authors@Fred Hutch

Pattacini, Laura

February 14, 2016 SCIENCE SPOTLIGHT 
 
1 Volume 6, Issue 2 | Fred Hutchinson Cancer Research Center 
 
Some rest is good 
February 14, 2016 
     L Pattacini 
 
 
 
 
A) Gating scheme used to identify 
regulatory T cells (Tregs) and B) 
frequencies of Tregs in controls and 
cases as percentages of total CD3+ or 
CD4+ T cells. 
 
Image provided by Dr. Laura Pattacini. 
 
 
More than thirty years into the HIV epidemic, we still do not know which kind of immunity can protect 
us from infection. The research is complicated by the fact that HIV infects CD4+ T cells, dendritic 
cells and macrophages, which are all part of the immune system, and is more likely to replicate in 
activated cells. Therefore, a vaccine that activates this part of the immune system might 
inadvertently increase the possibility of a productive infection. On the other hand, its lack of induction 
affects other compartments of the immune system, such as B and CD8+ T cell. How to solve this 
puzzle? Some individuals are exposed to HIV yet do not acquire the virus, defying the odds; 
therefore, looking at their immune asset may help identifying correlates of protection from infection. 
Starting from this idea, a team of researchers at Fred Hutch and University of Washington compared 
the immune responses of two groups of subjects, both highly exposed to HIV as a result of being in 
a relationship with an HIV-infected partner. One group included subjects that remained seronegative 
in the course of the study, while the other included only those that later seroconverted (although all 
samples were obtained before seroconversion). The groups were comparable for demographic 
characteristics as well as for their HIV exposure level, estimated by a score calculation based on an 
algorithm that takes into account several variables such as viral loads of the HIV-infected partner 
February 14, 2016 SCIENCE SPOTLIGHT 
 
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and frequency of unprotected sex (the algorithm was developed at the University of Washington and 
details can be found in Kahle E et al, Journal of acquired immune deficiency syndromes 2013). 
This collection of samples represents a unique opportunity to investigate factors that influence 
seroconversion in HIV exposed individuals. Two main hypotheses have been formulated to explain 
HIV resistance’: one is defined the immune quiescence hypothesis, which assumes that a state of 
general low immune activation prevents HIV infection by limiting the numbers of possible target cells, 
the second hypothesis supports a role for HIV-specific CD4+ and CD8+ T cell responses in 
protection from HIV. 
A study from Dr. Laura Pattacini and co-investigators (Vaccine and Infectious Disease Division), 
published in the Journal of Acquired Immune Deficiency Syndromes, reports that samples from 
individuals who did not acquire HIV were characterized by higher frequency of regulatory T cells 
(Tregs). Tregs play a fundamental role in controlling activation of several types of immune cells, in 
order to terminate the immune response after an infection is cleared as well as to avoid 
autoimmunity. On the other hand, no differences were found in HIV-specific immune responses 
detected by cytokine secretion upon in vitro stimulation with HIV-peptide pools. These findings, 
especially the increased frequency of Tregs in non-seroconverters support the immune quiescence 
hypothesis. 
The results open a path for further studies: "We plan to continue our investigations to identify novel 
mechanisms of protection from HIV-1 infection in HIV-1 exposed seronegative individuals, though 
we will next turn our focus to investigating the genital mucosal tissues, as a critically relevant tissue 
site for protection for individuals exposed to HIV-1 via the sexual route" said Dr. Jennifer Lund, 
principal investigator and coordinator of the project. Science Spotlight will surely follow-up with her in 
the future to report what happens at the sites of HIV infection. 
The study was supported by National Institutes of Health and the Bill and Melinda Gates foundation. 
Pattacini L, Baeten JM, Thomas KK, Fluharty TR, Murnane PM, Donnell D, Bukusi E, Ronald A, 
Mugo N, Lingappa JR, Celum C, McElrath MJ, Lund JM. 2015. Regulatory T-cell activity but not 
conventional HIV-specific T-cell responses are associated with protection from HIV-1 infection. 
Journal of acquired immune deficiency syndromes. doi: 10.1097/QAI.0000000000000919. [Epub 
ahead of print] 
 
 


English

Some rest is good

Neves, Alexandre

February 14, 2016 SCIENCE SPOTLIGHT 
 
1 Volume 6, Issue 2 | Fred Hutchinson Cancer Research Center 
 
TRS1 lives solely to antagonize PKR 
February 14, 2016 
     A Neves 
 
 
 
 
 
 
 
Schematic of the study. Wild-type (WT) 
Human cytomegalovirus (HCMV) can 
replicate efficiently in both wild-type 
(PKR+/+) and PKR-deficient cells (PKR 
0/0). in contrast HCMV mutants that 
either lacks the protein kinase R (PKR) 
antagonist TRS1 or expresses a TRS1 
mutant that cannot bind to PKR, 
replicate in PKR 0/0 but not PKR +/+ 
host cells. 
 
Image provided by Dr. Stephanie Child. 
 
 
The recent Zika virus outbreak illustrates the importance and complexity of virus-host interactions. 
To thwart viruses, host cells have evolved a wide variety of defense mechanisms. One well-
characterized innate defense pathway involves Protein Kinase R (PKR), which responds to double-
stranded RNA (dsRNA), a byproduct of viral replication. Activation of PKR results in phosphorylation 
of the eukaryotic initiation factor 2 (eIF2α), which in turn inhibits protein synthesis and prevents viral 
replication. Many viruses that infect human cells encode one or more PKR antagonists in their 
compact genomes. For example, human cytomegalovirus (HCMV) encodes two closely related 
proteins, TRS1 and IRS1, which can each can inhibit the PKR pathway. Previous studies from the 
Geballe Laboratory (Clinical Research and Human Biology Divisions) had demonstrated that a 
mutant HCMV lacking both TRS1 and IRS1 (HCMV [ΔI/ΔT]) was unable to replicate in cell culture. 
However, because both TRS1 and IRS1 participate in many other protein-protein interactions and 
may have other biochemical activities, it remains unclear which, if any, of these are important for 
HCMV replication. 
February 14, 2016 SCIENCE SPOTLIGHT 
 
2 Volume 6, Issue 2 | Fred Hutchinson Cancer Research Center 
 
A new Fred Hutch follow-up study from Dr. Adam Geballe’s Lab, led by former graduate student Dr. 
Jacquelyn Braggin and published in Virology, addressed this question by first identifying TRS1 
mutants that can no longer bind to PKR and then by generating PKR-deficient cells with CRISPR 
gene editing technology. To generate TRS1 mutants, the authors used a C-terminal charged-cluster-
to-alanine (CCTA) method, wherein charged clusters of amino acids, which are likely to be surface-
exposed, are mutated to alanine. After testing seven such mutants for PKR binding by utilizing a 
pulldown assay, the investigators successfully identified one CCTA mutant that could not bind to 
PKR, termed TRS-Mut1. Importantly, a recombinant HCMV expressing TRS1-Mut1 (HCMV [TRS1-
Mut1]) could not replicate in human fibroblasts, similar to HCMV [ΔI/ΔT] and in contrast to HCMV 
expressing wild-type TRS1 (HCMV [TRS1]). Next, the authors evaluated PKR activation by 
monitoring levels of both total and phosphorylated PKR and eIF2α by immunoblotting, which 
revealed that indeed HCMV [TRS1-Mut1] was unable to prevent PKR activation. Finally, they tested 
whether PKR inactivation could rescue the replication prowess of either HCMV [TRS1-Mut1] or 
HCMV [ΔI/ΔT]. To this end, the scientists knocked down and knocked out PKR by shRNA and 
CRISPR/Cas9 gene editing, respectively. Strikingly, both HCMV [TRS1-Mut1] and HCMV [ΔI/ΔT] 
were able to replicate in PKR-deficient cells. In summary, while TRS1 and IRS1 can inhibit the 
autophagy, RNAseL and PKR pathways and bind to PKR and dsRNA, only the PKR inactivation 
activity appears to be required to viral replication. Said Dr. Geballe: "Among the surprising results of 
this study was our finding the a mutant virus that has the TRS1 gene but not the IRS1 gene does not 
block the PKR pathway completely. We are conducting experiments now to try to distinguish 
whether a simple gene dosage effect (having two redundant PKR antagonists) accounts for these 
results or if TRS1 and IRS1 play complementary but non-redundant roles in blocking the PKR 
pathway. Our results also raise questions about the importance of other functions of TRS1 and IRS1 
during the CMV replication cycle. For example, TRS1 and IRS each block autophagy, a 
complicated "self-eating" cellular process that is proviral in some viral systems and antiviral in others. 
By eliminating the need for TRS1 or IRS to block the PKR pathway (by deleting PKR from the host 
cell) we can now explore whether the impact of TRS1 and IRS1 on autophagy is beneficial or 
harmful to the virus.  Finally, knowing that antagonizing PKR is the only essential function of TRS1 
and IRS1 will help push forward translational studies in which, in collaboration with investigator at 
University of Minnesota and Virginia Commonwealth University, we are attempting to develop a safe 
vaccine for use in preventing congenital transmission of CMV" 
Braggin JE, Child SJ, Geballe AP. 2015. Essential role of protein kinase R antagonism by TRS1 in 
human cytomegalovirus replication. Virology. 489:75-85. 
Funding for this work was provided by the National Institutes of Health. 


TRS1 lives solely to antagonize PKR.

Koch, Lori

March 21, 2016 SCIENCE SPOTLIGHT 
 
1 Volume 6, Issue 3 | Fred Hutchinson Cancer Research Center 
 
Cheaters allow cooperators to prosper 
March 21, 2016 
     L Koch 
 
In a population initially consisting entirely of cooperators (C; pink) (left graph), the number of defectors (D; blue) rises 
at about the same time in all locations due to the mutation of C to D. Because all locations are experiencing the same 
dynamics, D eventually causes the collapse of C and then goes extinct itself. In a population initially seeded with a 
mixture of C and D (right graph), the population dynamics are more heterogeneous, allowing C to escape to locations 
made empty by earlier D-induced extinctions. This allows the persistence of both populations and thus the survival of 
cooperation. 
 
Image provided by Dr. Adam Waite. 
There are two types of people in the world: those who continually give back to their community and 
those who don't.  Now over time you might imagine that those who take but never give, the 
defectors, would have the advantage while those who give back, the cooperators, would have less 
energy or resources to spend on securing their own survival and reproduction. In the long run, that 
often turns out to be true and leads to population collapse as the defectors, the professional 
moochers, take over and resources become harder to procure.  Interestingly however, former Fred 
Hutch graduate student Dr. Adam Waite and former UW undergraduate student Caroline Cannistra 
of the Shou Laboratory (Basic Sciences Division) uncovered several mechanisms in which the 
presence of defectors can actually increase the survival of populations of cooperators in the long 
run. The results of their study were recently published in PLOS Computational Biology. 
March 21, 2016 SCIENCE SPOTLIGHT 
 
2 Volume 6, Issue 3 | Fred Hutchinson Cancer Research Center 
 
Instead of experimentally measuring population dynamics, scientists can run simulations of 
populations with controlled parameters in silico.  These simulations can paint realistic pictures of 
large populations that would be extremely difficult and time-consuming to generate by observing 
large populations of living organisms.  Understanding population dynamics between groups of 
organisms, such as microbes, is important for understanding natural communities, such as those 
present in the human gut, as well as for bioengineering applications such as drug or biofuel 
production.  
Dr. Waite and his colleagues modeled dynamics of populations of yeast in silico, allowing them to 
occupy a collection of distinct locations (i.e. a population of populations or "metapopulation"), an 
arrangement that mimics the patchy distributions of individuals often seen in natural ecosystems. At 
each location, cooperators released a resource that could be used by all individuals in that 
location.  Defectors used the resource without giving back and cooperators and defectors competed 
for the released resource. Individuals could occasionally migrate to a different location. A cooperator 
could mutate into a defector, which commonly occurs through loss-of-function mutations, while the 
reverse phenomenon is only very rarely observed. Both cooperators and defectors could evolve to 
become better adapted to resource limitation. 
To make their predictions realistic, the scientists used parameters based on experimental 
measurements of yeast resource consumption and release as well as rates of birth and death of 
individual yeast cells. They varied other parameters such as mutation rate, migration rate, and the 
initial presence of defectors to test how these affected cooperator survival.  
  
The scientists found that when populations initially consisting of a mixture of cooperators (C) and 
defectors (D) were not allowed to migrate to different locations, defectors outgrew cooperators and 
eventually caused the collapse of the entire population.  However, when C and D were allowed a 
moderate rate of migration, the survival of cooperators was greatly improved.  This is because C 
could migrate to "empty" locations where D had caused the sub-population to collapse.  In contrast, 
individual migrating defectors could not "seed" these empty locations without the presence of 
cooperators.   
Intriguingly, Waite et al observed that populations beginning with a mixture of cooperators and 
defectors could sometimes outlive populations initially consisting entirely of cooperators.  This is 
because when defectors were initially absent, all locations shared a similar dynamic:  cooperators 
migrated and grew to high density, then cooperators mutated into defectors at a certain rate and the 
subsequent rise of defectors caused synchronous population collapse. In contrast, when defectors 
March 21, 2016 SCIENCE SPOTLIGHT 
 
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were initially present the population dynamics across different locations became heterogeneous; 
some sub-populations collapsed due to defectors, allowing for migrant cooperators to seed and grow 
with greater initial advantage there than migrant defectors.  This type of oscillation between C and D 
numbers at individual sub-populations appeared many times in simulations with different 
assumptions and initial conditions, suggesting that it is likely a very common phenomenon. Thus, 
both the ability to migrate into locations made empty by defector-induced collapse and the dynamic 
and stochastic nature of the competition between C and D leads to the survival of C over longer 
periods of time than would be observed without D.  
In addition to this, the scientists observed that the deteriorating environment created by defectors 
selected for mutant cooperators (C*) and defectors (D*) more suited to nutrient limitation. Since 
these high fitness mutants quickly dominated the entire metapopulation, they had the same effect as 
dramatically reducing population size. This favored the survival of cooperators (C*) in a way similar 
to the "chasing" dynamics (Supplementary Movie 2) observed between C and D in migrating 
populations.   
  
"Because they have a selective advantage over cooperators, defectors will always show up in 
cooperative systems. Usually, defectors are considered exclusively bad for cooperation," said Dr. 
Waite.   "Our work reveals several ways in which the poor environments created by defectors can 
end up favoring cooperators."   
   
Waite AJ, Cannistra C, Shou W. 2015. "Defectors can create conditions that rescue cooperation." 
PLOS Computational Biology. 11(12):e1004645. Doi:10.1371/journal.pcbi.1004645. 
Interview with Dr. Waite 
This research was funded by the W.M. Keck Foundation, the National Institutes of Health, the 
National Science Foundation Bio/Computation Evolution in Action Consortium (BEACON), and Fred 
Hutch.  
 
 


Cheaters allow cooperators to prosper

Herman, Jake

March 21, 2016 SCIENCE SPOTLIGHT 
 
1 Volume 6, Issue 3 | Fred Hutchinson Cancer Research Center 
 
Finding the goldilocks dose for new antivirals 
March 21, 2016 
     J Herman 
 
The left panels demonstrate heterogeneity of CD8 T cell densities in 200 genital tract micro regions in our 
mathematical model. Each region is intended to approximate a 5 mm diameter of tissue. The right panels 
demonstrate the reproductive number, which is inversely correlated with CD8+ T cell density. There is no potential for 
virus to spread extensively in grey or black regions, whereas green regions are not immunologically protected and 
virus has the potential to expand more extensively leading to lesions. The top two panels represent a simulated 
patient on no drug. The bottom two panels demonstrate a simulated patient on high dose pritelivir (75mg daily). 
While, there is an equivalent spatial distribution of CD8 T cell densities across the genital tract, the overall growth 
potential for virus is lower on high dose treatment: a higher percentage of regions are grey or black meaning that 
HSV reactivation would result in rapid viral elimination. 
 
Image provided by Dr. Joshua Schiffer. 
An often unappreciated facet of developing new therapeutics is the design and execution of clinical 
trials. Recently, predictive therapeutic biomarkers have become commonplace for trial design. By 
understanding the molecular drivers of disease researchers have developed drugs that target these 
molecular pathologies. Drugs like vemurafenib (for mutant BRAF in melanoma) are only beneficial to 
patients affected by specific genetic and molecular lesions. Identifying and enriching the clinical trial 
population for likely 'responders' results in a clear representation of drug efficacy. However, even 
with the best patient groups doctors must still identify optimal doses for a trial to demonstrate 
March 21, 2016 SCIENCE SPOTLIGHT 
 
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efficacy. Often these are estimated from results of human cultured cells and mouse models of 
disease. Doctors must understand the lifespan of the compound in the human body, but also the 
drug concentration required to achieve the desired activity. When doses are too high they can cause 
unnecessary adverse effects, when doses are too low therapies are not efficacious. Dr. Joshua 
Schiffer and other Fred Hutch researchers in the Clinical Research and Vaccine and Infectious 
Disease Divisions are using mathematical models to identify optimum patient dosing for antiviral 
therapies. In a recent Science Translational Medicine article, researchers developed a mathematical 
model of herpes simplex virus (HSV2) suppression using the novel helicase inhibitor, pritelivir. 
The model consists of three major sets of equations for the following: pharmacodynamics (PD), 
pharmacokinetics (PK), and viral kinetics in the absence of drug. PD equations represent the 
concentration dependent inhibition of pritelivir on HSV2 shedding, while PK equations model the 
distribution and serum lifespan of pritelivir. The FDA requires PK and PD data for licensure of all new 
therapeutics; however, in modeling and understanding HSV2 suppression, researchers found these 
equations alone could not explain results seen in clinical trials. "We identify that standard 
pharmacodynamics models do not accurately capture the true potency of antiviral drugs. To 
compute this potency in a dose response fashion requires synthesizing these models with equations 
that capture the natural features of infection," said Dr. Schiffer. A key component to modeling HSV2 
infection was to include equations representing the infectious cycle of the virus. In an infected 
individual, a herpes outbreak occurs when virus is released from a neuronal pool and infects the 
genital tract. As virus incubates in this initial site of genital infection, it can then spread locally 
ultimately becoming a lesion. Researchers modeled this using 200 'micro regions' where virus could 
spread depending on the location and T cell activity of each micro region.  
To validate this model, researchers compared its predictions to patient data from an in-depth phase 
II trial using pritelivir. In that trial patients were placed on four different pritelivir doses, then patients 
swabbed commonly infected areas every day for the one month trial. From these samples 
researchers measured HSV copy numbers and found that higher daily doses of pritelivir decreased 
viral shedding. The mathematical model was used to simulate over 100 trials that consistently 
predicted equivalent shedding rates to real patients at all four doses. The model also demonstrated 
that pritelivir decreased HSV shedding rates by indirectly decreasing viral cell-to-cell transmission. 
Upon a new outbreak there is a lag in the immune response required to clear the virus, if 
neighboring cells are infected during this lag time they will still be cleared, but if a spatially distant 
seeding occurs this will prolong the episode length. Thus by decreasing cell-to-cell transmission 
overall, pritelivir dramatically reduces these secondary ulcers and overall outbreak length. 
March 21, 2016 SCIENCE SPOTLIGHT 
 
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In generating this mathematical model researchers also identified important new measures for 
choosing doses in clinical trials. Most doses are estimated from EC50 measures established with 
cultured human cells that may not fully represent in vivo activity. Researchers identified an 'in vivo 
EC50' by testing many EC50 values (drug concentration in serum) in their model ranging from 0-100 
ng/mL and comparing the results to the patient shedding numbers. It was found that pritelivir doses 
from 50-70 ng/mL best matched patient data, importantly these values range from 2-15 fold higher 
than in vitro studies. The model also revealed that pritelivir levels are very stable in patients, which 
translates to understanding patient response even with missed doses, "a surprising finding was that 
total cumulative weekly dose is highly predictive of shedding rate in a clinical trial population. 
Therefore, if we knew the number of missed doses, we could extrapolate the predictive shedding 
rate" said Dr. Schiffer. 
While this model is specific to HSV2 and pritelivir, this approach can be adapted to other infectious 
diseases, as long as the viral kinetics are well understood. A model specific to one disease and drug 
combination would function very much like a therapeutic biomarker, and could increase the 
efficiency of clinical trials. "I am hopeful that our approach will allow the field to more accurately 
assess drug potency in vivo and therefore select dosing regimens more strategically. This could 
potentially lower expenditures on drug trials and also increase the likelihood of demonstrating 
therapeutic efficacy" said Dr. Schiffer. 
 
Schiffer JT, Swan DA, Magaret A, Corey L, Wald A, Ossig J, Ruebsamen-Schaeff H, Stoelben S, 
Timmler B, Zimmermann H, Melhem MR, Van Wart SA, Rubino CM, Birkmann A. 2016. 
Mathematical modeling of herpes simplex virus-2 suppression with pritelivir predicts trial outcomes. 
Sci Transl Med. 8(324): 324ra15. Doi: 10.1126/scitranslmed.aad6654. 
 
 


Finding the goldilocks dose for new antivirals

Zubair, Niha

February 14, 2016 SCIENCE SPOTLIGHT 
 
1 Volume 6, Issue 2 | Fred Hutchinson Cancer Research Center 
 
Breast cancer survivor? MRI screening offers 
benefits. 
February 14, 2016 
     N Zubair 
 
Shown is a 2cm breast cancer detected on MRI screening in a 63 year old woman with personal history of cancer 
(treated for invasive breast cancer 19 years earlier). Her screening mammogram was negative. 
 
Image provided by Dr. Savannah Partridge. 
Current guidelines from the American Cancer Society (ACS) and National Comprehensive Cancer 
Network (NCCN) recommend annual breast MRI screening (along with mammography) for women 
who are carriers or have first-degree relatives with BRCA or other cancer susceptibility genetic 
mutations, who have a greater than 20-25% lifetime risk of developing breast cancer based on family 
history, or who have had radiation to the chest. Recommendations for these individuals stem from 
studies in which MRI added to mammography resulted in an increased rate of identifying invasive 
breast cancer without an unacceptable increase in unnecessary follow up procedures, including 
biopsies. 
Women with a personal history of breast cancer also have an elevated risk of future breast cancer 
events. However, for these women, the American Society of Clinical Oncology (ASCO) recommends 
against MRI screening, while the ACS and NCCN give no recommendations.  A recent study led by 
Dr. Janie Lee, one of the co-authors of this study and Section Chief of Breast Imaging for University 
of Washington and Seattle Cancer Care Alliance (SCCA), showed that some breast cancer survivors 
are at higher risk of developing second breast cancers that are missed on screening mammography 
February 14, 2016 SCIENCE SPOTLIGHT 
 
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and that these women may benefit from more intensive screening with a test like MRI after 
completing their treatment (Lee JM et al. JNCI 2015). However, there is insufficient evidence 
supporting MRI’s use in this specific patient population and there exist concerns of morbidity and 
costs associated with additional imaging or biopsy of benign lesions identified as suspicious by MRI. 
To address these scientific gaps, Drs. Constance Lehman, Savannah Partridge, Janie Lee, and 
colleagues assessed the diagnostic performance of MRI screening of women with a personal history 
(PH) of treated breast cancer vs. those with a genetic or family history of breast cancer (GFH). They 
presented their results recently in Journal of the National Cancer Institute. 
The researchers used case-series registry data, collected at time of MRI and at 12-month follow-up, 
from the Consortium Oncology Data Integration Project, a board-approved solid tumor clinical 
database developed and maintained by the Fred Hutchinson Cancer Research Center in 
collaboration with the University of Washington. After satisfying inclusion and exclusion criteria, 
1,521 women 18 years and older underwent MRI for the sole clinical indication of screening from 
July 2004 to November 2011. Of these women, 915 had PH and 606 had GFH of breast cancer; 64 
women with PH and GFH were included in the GFH group.    
Compared to those with GFH, women in the PH group: were more likely to have had a prior breast 
MRI (54% vs. 21%, P < 0.001), had lower recall rates and biopsy recommendations (14% vs. 24% 
and 8% vs. 16%, P < 0.001 respectively), had lower false-positive MRI rates (12% vs. 22%, P 
<0.001), and had higher MRI specificity (94% vs. 86%, P < 0.001). Cancer detection rates and 
sensitivity of the MRI did not differ between the PH and GFH groups. To further explore, why the PH 
group had lower false-positive rates, the researchers conducted a logistic regression analysis and 
found older age, history of prior breast MRI, and clinical indication of PH (vs. GFH) were all 
individually associated with a women’s decreased risk for a false-positive interpretation on an MRI 
screening; however when these factors were combined in a multivariable model, only age and prior 
MRI remained statistically significant.  One explanation for the finding of lower false-positive rates in 
PH patients is that a prior MRI serves as a comparison MRI. Another explanation is that the 
treatment in the PH patient (whether mastectomy, radiation, or hormonal therapy) could render the 
MRI easier to interpret.  
Dr. Lehman, currently the Division Chief of Breast Imaging, Massachusetts General Hospital, 
describes the importance of the findings of this study: "the challenge to date has been that while 
women with a personal history of breast cancer are at increased risk for a future breast cancer 
diagnosis, the majority of research on screening MRI was performed in women at high risk based on 
family or genetic history, not personal history.  Our study shows that MRI detects as many cancers in 
February 14, 2016 SCIENCE SPOTLIGHT 
 
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women with a personal history as it does in high risk women, suggesting that breast cancer survivors 
are another group of women that may benefit from screening MRI." 
According to Dr. Lee, future research directions involve identifying "which women to screen with 
breast MRI  based on factors related to a woman’s clinical history, first tumor biology, treatment 
choices and imaging characteristics. At the SCCA, we are also investigating other new screening 
technologies that can complement mammography to identify cancers earlier. Detecting second 
breast cancers earlier while minimizing the potential harms of additional screening will help breast 
cancer survivors live longer, healthier lives." 
Lehman CD, Lee JM, DeMartini WB, Hippe DS, Rendi MH, Kalish G, Porter P, Gralow J, Partridge 
SC. 2016. Screening MRI in Women With a Personal History of Breast Cancer. J Natl Cancer Inst. 
108(3):1-8. doi: 10.1093/jnci/djv349. 
Funding for this study was provided by the National Institutes of Health and the National Cancer 
Institute. 
 
 


Breast cancer survivor? MRI screening offers benefits.

February 14, 2016 SCIENCE SPOTLIGHT 
 
1 Volume 6, Issue 2 | Fred Hutchinson Cancer Research Center 
 
Forget foraging - Designer proteins are 
becoming a reality 
February 14, 2016 
     L Koch 
 
 
 
 
 
 
 
 
 
 
Top (left) and side (right) views of the five structures of designed 
monomeric tandem repeat proteins chosen for purification. From top to 
bottom the proteins are of the 3- repeat, 6- repeat (two structures), 9- 
repeat and 12- repeat classes. The proteins are colored from blue to 
red from amino to carboxy terminus. 
 
Image provided by Dr. Phil Bradley. 
 
 
 
 
 
 
Protein modeling and design is a field that has made significant advances in recent years, advances 
that have enable researchers to pursue lofty and increasingly translational goals. Bioengineers aim 
to understand the range of protein structure "space" that can be designed, folded into a stable 
structure, purified and put to a good use.  Tandem repeat proteins are especially attractive as a 
design target for both aesthetic and bioengineering purposes, as they could potentially form stable 
vehicles or platforms for therapeutics. Tandem repeat proteins, which contain repeated segments of 
structural motifs, that could self-associate to form a closed structure are ideal for therapeutics as well 
as purification because they avoid the problem of oligomerization (the stable association of more 
than one protein in higher-order complexes).   
February 14, 2016 SCIENCE SPOTLIGHT 
 
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Dr. Phil Bradley (Public Health Sciences Division) wanted to put his structural know-how to the test 
and design and make tandem repeat proteins that "close" themselves unlike any of those found in 
the natural world to date. The product of this vision and close collaboration with structural 
biologist Dr. Barry Stoddard (Basic Sciences Division) was recently published in Nature.   
Dr. Bradley wrote an algorithm to design tandem repeat proteins that could be used within the 
Rosetta molecular modeling package developed in David Baker’s Lab (UW Biochemistry). Rosetta 
designs proteins through an iterative process and the algorithm Dr. Bradley wrote contains extra 
features which ensure the symmetry of the protein "backbone" as well as the regularity of the 
structure and spacing of each segment in these tandem repeat proteins.  Following structure 
prediction, the authors chose to focus on structures with left-handed bundles, as this architecture 
appears totally absent from the known protein structure database 
(http://www.rcsb.org/pdb/home/home.do).  They selected proteins with several different numbers of 
repeats, ranging from 3- to 12- repeated segments.   
Research technicians Lindsey Doyle, Jazmine Hallinan and Jill Bolduc (Stoddard Lab, Basic 
Sciences Division) purified and determined the structure of proteins representing four different 
classes of repeat proteins (3-repeat, 6-repeat, 9-repeat, and 12-repeat structures) by X-ray 
crystallography (see Figure).  The donut-shaped or toroid structures were solved to high resolution 
(2-3 Angstroms) and only varied slightly (0.6-1.1 Angstrom) from the predicted structures in root 
mean square (RMS) deviation of alpha carbon atoms.  The fact that it was possible to purify and 
characterize the structure of tandem repeat proteins with different numbers of repeats suggests that 
the central 'pore' of the protein could be manipulated by altering the number of 
repeats.  Researchers in the Stoddard Lab further characterized the proteins by size-exclusion 
chromatography and found that the 3- and 6-repeat proteins formed stable dimers in solution while 
the 9- and 12- repeat proteins formed monomers. The biochemical behavior of the dimers and 
monomers did not vary significantly over different protein or salt concentrations, suggesting their 
potential utility as stable therapeutic agents. 
To test the flexibility of the system, the scientists wondered if they could divide the 9-repeat structure 
into 3 equivalent pieces that would self-assemble and form the same size ring. In doing this, the 
authors discovered that the smaller subunits formed a completely different pattern when packed 
together in the crystal lattice.  Rather than forming a ring with 3 subunits, the proteins instead formed 
a larger ring with four subunits. Amazingly, this structure formed an interlocking set of rings 
resembling a chain.  This structure, as well as layers of 9- and 12- repeat proteins that form tubes, 
have the potential to be developed for novel drug delivery platforms or nanomaterials. In addition to 
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this, by changing the amino acids that line the central pore, it may be possible to build proteins that 
aid in catalysis or drug presentation.  "We are optimistic that these highly stable designed proteins 
will be useful as scaffolds for engineering new functional domains. For example, we are investigating 
whether we can insert multiple binding domains into these structures and create signaling proteins 
with high avidity for their receptors," said Dr. Phil Bradley.  The future of bioengineering is bright 
indeed.   
    
Doyle L, Hallinan J, Bolduc J, Parmeggiani F, Baker D, Stoddard BL, Bradley P.  2015.  "Rational 
design of alpha-helical tandem repeat proteins with closed architectures."  Nature.  528:585-8. 
This research was funded by the National Institutes of Health, a Swiss National Science Foundation 
Postdoc Fellowship and Human Frontier Science Program Long-Term Fellowship. 
 
 


Forget foraging - Designer proteins are becoming a reality

March 21, 2016 SCIENCE SPOTLIGHT 
 
1 Volume 6, Issue 3 | Fred Hutchinson Cancer Research Center 
 
New findings for teen smoking cessation 
March 21, 2016 
     N Zubair 
 
Experimental design and results of the Hutchinson study of high 
school smoking. 
 
Image provided by Dr. Arthur V. Peterson Jr. 
 
 
Cigarette smoking remains the primary cause of preventable, premature death in the United States 
today, killing more than 480,000 Americans every year and costing the nation more than $170 billion 
annually in health care. Smoking prevalence among teens and young adults places these young 
individuals at risk for a lifetime of medical problems and premature death.  
In 2009, the Hutchinson Study of High School Smoking, a 14-year study involving more than 2,000 
teen smokers in 50 Washington state high schools, was the first randomized trial to report beneficial 
effect in helping teen smokers quit, at one year after high school intervention. The next question was 
to what extent the positive effects from teen smoking cessation intervention would endure, without 
additional intervention, into young adulthood. To address this question Dr. Arthur V. Peterson Jr., 
Patrick Marek, and colleagues in the Public Health Sciences Division at Fred Hutch, undertook the 
first-ever long-term follow-up of the adolescent study participants into young adulthood, seven years 
post high school. The results from their study were recently published in PLoS One.  
The study included 2,151 high school smokers that were identified by a baseline survey of all juniors 
from 50 randomly selected Washington State high schools. Half of the high schools were randomly 
assigned to the experimental intervention. The intervention integrated two types of counseling: The 
first is known as motivational interviewing, a caring, nonjudgmental and respectful approach that 
emphasizes building motivation and confidence for quitting while the second, cognitive behavioral 
skills training, gives smokers the tools they need to learn how to quit. All baseline smokers in the 25 
experimental high schools were proactively recruited to the intervention (with parental consent 
obtained prior to first contact for those who were not yet 18). The intervention in the experimental 
schools was delivered by a team of trained counselors by telephone, when the smokers were high 
March 21, 2016 SCIENCE SPOTLIGHT 
 
2 Volume 6, Issue 3 | Fred Hutchinson Cancer Research Center 
 
school seniors. The remaining half of schools served as control/comparison schools; teen smokers 
from these schools did not participate in the telephone counseling intervention. 
Following up on the positive results at one year post-high school, the investigators undertook a new 
survey of the cohort at seven years post-high school, when most were in their mid-20s. The 
investigators found no evidence that the intervention impact lasted into young adulthood: among 
those in the experimental intervention, 14.2% achieved six-year prolonged smoking abstinence vs. 
13.1% in the control condition, P value = .61). In addition, there were no differences in smoking 
abstinence at six years among the subgroups of daily smokers or less-than-daily smokers, nor 
among any other a priori subgroups. Overall, two-thirds of those who had quit at one-year post high 
school relapsed. 
Despite the intervention’s lack of long-term effect, the trial provided new insights. First, teens liked 
the trial’s motivational interviewing-based telephone conversations; two-thirds of those invited 
participated in the intervention. "To get this large amount of participation by teen smokers was a 
terrific vote of confidence in telephone counseling that uses the motivational interviewing approach," 
said Dr. Peterson, lead author of the paper. "This result helped to overcome the long-term problem 
that teens have been historically difficult to recruit to interventions." Second, this proactive, 
personalized telephone intervention succeeded in helping teen smokers quit at one-year post high 
school.  According to Dr. Peterson, "this result was a milestone: the first-ever demonstration in a 
general population of teens of an intervention with a positive impact on teen’s quitting smoking."  
Overall, the results from this follow-up study suggest that to help teen smokers quit smoking and 
remain smoke-free, new extended interventions are needed. "It is well known that individual 
motivations, and pressures to smoke, change substantially in the critical period after high school," 
Dr. Peterson said. "So, a sustained intervention that sticks with them during this period of change, 
aimed at helping them address their changing motivations and new situations to help them avoid 
relapse to smoking, makes good sense."  
The National Cancer Institute funded the study, which also involved investigators at Group Health 
Research Institute in Seattle. 
Citation: 
Peterson AV, Jr., Marek PM, Kealey KA, Bricker JB, Ludman EJ, Heffner JL. 2016. Does 
Effectiveness of Adolescent Smoking-Cessation Intervention Endure Into Young Adulthood? 7-Year 
Follow-Up Results from a Group-Randomized Trial. PLoS One. 11(2): e0146459.  
March 21, 2016 SCIENCE SPOTLIGHT 
 
3 Volume 6, Issue 3 | Fred Hutchinson Cancer Research Center 
 
See also: 
Kealey KA, Ludman EJ, Marek PM, Mann SL, Bricker JB, Peterson AV. 2009. Design and 
Implementation of an Effective Telephone Counseling Intervention for Adolescent Smoking 
Cessation. Journal of the National Cancer Institute. 101(20): 1393-1405. 
Peterson AV, Kealey KA, Mann SL, Marek PM, Ludman EJ, Liu J, Bricker JB. 2009. Group-
Randomized Trial of a Proactive, Personalized Telephone Counseling Intervention for Adolescent 
Smoking Cessation. Journal of the National Cancer Institute. 101(20): 1378-1392. 
 
 


New findings for teen smoking cessation

May 16, 2016 SCIENCE SPOTLIGHT 
 
1 Volume 6, Issue 5 | Fred Hutchinson Cancer Research Center 
 
Economics of prostate cancer screening 
strategies 
May 16, 2016 
     N Zubair 
 
 
 
 
Cost-Effectiveness of Candidate Prostate-
Specific Antigen (PSA) Screening Strategies 
Under Contemporary and Selective 
Treatment Scenarios. The percentages 
describe the proportion of simulation runs in 
which the cost per quality-adjusted life-years 
(QALY) was less than or equal to the given 
willingness to pay. Results for strategies with 
a PSA threshold for biopsy referral of 10.0 
ng/mL in the Selective treatment scenario 
are not reported because cases detected by 
screening are unlikely candidates for 
conservative management with delayed 
curative treatment. 
 
Image provided by Dr. Joshua Roth. 
 
 
 
 
The US Preventative Services Task Force recently recommended against routine prostate-specific 
antigen (PSA) screening for prostate cancer. Interpreting the results from clinical trials, the Task 
Force concluded that PSA screening provides "at most modest benefit, with unacceptable costs in 
terms of overdiagnosis and overtreatment." As an alternative, experts recommend more 
personalized or smarter screening strategies that preserve benefit while reducing harm. 
Unfortunately, it is unlikely that these strategies will be evaluated in randomized clinical trials due to 
constraints in resources and logistics.  
May 16, 2016 SCIENCE SPOTLIGHT 
 
2 Volume 6, Issue 5 | Fred Hutchinson Cancer Research Center 
 
To address this concern, Drs. Joshua Roth, Roman Gulati, Ruth Etzioni, and colleagues, used 
simulation modeling to conduct comparisons of candidate screening approaches. Specifically, the 
investigators examined whether smarter PSA screening strategies have the potential to be effective 
and cost-effective relative to no screening. In addition, they evaluated the potential added value of 
projecting outcomes under selective treatment practices with increased use of conservative 
management (consisting of an annual office visit, annual PSA testing, and a biennial biopsy) among 
men with screen-detected, low-risk disease. The results from their study were recently published in 
JAMA Oncology. 
The investigators created a model of prostate cancer incidence and mortality and developed it as 
part of the National Cancer Institute’s Cancer Intervention and Surveillance Modeling Network 
Prostate Cancer Working Group. A simulated contemporary cohort of US men beginning at 40 years 
of age underwent 18 strategies for PSA screening (see Figure). Treatment strategies included (1) 
contemporary treatment practices based on age and cancer stage and grade observed in the 
Surveillance, Epidemiology, and End Results program in 2010 or (2) selective treatment practices 
whereby cases with a Gleason score (a system of grading prostate cancer tissue based on how it 
looks under a microscope) lower than 7 and clinical T2a stage (the cancer is in one half or less of 
only one side of your prostate) cancer or lower are treated only after clinical progression, and all 
other cases undergo contemporary treatment practices. They analyzed national and trial data on 
PSA growth, screening and biopsy patterns, incidence of prostate cancer, treatment distributions, 
treatment efficacy, mortality, health-related quality of life, and direct medical expenditure. Data were 
collected from March 18, 2009 to August 15, 2014, and analyzed from November 20, 2012 to 
December 11, 2015.  
Using the model, the investigators examined the following measures and outcomes: life-years (LYs), 
quality-adjusted life-years (QALYs), direct medical expenditure, and cost per LY and QALY gained. 
All 18 screening strategies were associated with increased LYs (range, 0.03-0.06) and costs ($263-
$1,371) compared with no screening, with the cost ranging from $7,335 to $21,649 per LY. With 
contemporary treatment, only strategies with biopsy referral for PSA levels higher than 10.0 ng/mL 
or age-dependent thresholds were associated with increased QALYs (0.002-0.004), and only 
quadrennial screening of patients aged 55 to 69 years was potentially cost-effective in terms of cost 
per QALY (incremental cost-effectiveness ratio, $92,446). With selective treatment, all strategies 
were associated with increased QALYs (0.002-0.004), and several strategies were potentially cost-
effective in terms of cost per QALY (incremental cost-effectiveness ratio, $70,831-$136,332).  
May 16, 2016 SCIENCE SPOTLIGHT 
 
3 Volume 6, Issue 5 | Fred Hutchinson Cancer Research Center 
 
"This study is important because it shows that when it comes to PSA screening, less is more," Dr. 
Roth states.  In addition, "more conservative PSA screening strategies (that is, those with less 
frequent screening and higher PSA level thresholds for biopsy referral) are more likely to be cost-
effective versus less conservative strategies. Additionally, we found that no strategy was likely to be 
of high value under contemporary treatment patterns where many men with low-risk prostate cancer 
(that is, those with a Gleason score lower than 7 and clinical T2a stage cancer or lower) receive 
treatment with surgery or radiation therapy, but several strategies were likely to be of at least 
moderate value with increased use of conservative management (that is, treating only after clinical 
progression) for low-risk, screen-detected cancers." 
According to Dr. Roth, "Future studies should evaluate the feasibility and comparative effectiveness 
of personalized screening strategies, and additional cost-effectiveness analyses should be 
conducted to evaluate if they offer good value. For example, current work by study authors is 
exploring tailoring screening based on BRCA germline status, which has been associated with 
elevated risk of prostate cancer."  Dr. Roth elaborates, "more personalized prostate cancer 
screening strategies are needed to tailor screening approaches to individual patients and further 
improve benefit-risk balance. By that, I mean rather than using a 'one size fits all' approach to 
screening, strategies should increasingly determine screening frequency, biopsy thresholds, and 
prostate cancer treatment approaches based on the specific characteristics of individual patients."  
Funding for this study was provided by the National Cancer Institute and the Centers for Disease 
Control and Prevention as part of the Cancer Intervention and Surveillance Modeling Network. 
Citation:  
Roth JA, Gulati R, Gore JL, Cooperberg MR, Etzioni R. 2016. Economic Analysis of Prostate-
Specific Antigen Screening and Selective Treatment Strategies. JAMA Oncol. doi: 
10.1001/jamaoncol.2015.6275. [Epub ahead of print] 
 


Economics of prostate cancer screening strategies

January 17, 2016 SCIENCE SPOTLIGHT 
 
1 Volume 6, Issue 1 | Fred Hutchinson Cancer Research Center 
 
Transcription program differentiates AML cells, 
halts proliferation 
January 17, 2016 
     J Herman 
 
KLF4 decreases leukemia cell 
proliferation by regulating transcription 
of multiple gene targets. KLF2 and 
KLF4 expression slows the proliferation 
rate in both (A) CML and (B) AML cell 
lines, and is not reversed by miR-150 
inhibition. (C) KLF4 expression reduces 
MYC expression, alters expression of 
MYC targets associated with 
metabolism, and stimulates MXD1 
transcription, a combination that is 
known to limit proliferation. (D) 
Schematic of a gene network regulated 
by KLF4 to limit proliferation and 
promote differentiation. 
 
Image provided by Dr. Valerie Morris 
 
In acute myeloid leukemia (AML) myeloid progenitor/stem cells fail to differentiate and instead 
remain in an immature and hyper-proliferative state. Traditional therapies used to treat AML and 
other types of cancer have been cytotoxic agents that induce cell death or apoptosis. As our 
understanding of these malignancies grows new therapeutic approaches become viable. In the case 
of AML, researchers are also exploring the possibility of differentiation therapy. In this approach 
leukemic cells are forced to differentiate into a more mature cell type, essentially converting the 
proliferating leukemia cells into a benign cell with a limited life span. Central to the differentiation 
process are transcription factors, which have been well studied in AML. Of recent interest has been 
the family of Krüppel-like factors (KLF). In normal hematopoiesis KLF2 and KLF4 drive myeloid cell 
differentiation and limit proliferation by increasing expression of the tumor suppressor and cyclin-
dependent kinase inhibitor p21. Moreover, it was recently shown in a small number of AML patient 
samples that KLF4 expression is silenced. In a study from the Oehler Laboratory (Clinical Research 
Division) published in Molecular and Cellular Biology, researchers elucidated a network of microRNA 
and proteins downstream of KLF4 that regulate proliferation and differentiation in AML cells.  
January 17, 2016 SCIENCE SPOTLIGHT 
 
2 Volume 6, Issue 1 | Fred Hutchinson Cancer Research Center 
 
First, the scientists confirmed that KLF4 expression is commonly decreased in AML cells using 
publicly available microarray data sets of AML patient samples. KLF4 expression was significantly 
reduced in most AML samples compared to CD34
+
 hematopoietic progenitors from healthy patients, 
though patient-to-patient heterogeneity was common. Researchers further verified that KLF4 and to 
a lesser extent KLF2 expression induced differentiation and reduced proliferation of the AML patient 
derived cell line THP-1. Surprisingly, gene expression changes in KLF4 transduced cells were 
similar to changes observed when cells are transduced with the microRNA, miR-150. Decreased 
expression of miR-150 has also been commonly observed in AML and re-expression of this miRNA 
in a murine leukemia model inhibited cancer cell growth. These findings led researchers to explore if 
expression of miR-150 is regulated by KLF4. The first step was to identify the promoter region of 
miR-150 and then to predict transcription factor binding sites, which among a dozen sites included 
two GC-rich elements that commonly bind KLF transcription factors. Using the miR-150 promoter to 
drive luciferase expression it was shown that KLF2 and KLF4 increased luciferase production, and 
mutation to either of the GC-rich elements prevented this activity. Thus KLF4 and KLF2 positively 
regulate miR-150 transcription. 
The initial hypothesis from these results was that KLF4-mediated differentiation in AML was the 
result of miR-150 expression. However, genomic knock out of miR-150 with CRISPR/Cas9 did not 
prevent KLF4-mediated differentiation. KLF4 also activates the tumor suppressor p21 (also called 
CDKN1A), so researchers performed a double knockout of miR-150 and CDKN1A in KLF4 
expressing cells. The double knockout also failed to prevent differentiation, suggesting KLF4’s 
effects on differentiation are the result of other uncharacterized downstream regulators. To identify 
these unknown KLF4 targets microarray expression profiles were determined for KLF4 positive cells 
expressing three unique CRISPR sgRNA: non-targeting control, miR-150, and CDKN1A. From these 
gene expression profiles, researchers identified alterations that were common to all three conditions, 
as each condition is anti-proliferative and differentiates. Numerous cytokines and transcription 
regulators were included in the group, such as TNF, NFkB, and E2F2; however, of most interest was 
the inverse relationship between MYC and MXD1. Elevated KLF4 levels repressed MYC and 
activated MXD1 expression. These two related transcription factors have opposing effects on cells: 
MYC activates proliferation and metabolism, while MXD1 promotes cell cycle arrest. KLF4 
expression also decreased BTK levels, which is overexpressed in AML and also contributes to MYC 
activation. This network of KLF4 regulated proteins can now be further dissected to understand how 
they contribute to differentiation and cell cycle arrest. Moreover, targeting genes in this network 
could be exploited as a therapeutic strategy. While this research focused on AML, Dr. Valerie Morris 
(Clinical Research Division) commented that such a therapy may apply to other cancers as well, 
"KLF4 has been described as a tumor suppressor regulating proliferation, apoptosis and 
January 17, 2016 SCIENCE SPOTLIGHT 
 
3 Volume 6, Issue 1 | Fred Hutchinson Cancer Research Center 
 
differentiation in other blood cancers, including both T-cell acute lymphoblastic leukemia and B-cell 
lymphomas, as well as various solid tumors, including colon and lung cancer." Such a therapy could 
be even further reaching and benefit any cancer with low KLF4 levels, "KLF4 expression is low or 
absent in a number of other malignancies including various gastrointestinal malignancies, brain 
tumors and lung cancer." Said Dr. Vivian Oehler (Clinical Research Division). 
Funding for this research was provided by the Southwest Oncology Group Hope Foundation. 
Morris VA, Cummings CL, Korb B, Boaglio S, Oehler VG. (2015). Deregulated KLF4 expression in 
myeloid leukemias alters cell proliferation and differentiation through microRNA and gene targets. 
Mol Cell Biol. doi: 10.1128/MCB.00712-15.  [Epub ahead of print.] 
 
 


Transcription program differentiates AML cells, halts proliferation

May 16, 2016 SCIENCE SPOTLIGHT 
 
1 Volume 6, Issue 5 | Fred Hutchinson Cancer Research Center 
 
Genetically distinct flu viruses grow better 
together in vitro 
May 16, 2016 
     L Koch 
 
 
 
Two influenza variants, differing only by 
one mutation, can cooperate to improve 
viral growth in cells in culture. 
 
Image provided by Katherine Xue. 
 
 
 
To replicate effectively, viruses must be able to enter and exit host cells.  Neuraminidase (NA) is an 
enzyme on the influenza virus capsid that mediates exit from host cells by cleaving the virion from 
receptors on the host cell surface.  A specific amino acid residue in the NA enzyme catalytic site, 
aspartic acid 151 (D151), has been shown to be required for cleavage activity.  Interestingly 
however, different amino acids have been observed at this position in H3N2 influenza viruses 
sequenced since 2007.  The point mutations D151G and D151N, as well as an "ambiguous" 
nucleotide at that position, have been documented in the GISAID EpiFlu database since 
2007.  Isolated strains of influenza from clinical samples are usually passaged through cells cultured 
in vitro to grow enough virus for sequencing.  Because having a glycine (G) or asparagine (N) rather 
than aspartic acid (D) at residue 151 of NA should prevent cleavage activity and prevent the virus 
from exiting cells, scientists have suggested that the increased frequency of the G151 and N151 flu 
variants is likely an artifact of passaging the virus in vitro.  However, researchers in Dr. Jesse 
Bloom’s laboratory (Basic Sciences) hypothesized that genetically distinct flu viruses might work 
together to improve viral growth.  Their research, recently published in eLife, provides evidence that 
cooperation occurs when two particular viral variants are grown in cell culture.  It also suggests that 
cooperation between viruses could be an unappreciated facet of influenza evolution and infection in 
natural populations.    
May 16, 2016 SCIENCE SPOTLIGHT 
 
2 Volume 6, Issue 5 | Fred Hutchinson Cancer Research Center 
 
The scientists genetically engineered strains of human H3N2 virus with either aspartic acid or 
glycine (D or G) at position 151 of the NA enzyme.  They inoculated human cells in culture with pure 
populations of either variant alone or a mixture of the two viral variants and they measured viral titer 
(growth).  Interestingly, they found that inoculating cells with a mixture of D151/G151 viruses led to 
higher viral titers than those seen with either variant on its own.  If the two H3N2 variants cooperate, 
one would expect a stable proportion of the two to appear and then be maintained over time.  Thus 
the scientists performed serial passaging experiments with pure D151, G151, and mixed D151/G151 
viruses and sequenced viruses after each passage.  They found that cells initially infected with D151 
virus alone accumulate G151 viruses over time and vice versa for cells starting with G151 
virus.  When beginning with a 50/50 mixture of D151/G151, the researchers observed that the 50/50 
proportion of D151 and G151 was maintained over time, through 5 serial passages.  It is of note that 
the G151 variant did not continue to increase in frequency through serial passage, suggesting it is 
not simply an adaptation to the artificial lab conditions. 
Each influenza virus can only have one copy of the neuraminidase (NA) gene.  Therefore, the 
scientists knew that if cooperation among viruses was actually happening, it should depend on the 
concentration of viruses, called multiplicity of infection (MOI).  In agreement with this, the scientists 
found that during an infection with low MOI (fewer viruses), the mixed D151/G151 viruses grew 
indistinguishably from pure D151 H3N2 viruses.   
Given the appearance of the G151 NA variant in H3N2 viruses since 2007, the scientists 
hypothesized that compensatory mutations in other viral genes led to the rise of G151 NA. They 
postulated that the G151 NA variant, which should bind but not cleave host cell surface receptors, 
might be adaptive for viruses with mutations that weaken receptor binding.  Therefore, they cloned 
the sequence of the influenza receptor binding protein hemagglutinin (HA) from H3N2 viruses 
isolated in 2005 and 2007.  As before, a mixture of D151/G151 NA viruses had enhanced viral 
growth when the viruses had the 2007 HA sequence.  Intriguingly however, they found that the 
mixture of D151/G151 NA viruses did not grow better than the D151 variant in combination with the 
HA sequence from 2005.  Finally, to test whether a 50/50 mixture of D151/G151 virus could perform 
both the receptor binding and cleaving functions of H3N2, they measured whether this population of 
viruses could grow when the HA protein could not bind receptors.  Strikingly, they found that the 
D151/G151 NA virus population could grow well even in the absence of functional HA, while pure 
populations of either D151 or G151 NA virus could not grow when HA was inactivated.   
So why don’t we see more mutations at position 151 of NA in unpassaged flu viruses?  The 
scientists hypothesize that the sensitivity of sequencing is not always sufficient to detect low-copy 
May 16, 2016 SCIENCE SPOTLIGHT 
 
3 Volume 6, Issue 5 | Fred Hutchinson Cancer Research Center 
 
variation in the population.  To this end, they are "working now on sequencing flu from patient 
samples to try to understand whether cooperation is something that occurs in clinical infections," 
said author Katherine Xue.  
This research was funded by the National Institute of General Medical Sciences (NIGMS), the 
National Science Foundation (NSF), and the Hertz Foundation. 
Xue KS, Hooper KA, Ollodart AR, Dingens AS, Bloom JD.  2016.  "Cooperation between distinct viral 
variants promotes growth of H3N2 influenza in cell culture." eLife. 2016; 5:e13974. 
 
 


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