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Keyword: musc hollings cancer center

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.”

Summary: Boosting the immune system’s cancer-fighting ability is the aim of adoptive T cell immunotherapy, in which the patient’s T cells are harvested, expanded, and then reinfused. Ensuring T cell persistence after reinfusion has been a challenge. In the October 15, 2016 Cancer Research, investigators at the Medical University of South Carolina and Loyola University report that culturing T cells in N-acetyl cysteine (NAC) significantly improves persistence. Adding NAC to melanoma immunotherapy protocols could improve outcomes.

A collaborative team of investigators at the Medical University of South Carolina (MUSC) and Loyola University have demonstrated for the first time that culturing T cells in N-acetyl cysteine (NAC) before they are infused as immunotherapy improves effectiveness and outcomes in a preclinical model of melanoma. These findings were reported in the October 15, 2016 issue of Cancer Research.

Both incidence and mortality rates for metastatic melanoma continue to rise. Only about 15% of Stage IV melanoma patients receiving standard treatment can expect to survive for five years. By contrast, clinical trial data show that up to 40% of Stage IV melanoma patients survive for five years when treated with adoptive cell therapy (ACT), a form of immunotherapy that calls for infusion of autologous, melanoma-specific T cells.

ACT aims to boost a patient’s own immune responses against the cancer. To do this, the patient’s own T cells are harvested, genetically modified with a therapeutic T cell receptor, activated, and then rapidly expanded to generate large numbers of T cells for therapeutic re-infusion. Unfortunately, patient responses vary. Better outcomes are positively correlated with persistence of the transferred cells. The rapid expansion of harvested T cells before reinfusion increases their susceptibility to activation-induced cell death (AICD), prompting the authors to hypothesize that AICD reduces ACT’s overall effectiveness.

Researchers have long known that factors limiting T cell persistence also limit ACT efficacy but, until now, no one knew that something as simple as changing the culture condition by supplementing with NAC could improve survival of the reinfused T cells. The research team showed that adding NAC to the in vitro T cell expansion culture prevents increases in the DNA damage marker ?H2AX and significantly improves T cell persistence and immunotherapy outcomes, including reduced tumor growth and enhanced survival.  

The team found that nearly 40% of NAC-cultured T cells were detectable in tumors after transfer compared to approximately 1.2% of standard-culture T cells. They also found that mice receiving NAC-cultured cells experienced significantly delayed tumor growth compared to mice receiving standard-culture cells (P<.0001).

"We were really surprised by the number of adoptively transferred T cells we saw in the tumor,” said Christina Voelkel-Johnson, Ph.D., Associate Professor of Microbiology and Immunology at MUSC's Hollings Cancer Center and the senior author on the article. “Given the harsh environment T cells encounter within tumors, we did not expect that the number of NAC-cultured T cells would be 33-fold higher than T cells not cultured in NAC.”

Adding NAC to existing protocols should pose little risk to patients, according to Voelkel-Johnson, because NAC is already in clinical use for many indications and the culturing of T cells in NAC would occur outside of the patient. “The only difference would be a change to the cell culture protocol in an effort to generate cells with an improved phenotype," said Voelkel-Johnson.

The road to discovering the protective role of NAC began with the MUSC/Loyola research team hypothesizing that preventing T cells from becoming susceptible to AICD during in vitro expansion might improve their persistence and effectiveness upon reinfusion.

The team started by examining the role of p53, which is crucial for coordinating cellular stress responses and determining the fates of damaged cells (i.e., whether they will be repaired or allowed to die). While it is known that inhibiting p53 protects T cells from AICD, the molecular-level mechanisms that provide this protection were unknown. The investigators created conditions similar to those that occur during in vitro T cell expansion by re-stimulating the T cell receptors (TCR) to assess p53 status and cell death. They found that AICD following TCR re-stimulation is accompanied by phosphorylation of p53 on Ser15 and accumulation of p53 in the cell nucleus.

The next set of experiments clarified that the PI3K-like serine kinase, ataxia telangiectasia mutated (ATM), is necessary for the phosphorylation of p53 on Ser15 after TCR restimulation. They also found that inhibiting ATM almost completely prevents cell death (99%) after TCR restimulation. Thus, ATM appears to be a required upstream factor for AICD onset.

While activation of ATM and p53 are parts of a known DNA damage response pathway, ATM also responds to oxidative stress or hypoxia. So, the team needed to determine whether the changes in ATM and p53 were being caused by DNA damage or oxidative stress/hypoxia. They looked at two established DNA damage markers, gH2AX and p-SMC-1, and found that, within 15 minutes of TCR restimulation, both gH2AX and p-SMC-1 increased three-fold. This suggested that DNA damage and ATM activation occur in parallel, leading the authors to conclude that TCR restimulation causes DNA damage, which then triggers the ATM/p53 DNA damage response pathway.

Because it is known that AICD depends on the generation of reactive oxygen species (ROS), the investigators focused on ROS as a potential cause of DNA damage. They incubated some T cells with the antioxidant NAC prior to restimulation and compared them to T cells that were expanded using a standard protocol. Results showed that ROS levels were significantly lower in the NAC cultures than in the standard cultures. In addition, pretreatment with NAC reduced activation of the DNA damage markers, ?H2AX and p-ATM, by 94% and 69%, respectively. These results confirmed that ROS generated during TCR restimulation play a central role in damaging the DNA.

This is the first study to show that expanding therapeutic T cells in the presence of NAC prior to adoptive transfer improves their ability to resist AICD. Most importantly, using these "AICD-resistant" T cells improves therapeutic outcomes in a preclinical model by enhancing T cell persistence, increasing tumor control, and improving survival.

"Now we are looking at studies to help us understand exactly how NAC changes the phenotype of T cells,” said Voelkel-Johnson. “How does it make these cells survive? How is trafficking to tumors improved? There may be benefits to culturing T cells in NAC aside from generating AICD resistance that we haven't yet recognized.”

image of scannerMUSC Health has brought the latest in Positron Emission Tomography (PET)/CT scanner technology to the U.S. with the Department of Radiology’s new scanner that offers 128-slice CT.  The mCT 128 PET/CT system (Siemens Healthcare, Erlangen, Germany) enables nuclear medicine specialists to see anatomical images, such as lung nodules, and physiologic functions, such as coronary blood flow, in greater detail. This diagnostic advantage will be especially beneficial in three clinical areas, according to Leonie L. Gordon, M.B., ChB, Director of Nuclear Medicine. These specialists will use the scanner to interpret images in (1) cancer diagnosis (to locate tumors and metastases to other organs and bones) and treatment adjustment (with clearer pictures of the tumor’s borders, they can better advise surgeons planning to resect cancerous tissue or help medical oncologists change chemotherapy if it has been ineffective ); (2) heart disease diagnosis and treatment (to better assess cardiac muscle viability and the degree of blockage in coronary vessels, for example); and (3) neurological disease (to confirm the diagnosis of dementia, identify epilepsy seizure locations, and assess treatment effectiveness in certain  brain tumors). 

Furthermore, this scanner is more patient-friendly. Scanning time is reduced from an average of 45 minutes to 15 to 20 minutes, a feature especially advantageous for patients from MUSC Children’s Hospital. “Because the scanner is quicker, children may not need to be sedated,” says Gordon.

Combined PET/CT scanning has been commercially available since 2001. Its advantage is that it fuses the PET and CT information into one image and almost complete eliminates the false-positive and false-negative PET findings. The mCT 128 PET/CT system is different in that the CT provides 128 detector rows (slices) of anatomical images, as opposed to the 64 slices or fewer in older technology. This system has been available in Europe for 2 to 3 years. Siemens chose MUSC Health as the first hospital in the U.S. to receive the system because of its history with MUSC Health specialists’ clinical development and evaluation of Siemens equipment. This system is FDA-approved, but MUSC Health will be evaluating further the clinical utility of putting such a high-resolution CT scanner with a PET scanner. Gordon predicts the system will be of particular benefit to the patients at MUSC Hollings Cancer Center and MUSC Health Heart & Vascular Center.

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