.

Another series of experiments were undertaken to clarify exactly how Dab2 functions to prevent autophagy and promote apoptosis. These findings show that Dab2 inhibits TGF-beta–induced autophagy by blocking the Vps-Beclin-1 interaction and promotes apoptosis by attenuating ERK-Bim interactions.

Finally, the team used the chemotherapeutic agent doxorubicin (DOXO) to determine whether the role of Dab2 in inhibiting autophagy might affect tumor cell chemo-sensitivity. They found that cells in which CTSB was overexpressed had increased survival in the presence of DOXO. However, cells with high Dab2 levels due to CTSB inhibition or expression of the CTSB-resistant Dab2 mutant were more chemo-sensitive and underwent apoptotic changes. Thus, Dab2 was shown to promote chemotherapeutic drug–induced cell death by attenuating drug-induced autophagy. In vivo tumor studies in mice further found that Dab2 both enhanced DOXO-mediated cell death and attenuated tumor cell metastasis.   

These direct insights into molecular mechanisms supporting tumor cell survival and death are crucial for maximizing the effectiveness of existing chemotherapeutic agents. "This is important because there aren't a whole lot of drugs out there," explains Howe. "Most of what we use today has been around for 20 or 30 years because of a lack of investment in basic science." 

The team's next steps are to investigate in vivo models for combination therapies using DOXO and a CTSB inhibitor to further illuminate the potential for targeting Dab2 as a means of reducing tumor recurrence and metastasis.

Image Caption: In the absence of Disabled-2 (Dab2), Vps34/Beclin-1 Interactions are maintained. Immunofluorescence analysis to detect the expression and localization of Vps34 and Beclin-1 in cathepsin B overexpressing mouse mammary epithelial (CTSB/OE cells) treated -/+ TGF-beta for 7 days. Photos were taken by confocal microscope. Scale bars, 10 mm. The data show that in the absence of Dab2, due to CTSB overexpression, Vsp34/Beclin-1 interactions are maintained and autophagy is initiated. Adapted from a figure originally published in an article by Jiang Y, Woosley AN, Sivalingam N, Natarajan S, and Howe PH in Nature Cell Biology (doi: 10.1038/ncb3388).

 

SUMMARY: A genomics approach at the Medical University of South Carolina (MUSC) has unmasked genetic signatures in breast cancer cells that predict their sensitivity to certain drugs. The findings, published in the May 2, 2016 issue of Oncotarget, provide proof of concept for personalized pharmaceutical therapies that target the genes responsible for driving tumor growth.

Drug treatments for breast cancer patients might soon be designed based on the unique genetic autograph of their tumor.

A genomics approach at the Medical University of South Carolina (MUSC) has unmasked genetic signatures in breast cancer cells that predict their sensitivity to certain drugs. The findings, published in the May 2, 2016 issue of Oncotarget, provide proof of concept for personalized pharmaceutical therapies that target the genes responsible for driving tumor growth.

Dr. Stephen EthierCertain oncogenes drive solid tumor growth in some breast cancer patients but are just passenger genes in others—expressed but not essential for growth. As a result, tumors in different breast cancer patients may respond differently to the same treatment depending on which oncogenes are active and which are just along for the ride. Identifying the panel of active genes in a patient’s tumor—called the functional oncogene signature—could help an oncologist select therapies that target its growth, according to Stephen P. Ethier, Ph.D., Interim Director of the Center for Genomic Medicine at MUSC and senior author on the study.  

“In order to move the field forward, we now need to be able to use oncogene signatures to tailor therapies using combinations of targeted drugs so that multiple driving oncogenes can be targeted at once,” said Ethier.  “Doing this successfully requires the identification of the oncogenes to which the cancer cells are addicted, as this allows the use of targeted drugs at very low doses. Low doses are essential if we are to use combinations of different targeted drugs.”

Ethier’s group identified unique functional oncogene signatures in four different human breast cancer cell types. Using next-generation genome sequencing and genome silencing as each cancer cell type grew and multiplied, they assembled a list of genes for each cell type’s functional oncogene signature—those genes that were copy number amplified or point mutated, and most essential to cancer cell survival. Although thousands of candidate oncogenes were screened during experimentation, only a handful made the list—fewer than 20 for each cell type.

The brevity of each list facilitated selection of the best oncogene for pharmaceutical targeting. Because lower doses of targeted drugs can be highly effective, side effects could be reduced. For example, Ethier’s group found that targeting two or more members of a signature with much lower total drug concentrations in combination still killed cancer cells better than one higher-concentration drug alone.

Remarkably, a BCL2L1-targeted drug  that worked in one cell line also then worked in a fifth breast cancer cell line with a similar oncogene signature containing BCL2L1, an oncogene not normally associated with breast cancer. This work demonstrates that one signature-targeting treatment can be extended to more than one cancer cell type. This means that patients with other types of cancer who have a similar functional oncogene signature might benefit from drugs that target BCL2L1, which are already in development.

Ethier thinks that oncogenes identified in a tumor biopsy might one day soon provide a rational and individualized approach to pharmaceutical treatment with targeted drug combinations. Meanwhile, these findings from his laboratory—showing the importance of considering a patient’s functional oncogene signature before testing a new drug— could provide a rationale for redesigning clinical trials for breast cancer.

Stephen T. Guest, Ph.D., of the MUSC Department of Pathology & Laboratory Medicine, was first author on the study.

Reb-1Researchers at the Medical University of South Carolina and elsewhere resolve the first protein structure in a family of proteins called transcription terminators that could provide insight into aging and cancer. The work reveals the protein Reb1 to be a traffic signal for coordinating transcription and gene replication, rather than a passive roadblock as previously thought.

Image Caption: Space-filling model of Reb1 bound to DNA. Reused with permission from PNAS.

 

In a study published on 28 March 2016 in the Proceedings of the National Academy of Sciences, researchers at the Medical University of South Carolina (MUSC) and Virginia Commonwealth University have resolved the first protein structure in a family of proteins called transcription terminators. The crystal structure of the protein, called Reb1, provides insight into aging and cancer, according to Deepak Bastia, Ph.D., Endowed Chair for Biomedical Research in the MUSC Department of Biochemistry and Molecular Biology and co-senior author of the study.

During transcription, large molecular machines read genes by traveling along double-stranded DNA. This machinery simultaneously reads out the gene code in continually lengthening chains of single-stranded RNA. The RNA code is then used to assemble proteins that cells use for growth and division. At certain times during the life of a cell, transcription must be stopped–in order to conserve cellular energy or prevent uncontrolled growth, for example. At other times, cells may be preparing to divide, during which period trouble can arise.

Before a cell can divide, the DNA must be exactly replicated for use in the new cell. During part of this process, two types of machinery are now moving along the DNA strand–transcriptional machinery and replication machinery. In regions where the two machines are moving in opposite directions, collisions can occur and DNA broken, causing mutations. Harmful gene mutations can be passed into the new cell. That’s where Reb1 comes in.

One way to prevent genome instability is to prevent replication from colliding with transcription,” says Bastia. That’s what these terminator proteins do.”

Bastia’s group knew that there are specific sites on the DNA strand called terminator regions to which Reb1 binds itself. Reb1 was thought of as a simple physical barrier that sits on DNA and blocks both the transcriptional and replication machinery from moving further along the DNA strand and colliding with each other. Then Bastia’s group did an experiment to cut the transcription terminator region (tail) off of Reb1. Intriguingly, Reb1 was no longer able to halt the transcription machinery without its tail but was still able to bind to DNA. Therefore, the simple roadblock theory couldn’t be correct.

The insight came when they solved the crystal structure–a laborious process during which Carlos R. Escalante, Ph.D., Bastia’s co-senior author from Virginia Commonwealth University, made monthly drives transporting freshly made crystals from MUSC to the X-ray crystallography facility at Brookhaven National Laboratory in New York. The crystal structure showed that, when bound to DNA, the transcription terminator tail of Reb1 can interact with a specific part of the transcriptional machinery, acting as a tether between the two.

The work illuminates Reb1 as a traffic signal for coordinating transcription and gene replication, rather than as a simple roadblock as previously thought.

Though the tether between Reb1 and the transcriptional machine is clear, the team is still not sure exactly how terminator proteins stop transcription, a question which drives their current work. And the connection between terminator proteins and colorectal cancer has been made, but work in other cancers and in aging has yet to be undertaken.

Still, Bastia suspects that this coordination prevents the type of gene errors that lead to many types of cellular aging and tumor growth, both of which are processes that result from uncontrolled transcription and replication. The group is currently researching another type of terminator protein, work which Bastia hopes will lend further knowledge to the diseases of aging.

 

Accumulation of DihydroceramidesSummary: Sphingosine kinase inhibitors are a new category of drugs that act on specific enzymes involved in sphingolipid metabolism to reduce the formation of a pro-cancer, pro-inflammatory lipid signaling molecule known as sphingosine-1 phosphate (S1P). Preclinical studies led by immunologist Christina Voelkel-Johnson, Ph.D., of the Medical University of South Carolina showed that a first-in-class sphingosine kinase 2 inhibitor slowed growth of aggressive prostate cancer cells.

A first-in-class sphingosine kinase 2 inhibitor slowed the growth of castration-resistant prostate cancer cells, in part by inhibiting the enzyme dihydroceramide desaturase (DEGS), but did not kill them, according to the results of preclinical in vitro and in vivo studies published in the December 2015 issue of Molecular Cancer Therapeutics by researchers at the Medical University of South Carolina (MUSC) and others.

Christina Voelkel-Johnson, Ph.D., Associate Professor of Microbiology and Immunology at MUSC, led the study, which was funded by a pilot grant from MUSC Hollings Cancer Center. Co-authors include Charles D. Smith, Ph.D., who developed the compound and led an earlier phase 1 trial at MUSC Hollings Cancer Center; oncologist Michael Lilly, M.D., a prostate cancer specialist; and Richard Drake, Ph.D., director of the Proteomics Core at MUSC, who has developed techniques to use MALDI imaging mass spectrometry to measure sphingolipid levels.

Sphingosine kinase inhibitors are a new category of drugs that reduce the generation of sphingosine-1-phosphate. This lipid signaling molecule promotes cancer cell growth and survival, thereby supporting the development of resistance to chemotherapy and radiation by cancer cells.

The study reported in Molecular Cancer Therapeutics showed that the compound YELIVA™ (ABC294640; RedHill Biopharma Ltd.; Tel Aviv, Israel) slowed prostate cancer cell proliferation by inhibiting sphingosine kinase 2, but also that it did something unexpected. “By inhibiting a second sphingolipid enzyme (DEGS), the compound increases levels of another class of lipids - dihydroceramides - which may contribute to the growth suppressive effects of the drug,” says Voelkel-Johnson. This study is the first to show activity for this compound against DEGS and to potentially link inhibition of DEGS to slowing the growth of castration-resistant prostate cancer cells. Treatment with YELIVA™ (ABC294640) increased dihydroceramide levels even in the absence of sphingosine kinase 2. 

The MUSC team conducted both in vitro and in vivo studies with YELIVA™ (ABC294640) in castration-resistant prostate cancer, relying on the MUSC Lipidomics Shared Resource for measurement of sphingolipid levels and the MUSC Proteomics Center for MALDI imaging mass spectrometry.

 In vitro studies conducted with castration-resistant mouse prostate cancer cells (TRAMP-C2) showed that treatment with YELIVA™ (ABC294640) reduced expression of the androgen receptor and the oncogene c-Myc, both important therapeutic targets for prostate cancer. Although many existing prostate cancer therapies target the androgen receptor, none directly target c-Myc.

To test in vivo response, one million TRAMP-C2 cells were injected under the skin of mice with an intact immune system, which were then treated with YELIVA™ (ABC294640) three days later. MALDI imaging mass spectrometry showed the presence of YELIVA™ (ABC294640) within murine tumors and confirmed in vitro findings of increased dihydroceramide levels.

“The significance of these findings is that this compound might be a novel therapeutic for advanced prostate cancer,” says Voelkel-Johnson, who believes that combination regimens of YELIVA™ (ABC294640) and focal radiation in this difficult-to-treat patient population deserve further study.

See full EurekAlert! release at http://www.eurekalert.org/pub_releases/2016-01/muos-anc012816.php

Image Caption:

The signal for ABC294640 is detected only when the drug but not the vehicle was administered (upper panel). The intensity for two different dihydroceramides is shown in the middle panel (dhC16-cer) and lower panel (dhC18-cer). A color bar indicates the signal intensity. Adapted with permission from the American Association for Cancer Research : Venant H, et al. The Sphingosine kinase 2 inhibitor ABC294640 reduces the growth of prostate cancer cells and results in accumulation of dihydroceramides In vitro and In vivo. Molecular Cancer Therapeutics; 2015 Dec; 14(12):2744-52. doi: 10.1158/1535-7163.MCT-15-0279.

It is now accepted that our immune system is capable of mounting an attack against cancer. However, tumors have devised ways to elude detection and to render tumor reactive or “effector” T cells indolent. Cancer immunotherapies such as adoptive T cell transfer (ACT) seek to reinvigorate and reinforce tumor-reactive cells so that they can effectively target tumor.

In ACT therapy, exhausted T cells in the vicinity of a tumor are harvested; expanded, conditioned, and sometimes genetically reengineered to better recognize and target the tumor; and then reinfused. T cell growth factors, such as interleukin (IL) 2 and IL-15, are often administered to promote proliferation of the reinfused T cells that have been trained to target the patient’s tumor. IL-15 is a more recently discovered cytokine and seems to promise some advantages over IL-2, which is toxic at therapeutic doses and which can stimulate regulatory T cells that blunt the effect of effector T cells.  One limitation of translating this therapy to the clinic is that preconditioning with chemotherapy or radiation is required for best results. Chemotherapy and radiation are expensive, associated with substantial adverse effects, and require hospital admission.T cells expressing IL-2 receptor alpha outcompete host cells for IL-2

In an article published in the October 28, 2015 issue of Science Translational Medicine, senior author Mark P. Rubinstein, Ph.D., and his colleagues in the Department of Surgery and the Department of Microbiology and Immunology at the Medical University of South Carolina and his collaborators at the University of California San Diego report the surprising finding that curative responses were achieved with ACT in a mouse model of melanoma without lymphodepletion when IL-2 but not IL-15 was co-administered. These findings are important because they suggest that low-dose IL-2 could be used as an alternative to chemotherapy and radiation as a preconditioning regimen for ACT therapy.

Host cells are thought to outcompete the reinfused or “donor” cells for T cell growth factors such as IL-2 and IL-15. Chemotherapy and radiation knock down the number of host cells so that the donor cells can more effectively compete for IL-2 or IL-15.  Rubinstein and his colleagues found that curative responses were achieved with IL-2 without lymphodepletion when the effector T cells were engineered to express elevated levels of IL-2 receptor alpha (IL-2R?). The presence of IL-2R? on the surface of the effector T cells enabled them to outcompete host cells for IL-2. This suggests that, if the effector T cells harvested from patients are engineered to express high levels of IL-2R?, then low doses of IL-2 may be adequate to achieve significant clinical response, making preconditioning with chemotherapy or radiation unnecessary.

The study authors also describe a novel mechanism that helps account for the ability of IL-2 to mediate curative responses in the absence of chemotherapy or radiation. They found that, in T cells expressing IL-2R?, IL-2 does not degrade as expected after being taken up by the effector T cell. Instead, IL-2R? rescues IL-2 from being degraded, thereby enabling IL-2 to be recycled so it can continue to optimize effector T cell response. “The ability of IL-2R? to sustain IL-2 signaling provides a molecular mechanism to explain how IL-2 therapy may be particularly useful clinically,” says Rubinstein. “This mechanism could provide a novel way to enhance the tumor-killing potential of T cells transferred to patients in the absence of prior chemotherapy or radiation.”

 The lead researchers in the Rubinstein laboratory for this study were Ee Wern Su, Ph.D.,and Caitiln Moore. Ms. Moore is currently in medical school at MUSC. For a complete list of authors, please view the article's abstract.

photo of a box of fruits and vegetablesA recent study led by MUSC professor David P. Turner, Ph.D. finds that lifestyle habits such as diet and exercise could affect the progression of cancer and the rate of survival, but so could race. According to the study published in Cancer Research in May, our bodies have to metabolize food to obtain the sugars we need, thus leaving behind a reactive-metabolite waste product. These leftovers are referred to as advanced glycation end-products (AGE), and this study addresses the apparent correlation between AGE levels and the prevalence of age-related diseases among non-Hispanic whites and African Americans. 

High levels of AGE are associated with diabetes, cardiovascular disease, Alzheimer’s, and cancer. These levels are highest in African American men with prostate cancer—they are 1.5 times more likely to be diagnosed with this cancer and twice as likely to die from it than non-Hispanic whites. Consumption of sugar and processed food can contribute to AGE levels. Food preparation (i.e., browning) also plays a large role in these levels. They are higher in the West, where the diet commonly consists of red meat, refined grains, and high sugar and fatty foods.

 When analyzing serum from cancer patients, Turner found that AGE levels were significantly higher in patients with cancer than those without. Breast and prostate immortalized cancer cell lines grew more, migrated farther, and invaded more when treated with AGE. In conjunction with higher AGE levels, African Americans have more C-reactive protein (CRP), making them more susceptible to chronic inflammation. Chronic inflammation is one of the key factors implicated in the development of cancer, along with oxidative stress, an increased immune response, and the presence of AGE.

AGE cannot be completely eliminated, but levels of circulating AGE can be lowered. Simply changing lifestyle habits can slow down the accumulation of AGE in the body. Avoid food with high protein, sugar, and fat, as well as processed foods. Then increase your intake of natural grains, fruits, and vegetables. Change the way you prepare your food by cooking meats at a lower temperature for a longer period of time, skipping the browning step of a dish. You can also replace high-sugar, oil-based marinades with lemon juice, vinegar, and tomato juice. The last big step of lowering your AGE levels is exercise. A sedentary lifestyle only allows for more AGE to accumulate.

Dr. Robert StuartDr. Azizul Haque

The body’s own immune system could be a potent weapon in the war on cancer if the cloaking mechanisms tumor cells use to elude it could be deactivated. In an article published in the February 15 issue of the Journal of Immunology, one of those cloaking mechanisms was identified in B cell tumors by a team of MUSC immunologists led by Azizul Haque, PhD (above right), MUSC Health hematologist/oncologist Robert Stuart, M.D. (above left), and their colleagues at the University of Indiana and German Research Center for Environmental Health. They reported that overexpression of the c-MYC protein, one of the most commonly activated genes in human cancers that is implicated in the cancer-related deaths of about 100,000 people worldwide, is linked to the ability of B cell tumors to “hide” from the immune system.1 Specifically, they showed for the first time that overexpression of the c-MYC protein in Burkitt’s lymphoma interferes with human leucocyte antigen (HLA) class II antigen presentation. T cells can mount an immune response against antigens only if they can “see” them; they “see” them when TCRs (T cell receptors) on their surface recognize antigen fragments bound to HLA class II molecules on the surface of antigen-presenting cells. When tumor antigen is not presented properly due to c-MYC overexpression, it remains invisible to the T cells. The article also provided evidence that treatment of c-MYC-overexpressing cells with a c-MYC inhibitor decreased c-MYC expression and partially restored HLA class II-mediated antigen presentation. These results suggest that c-MYC inhibitors could help “unmask” B cell lymphomas and promote a more robust immune response. According to Haque, “This study uncovers a mechanism by which c-MYC impairs immune detection of malignant tumors, which could be targeted in future treatments for B cell lymphomas and other malignancies.” The article by Haque and colleagues was highlighted in the “In This Issue” section of the Journal of Immunology, reserved for the top 10% of articles published in the journal.

References

1 God JM, Cameron, C, Figueroa J, Amria S, Hossain A, Kempkes B, Bornkamm GW, Stuart RK, Blum JS, Haque A. Elevation of c-MYC Disrupts HLA Class II–Mediated Immune Recognition of Human B Cell Tumors. The Journal of Immunology 2015;194:1434–1445.

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