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Friday, September 23, 2016

​​Researchers looking for a missing cause of a common type of mutation in breast cancer cells have uncovered the biochemical culprit and found that it may also be a general source of mutation in other cancer types.

Reporting in the scientific journal Nature Communications (DOI: 10.1038/ncomms12918) University of Minnesota researcher Reuben Harris and colleagues found that an enzyme known as APOBEC3H-I is the most likely cause of these previously unexplained mutations. Harris is a professor in the University's College of Biological Sciences, a Howard Hughes Medical Institute Investigator, and a member of the Masonic Cancer Center.

In breast cancer the solution became apparent in tumors lacking a related enzyme called APOBEC3B. All breast tumors with an APOBEC mutation footprint have APOBEC3B and, if they lack this enzyme due to a naturally occurring deletion, they invariably have APOBEC3H-I. The mutational contribution of APOBEC3H-I was also clear in lung cancer, in addition to expected mutational footprints from tobacco smoke and aging.

These findings are important because they provide a molecular explanation for a major source of mutation in many different types of cancer. The results point the way to fine-tuning the treatments of these cancers by inhibiting these enzymes in order to limit the development of mutations that undermine many current cancer therapies.

"Our results encourage the development of new cancer treatments that work by combining existing therapies and an APOBEC inhibitor to stop tumor cells from evading therapy by developing resistance mutations," Harris said.

The findings are based on the fact that cancer begins when the genetic material inside a cell mutates, causing the cell to change and indiscriminately multiply. The processes that cause these mutations leave a characteristic signature or footprint. The second-most common mutation type (behind aging) is that caused by the APOBEC enzymes. These enzymes are normally beneficial by helping to kill viruses, but the same DNA-mutating trait that makes them good at this job can also give them the ability to contribute mutations to tumor evolution if they become too abundant and/or misregulated.

Past research led by Harris showed that APOBEC3B is a source of mutations in breast cancers with APOBEC footprint. However, Harris and others also found that some breast tumors still have an APOBEC footprint but no APOBEC3B. To find out why, Harris, the lead author Gabriel Starrett, and collaborators from the University of Minnesota Masonic Cancer Center, the Howard Hughes Medical Institute, and the University of Saskatchewan began looking at variants of another APOBEC family member known as APOBEC3H. They were surprised to find that what was thought to be a relatively unstable, inactive variant known as APOBEC3H-I is likely the culprit. This protein is found in tumor cells with APOBEC signatures, and it is also much more active than expected from prior studies. APOBEC3H-I can access the nuclear DNA of the cell, providing a molecular explanation for its role in cancer mutagenesis.

Armed with the knowledge of APOBEC3H-I's role in cancer, Harris is now looking to learn more about how this enzyme actually induces mutations. "We would like to better understand the underlying molecular mechanism," he said. "As with any new process, a better understanding of the molecular details will provide additional angles for future therapies."

Thursday, September 22, 2016

​​A multi-institutional academic and industry research team led by investigators from Massachusetts General Hospital (MGH) and the Harvard Stem Cell Institute has identified a promising new approach to the treatment of acute myeloid leukemia (AML).

In their report published online in Cell, the investigators identify a crucial dysfunction in blood cell development that underlies AML and show that inhibiting the action of a specific enzyme prompts the differentiation of leukemic cells, reducing their number and decreasing their ability to propagate the cancer.

"AML is a devastating form of cancer; the 5-year survival rate is only 30 percent, and it is even worse for the older patients who have a higher risk of developing the disease," said David Scadden, MD, Director of the MGH Center for Regenerative Medicine (MGH-CRM), co-director of the Harvard Stem Cell Institute (HSCI), and senior author of the Cell paper. "New therapies for AML are extremely limited -- we are still using the protocols developed back in the 1970s -- so we desperately need to find new treatments."

In AML, the normal process by which myeloid stem cells differentiate into a specific group of mature white blood cells is halted, leading to the proliferation of immature, abnormal cells that crowd out and suppress the development of normal blood cells. A wide range of genetic changes occurs in AML, but the authors proposed that the effects on differentiation had to funnel through a few shared molecular events. Using a method created by lead author David Sykes, MD, PhD, of the MGH-CRM and HSCI, the team discovered that a single dysfunctional point in the pathway common to most forms of AML could be a treatment target.

Previous studies had shown that the expression of a transcription factor called HoxA9 -- which must be shut down for normal myeloid cell differentiation to proceed -- is actually maintained in 70 percent of patients with AML. Since no inhibitors of HoxA9 had been identified, the researchers pursued a novel approach to screening potential inhibitors based not on their interaction with a particular molecular target but on whether they could overcome the differentiation blockade characteristic of AML cells.

They first set up a cellular model of AML by inducing HoxA9 overexpression in mouse myeloid cells genetically engineered to glow green if they reached maturity. The team then screened more than 330,000 small molecules to find which would produce the green signal in the cells, indicating that the HoxA9-induced differentiation blockade had been overcome. Only 12 compounds produced the desired result, 11 of which were found to act by suppressing a metabolic enzyme called DHODH, which was not previously known to have a role in myeloid differentiation. Further experiments showed that DHODH inhibition could induce differentiation in both mouse and human AML cells.

The team then tested a known DHODH inhibitor in several mouse models of AML and identified a dosing schedule that reduced levels of leukemic cells and prolonged survival with none of the adverse effects of normal chemotherapy. While 6 weeks of treatment did not prevent eventual relapse, treatment for up to 10 weeks appears to have led to long-term remission, including a reduction of the leukemia stem cells that can lead to relapse. Similar results were seen in mice into which human leukemia cells had been implanted.

"Drug companies tend to be skeptical of the kind of functional screening we used to identify DHODH as a target, because it can be complicated and imprecise. We think that with modern tools, we may be able to improve target identification, so applying this approach to a broader range of cancers may be justified," said Scadden, who is Chair and Professor of Stem Cell and Regenerative Biology and Jordan Professor of Medicine at Harvard University. Additional investigation of the mechanism underlying DHODH inhibition should allow development of protocols for human clinical trials.

Tuesday, September 20, 2016

Researchers at University of California San Diego School of Medicine and Moores Cancer Center have identified a strategy to maximize the effectiveness of anti-cancer immune therapy.

The researchers identified a molecular switch that controls immune suppression, opening the possibility to further improving and refining emerging immunotherapies that boost the body's own abilities to fight diseases ranging from cancer to Alzheimer's and Crohn's disease. The findings were published in the September 19 online issue of Nature (DOI: 10.1038/nature19834).

"Immunotherapies, such as T cell checkpoint inhibitors, are showing great promise in early treatments and trials, but they are not universally effective," said Judith A. Varner, PhD, Professor in the Departments of Pathology and Medicine at UC San Diego School of Medicine. "We have identified a new method to boost the effectiveness of current immune therapy. Our findings also improve our understanding of key mechanisms that control cancer immune suppression and could lead to the development of more effective immunotherapies."

In cancer, highly abundant microphages express anti-inflammatory cytokines that induce immune suppression, effectively stopping the healing process. Varner and colleagues pinpoint a key, suspected player: an enzyme in macrophages called PI-3 kinase gamma (PI3Ky). In mouse studies, they found that macrophage PI3Ky signaling promotes immune suppression by inhibiting activation of anti-tumor T cells. Blocking PI3Ky activated the immune response and significantly suppressed growth of implanted tumors in animal models. It also boosted sensitivity of some tumors to existing anti-cancer drugs and synergized with existing immune therapy to eradicate tumors. Researchers also identified a molecular signature of immune suppression and response in mice and cancer patients that may be used to track the effectiveness of immunotherapy.

"Recently developed cancer immunotherapeutics, including T cell checkpoint inhibitors and vaccines, have shown encouraging results in stimulating the body's own adaptive immune response," said co-author Ezra Cohen, MD, who heads the cancer immunotherapy program at Moores Cancer Center. "But they are effective only on a subset of patients, probably because they do not alter the profoundly immunosuppressive microenvironment created by tumor-associated macrophages. Our work offers a strategy to maximize patient responses to immune therapy and to eradicate tumors. "

The Nature paper builds upon other work by Varner and colleagues. In a paper first published online in May in Cancer Discovery, Varner's team reported that blocking PI3Ky in tumor-associated macrophages stimulated the immune response and inhibited tumor cell invasion, metastasis and fibrotic scarring caused by pancreatic ductal adenocarcinoma (PDAC) in animal models.

"PDAC has one of the worst 5-year survival rates of all solid tumors, so new treatment strategies are urgently needed," said Megan M. Kaneda, PhD, an Assistant Project Scientist in Varner's lab and collaborator on all of the papers.

In a December 2015 paper published online in Cancer Discovery, Varner and colleagues described animal studies that revealed how disrupting cross-talk between B cells and tumor-associated macrophages inhibited PDAC growth and improved responsiveness to standard-of-care chemotherapy.

Specifically, that research team, which included scientists in San Francisco, Oregon, and Switzerland, reported that inhibiting Bruton tyrosine kinase, an enzyme that plays a crucial role in B cell and macrophage functions, restored T cell-dependent anti-tumor immune response. In other words, it reactivated the natural, adaptive immune response in tested mice.

Monday, September 19, 2016

Active monitoring is as effective as surgery and radiotherapy, in terms of survival at 10 years, reports the largest study of its kind, funded by the National Institute for Health Research (NIHR).

Results published in New England Journal of Medicine (DOI: 10.1056/NEJMoa1606220) show that all three treatments result in similar, and very low, rates of death from prostate cancer. Surgery and radiotherapy reduce the risk of cancer progression over time compared with active monitoring, but cause more unpleasant side-effects.

The ProtecT trial, led by researchers at the Universities of Oxford and Bristol in nine UK centres, is the first trial to evaluate the effectiveness, cost-effectiveness, and acceptability of three major treatment options: active monitoring, surgery (radical prostatectomy), and radiotherapy for men with localised prostate cancer.

Chief investigator Professor Freddie Hamdy from the University of Oxford, said: "What we have learnt from this study so far is that prostate cancer detected by PSA blood test grows very slowly, and very few men die of it when followed up over a period of 10 years, -- around 1% -- irrespective of the treatment assigned. This is considerably lower than anticipated when we started the study.

"However, treating the disease radically, when found, reduces the number of men who develop spread of prostate cancer, but we do not know yet whether this will make a difference to them living longer or better, and we have been unable to determine reliably which disease is lethal, and which can be left alone."

Between 1999 and 2009, 82,429 men aged 50-69 across the UK were tested and 1,643 diagnosed with localized prostate cancer agreed to be randomised to active monitoring (545), radical prostatectomy (553), or radical radiotherapy (545). The research team measured mortality rates at 10 years, cancer progression and spread, and the impact of treatments reported by men.

The research team found that survival from localized prostate cancer was extremely high, at approximately 99 percent, irrespective of the treatment assigned.

The rate of cancer progression and spread was reduced by more than half in men in the surgery and radiotherapy groups, compared with active monitoring; cancer progression occurred in one in five in the active monitoring group, as opposed to less than one in 10 in the surgery and radiotherapy groups. However, surgery and radiotherapy caused unpleasant side-effects, particularly in the first year after treatment.

There was some recovery from side-effects over 2 to 3 years. But after 6 years, twice as many men in the surgery group still experienced urine leakage and problems with their sex life, in comparison with those in the active monitoring and radiotherapy groups. Radiotherapy caused more bowel problems than surgery or active monitoring.

Overall quality of life, including anxiety and depression, were not affected by any treatment at any time. Half of the men stayed on active monitoring over the 10-year period and avoided treatment side effects.

"This is the first time radiotherapy, surgery and active monitoring treatments for prostate cancer have been compared directly. The results provide patients and clinicians with detailed information about the effects and impacts of each treatment so that they can make an informed decision about which treatment to have," said co-investigator Professor Jenny Donovan, from the University of Bristol. "Each treatment has different impacts and effects, and we need longer follow up to see how those balance out over the next 10 years."

Professor David Neal, a co-investigator from the University of Oxford, said: "Interestingly, we saw that disease spread was reduced by half in men who were assigned to radical treatment, but no difference in survival outcomes with either surgery or radiotherapy, and no progression of the disease in three quarters of the men in the active monitoring treatment group, over the 10 years. We need to continue to study these men to find out whether prevention of cancer progression by surgery or radiotherapy leads to better cancer control and survival in the longer term."

Professor Freddie Hamdy added: "Longer follow-up is now required to determine the 'trade-off' that patients need to make between cancer outcomes and quality of life, and further research to understand how we can distinguish lethal from non-lethal disease.

"It is important that this research was conducted and that wouldn't be possible without the NIHR and its infrastructure enabling large scale randomised clinical trials to be carried out across the NHS."

The findings of the study will play a key part in the decision to screen for prostate cancer, and are being used as part of a study investigating the effectiveness and cost-effectiveness of prostate-specific antigen (PSA) testing for screening for prostate cancer, the CAP study.

Anne Mackie, Director at Public Health England Screening said: "The National Screening Committee has been following the ProtecT trial closely. The results of this study will provide men and their doctors with key information needed to manage localised prostate cancer."

Friday, September 16, 2016

Cancer stem cells resist therapy and are a major cause of relapse, long after the bulk of a tumor has been killed. A University of Colorado Cancer Center study published in the Journal of the National Cancer Institute (doi: 10.1093/jnci/djw189) provides the most comprehensive picture to date of head and neck cancer stem cells, identifying genetic pathways that cancer stem cells hijack to promote tumor growth and visualizing the process of "asymmetric division" that allows a stem cell to create tumor tissue cells while retaining its own stem-like profile.

The study is the result of 7 years of research and innovation, including the development of novel techniques that allowed researchers to identify, harvest, and grow these elusive stem cells into populations large enough to study. This major body of work provides specific targets for the development of new cancer therapeutics.

"We wanted to determine the relationships between key genetic alterations and how head and neck cancer stem cells harness those alterations to drive initiation and growth," said CU Cancer Center investigator Antonio Jimeno, MD, PhD, the Daniel and Janet Mordecai Endowed Professor for Cancer Stem Cell Research, Director of the University of Colorado School of Medicine's Head and Neck Cancer Clinical Research Program, and the paper's senior author.

The current project was performed in collaboration with the Gates Center for Regenerative Medicine of which Jimeno is a faculty member. Jimeno started his work with cancer stem cells as a post-doc at Johns Hopkins University, but as he explained, "I focused on head and neck cancer stem cells because there has been an increase in head and neck cancer incidence of about 50 percent over the past 10 years in the U.S. and we need to better understand what is at the root of this disease."

Previously, a major challenge in characterizing cancer stem cells has been gathering a cell population large enough to study.

"There is a lot of 'noise' in cells and you need a lot of them because with only a few cells, it's impossible to tell which of these genetic differences are meaningful features of cancer stem cells and which are just genetic noise," said first author Stephen Keysar, PhD, Assistant Research Professor in the Jimeno lab.

To solve this problem, the group first gathered tumor samples from a larger number of head and neck cancer patients -- 10 patients in all -- more than in any previous study. These samples represented both tumors associated with alcohol and tobacco use and tumors caused by the human papilloma virus (HPV).

"It is important to always remember that we were able to make a difference thanks to the generosity of our patients, who enabled us to work with representative cancer models," Jimeno said.

These tumors were then grown in mice. Subsequently, the group undertook the painstaking process of isolating enough cells for genetic studies and one-by-one transplanting these patient-derived tumor samples onto new mice to study how cancer stem cells initiate tumor growth. "Sometimes it took a year just to get enough cells to study," Keysar noted.

"Antonio is a great example of perseverance," said Dennis Roop, PhD, Director of the Gates Center and also an investigator at the CU Cancer Center and the individual whom Jimeno credits with 'much of the philosophy behind this work.' "Antonio was submitting all these grants, and the reviewers were saying, 'there's no way you can do this; there's no way you'll get enough cells to characterize.' He simply found ways to prove them wrong."

This included leveraging private research funding, primarily from the Gates Center for Regenerative Medicine, the Daniel and Janet Mordecai Foundation and the Peter and Rhondda Grant Fund.

"Private funding allowed Antonio to do the groundwork and develop the techniques that eventually made his proposals to the NIH so compelling that he was able to get support. In the case of those of us who are driven to do what we do, you just find a way to get these things accomplished. This is a great example of how bridge funding from the private sector can move research forward," Roop said.


Study Results

Head and neck cancer stem cells are, in fact, distinct from the rapidly dividing cells that form the bulk of tumors, and there is little difference between cancer stem cells in HPV- and HPV+ cancers. Both are marked by CD44 expression and aldehyde activity, and both use the key pathway PI3K to drive their survival, growth and resistance to anti-cancer therapies.

The group found that the PI3K pathway, which is the most common alteration in head and neck cancer, then deploys SOX2, a transcription factor, to activate programs that modulate 'stemness' within the cell's nucleus. For example, SOX2 was found to control aldehyde activity, which is a common cancer stem cell marker and a well-known driver of cancer stem-cell-mediated tumor growth.

"In normal cells, PI3K is used as a sensor for energy," Jimeno explained. "For a cancer cell to act cancerous, it needs metabolic flexibility -- it needs to be able to over-use energy -- and so this 'energy sensor' is a pathway it wants to hijack. After chemo, PI3K helps the cell shut down and weather the storm. Then when the chemo is gone, PI3K helps cancer stem cells start back up again."

Chemotherapies kill rapidly-dividing cells. PI3K shuts down a cancer stem cell's metabolism, placing the cell in a dormant state. This gives cancer stem cells the ability to evade the trap of chemotherapy.

So what happens when you remove this ability? When the group eliminated SOX2 in mouse models of head and neck cancer, tumors became sensitive to therapies that previously had failed. But when the group amplified SOX2, tumors became even more resistant.

"This molecular thread from PI3K to SOX2 to aldehyde was responsible for all the features that define cancer stem cells," Keysar said. Further, "since SOX2-expressing cells fully behave like cancer stem cells, we now have a new laboratory tool to study cancer stem cell biology and therapeutics."

The work also allowed the group to witness an event of the stem cell cycle that had, at best, been only partially characterized in head and neck cancer.

"It was like the snow leopard of the Himalayas," Jimeno said. "We knew it existed because of the tracks, but no one had taken a picture of it -- that is, until someone patiently perched on a frozen ridge for 2 years with a camera. We did just that."

The event Jimeno refers to is "asymmetric division" of cancer stem cells. When a normal cell divides, it creates two identical copies of itself. However, if stem cells divided symmetrically, it would result in two stem cells but no differentiated cells, or two differentiated cells with the loss of the original stem cell. In either case, symmetrically dividing stem cells would not be able to promote tumor growth while also retaining their stemness.

The group was able to document that when cancer stem cells divide, "they don't divide into two of the same," Jimeno said. "One cell retains a stem profile, and the other goes a step beyond into differentiation."

Overall, this 7 year line of inquiry offered three major advances: it characterized head and neck cancer stem cells; it documented asymmetric division in head and neck cancer stem cells; and it identified genetic mechanisms that allow these cancer stem cells to grow and resist therapy. Importantly, identifying these genetic mechanisms of resistance may also help researchers and doctors overcome it.

"SOX2 and aldehyde inhibitors are now under exploration, and we've also done trials of early PI3K inhibitors here at CU Cancer Center," Jimeno said.

"This has been an excellent example of team science," Roop concluded. "You have Antonio -- a brilliant young clinician-scientist -- leading a group that includes basic scientists, pathologists, bio-informaticians, and statisticians, and their expertise can combine to attack a problem in a way that no individual would be able to do on their own. This work will provide the basis for the development of new therapeutic strategies."

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