authors@Fred Hutch

Pattacini, Laura

December 20, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 12 | Fred Hutchinson Cancer Research Center 
 
Conquering the outsides: an antibody story 
December 20, 2015 
     L Pattacini 
 
 
Immunohistochemistry staining of an 
inner foreskin sample, stained for 
CD138 (green, expressed by plasma 
cells and epithelial cells, and IgG (red), 
expressed by B cells and soluble in the 
tissue. 
 
Image provided by Dr. Maria Lemos 
 
 
This week, UNAIDS highlighted the need for encouraging a greater positive engagement of men in 
all aspects of the AIDS response and in advancing gender equality. Men and adolescent boys 
account for 52% of all new adult HIV infections. Around 60% of the 1.2 million people who died of 
AIDS-related illness in 2014 were male. A crucial site of HIV transmission in men is the foreskin, as 
confirmed by the observation that circumcision is protective against HIV acquisition.  
 
The immune response at this site, and especially antibody production and transportation from the 
blood to the foreskin is poorly characterized. Antibodies, central molecules for our immune 
responses, are large Y-shaped proteins with antigen-binding and functional domains. Depending on 
the characteristics of this fraction we can distinguish different antibody isotypes: IgA, IgM, IgE and 
four types of IgG (IgG1, IgG2, IgG3 and IgG4), characterized by distinct functions. Each of these 
isotypes has a different capacity to neutralize pathogens, or activate complement, phagocytic cells, 
cytotoxic cells or NK cells. 
 
Although the type of antibody response required for HIV protection is not completely understood, the 
HIV vaccine clinical trial RV144 or “ Thai trial”  showed that risk for HIV infection directly correlates 
with the concentration of circulating IgA, whereas it is inversely correlated with IgG3 specific for Env 
V1V2 domain. Based on this finding, it is likely that a protective vaccine for HIV should be able to 
induce the production of such antibodies at the sites of HIV transmission. Two relevant aspects for 
vaccine design would be 1) to understand which antibody types are present at the mucosal site and 
December 20, 2015 SCIENCE SPOTLIGHT 
 
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where they are produced and 2) to compare the antibody pattern in the blood and different mucosal 
sites, and to identify the tissues that should be studied during a vaccine trial. Research associate Dr. 
Maria Lemos, from the Vaccine and Infectious Disease Division at Fred Hutch, decided to take the 
challenge and answer these questions. The results of the study have been published in the 
November issue of Mucosal Immunology.  
 
An interesting finding coming out from Dr. Lemos’ work is that antibody distribution and 
concentration in the colon is not a proxy for foreskin, as it might not be for other mucosal tissues. In 
particular, IgA concentration is fourfold greater in colon than in the foreskin, whereas IgG 
concentration is higher in the foreskin as compared to colon. 
Foreskin antibodies derive in part from the blood, and in part are locally produced by IgM-, IgG- and 
IgA-secreting cells located in the foreskin dermis, which are detectable (as shown in the Figure) as 
single cells but not organized in cell follicles. Since part of the antibodies detected in the foreskin is 
in the transudate from blood, it is possible that vaccination-produced antibodies can reach the 
epidermal layer of the foreskin, where the contact with the virus is more likely to happen prior to 
actual infection. Although the majority of antibody isotypes were primarily located in the dermal layer 
and did not reach the epidermis, IgG1, IgG2 and IgG3 ratios were comparable between the two 
layers. This is especially interesting considering that Env-specific IgG3 have been shown protective 
against HIV acquisition in RV144. To evaluate whether it would be possible to characterize antigen-
specific responses in the different foreskin layers, the presence of Adenovirus 5-specific antibodies 
was evaluated. Ad5 antibodies were present in the inner foreskin epidermis, and were primarily 
derived from the blood. 
 
"I think there are two important lessons highlighted in this paper that contribute to vaccine design" 
said Dr. Lemos. "The first is that antibody immunity at the gut does not necessarily represent 
immunity at genital surfaces of the penis, so upon vaccination, it might be useful to characterize the 
two compartments.  The second is that some antibodies do appear to locate in the epidermis of the 
inner foreskin, which suggest they might contribute to protection at this site." The results obtained 
are encouraging in view of the design of a vaccine that can generate mucosal responses in a site of 
HIV transmission.  
The research was supported by grants from the National Institute of Allergy and Infectious Disease 
of the National Institutes of Health and from the University of Washington Center for AIDS Research. 
 
Lemos MP, Karuna ST, Mize GJ, Fong Y, Montano SM, Ganoza C, Lama JR, Sanchez J, McElrath 
MJ. 2015. In men at risk of HIV infection, IgM, IgG1, IgG3, and IgA reach the human foreskin 
epidermis. Mucosal immunology. doi: 10.1038/mi.2015.103. [Epub ahead of print.] 


English

Conquering the outsides: an antibody story

Herman, Jake

December 20, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 12 | Fred Hutchinson Cancer Research Center 
 
No stone unturned: rare mutation in CSF3R may 
inform therapy 
December 20, 2015 
     J Herman 
 
 
 
 
 
T604N mutation increases CSF3R dimerization 
(A) Schematic of co-immunoprecipitation 
experiment to quantitate CSF3R dimerization. 
(B) The T640N mutation has increased 
dimerization relative to WT CSF3R. Flag and 
V5-tagged CSF3R constructs are co-transfected 
into cells, then an antibody to flag is used to 
immunoprecipitate CSF3R complexes. Co-
immunoprecipitated V5 tagged CSF3R is 
detected by immunoblotting and is normalized to 
the input. 
 
Image provided by Dr. Julia Maxson (Clinical 
Research Division). 
 
 
 
Cancer is the second leading cause of death in the U.S.A. with over 1.5 million individuals being 
diagnosed every year. While cancer is often discussed as a single disease, it is an expansive and 
diverse set of diseases each requiring a unique treatment. In the face of these staggering numbers 
of patients and pathologies, rare cancers are often overlooked due to challenges in funding. 
Recruiting sufficient numbers of patient samples for clinical trials presents another challenge. 
Chronic neutrophilic leukemia (CNL) and atypical chronic myeloid leukemia (aCML) are two such 
rare cancers; they are characterized by high levels of neutrophils or neutrophil precursors and lack 
molecular signatures seen in most CML patients, such as BCR-ABL1 fusion, or rearrangements of 
PDGFR or FGFR. Instead, recent studies have identified colony stimulating factor 3 receptor 
(CSF3R) as a common oncogenic driver in CNL affecting approximately 80% of patients. In these 
December 20, 2015 SCIENCE SPOTLIGHT 
 
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patients the most common mutation is a substitution, T618I, which is sufficient to drive oncogenesis 
in multiple in vitro models. This mutation causes ligand-independent dimerization of CSF3R and 
results in constitutive signal transduction through the JAK pathway. CSF3R bearing the T618I 
mutation is also hypo-glycosylated, however, the relationship between glycosylation and 
dimerization remains unknown. 
In a recent study published in Clinical Cancer Research, Dr. Julia Maxson, a postdoctoral fellow in 
the Radich Laboratory (Clinical Research Division), identified a CSF3R mutation that has never been 
observed in CNL and aCML patients. Sequencing the CSF3R gene in two patient samples revealed 
the mutation, T640N, which was first identified in rare cases of AML. While this study focused on 
findings in CNL and aCML, Dr. Maxson has observed this mutation in other cancer types, "I'm also 
collaborating with Soheil Meshinchi on a project looking at CSF3R mutations in pediatric AML. 
Interestingly, the CSF3R T640N mutation was also found in two of these patients." In the current 
study the authors demonstrated that the T640N conferred many oncogenic behaviors to 
hematopoietic cells including the ability to grow independent of cytokine stimulation, and colony 
formation independent of the CSF3R ligand. These results suggested that T640N was acting 
through the same mechanism as the common T618I mutation –  ligand independent dimerization. 
Molecular modeling from a previous publication predicted the T640N mutation would drive 
dimerization through increased hydrogen bonding. As predicted, the mutation increased ligand-
independent receptor dimerization, however, this unique mutation provided a novel insight into the 
relationship of glycosylation and dimerization. T640N, unlike T618I, resides in the transmembrane 
region of CSF3R, which should preclude any physical interaction with glycosyltransferase enzymes. 
Interestingly, just like the common T618I mutation, T640N also decreased total levels of 
glycosylation on CSF3R. These findings raise the possibility that decreased glycosylation is a result 
of increased receptor dimerization, where glycoslytransferases are sterically occluded from the 
residues they normal act on. 
These researchers verified the oncogenic nature of the T640N mutation in a mouse leukemia model, 
demonstrating that at the pathological level it behaved equivalent to the T618I mutation. Cells 
harboring the T618I mutation in CSF3R are known to activate the Janus Kinase (JAK) protein family, 
and these cells demonstrate sensitivity to JAK inhibitors. Excitingly, the authors find that the T640N 
mutation induces a greater sensitivity to this class of inhibitors. This work makes a strong case for 
using both mutations as biomarkers to identify patients who will respond well to JAK inhibitors, 
however, such clinical studies have not yet been performed. 
December 20, 2015 SCIENCE SPOTLIGHT 
 
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The use of personalized genetic analysis to improve treatment options is a common theme in Dr. 
Maxson's research. In a soon to be published article in Cancer Research, she uses these 
approaches to identify new therapeutic targets for leukemia and expand those findings into solid 
tumors. This work is among a new class, demonstrating that personalized medicine cannot rely only 
on genomic and expression data sets, but must also be approached with a functional perspective.  
Funding for this research was provided by the National Cancer Institute, The Leukemia and 
Lymphoma Society, V Foundation for Cancer Research, Gabrielle’s Angel Foundation for Cancer 
Research, and the Charles and Ann Johnson Foundation. 
Maxson JE, Luty SB, MacManiman JD, Paik JC, Gotlib J, Greenberg P, Bahamadi S, Savage SL, 
Abel ML, Eide CA, Loriaux MM, Stevens EA, Tyner JW. 2015. The colony stimulating factor 3 
receptor T640N mutation is oncogenic, sensitive to JAK inhibition, and mimics T618I. Clin Cancer 
Res. doi: 10.1158/1078-0432.CCR-14-3100. [Epub ahead of print.] 
 
 


No stone unturned: rare mutation in CSF3R may inform therapy

Kocarnik, Jonathan

February 16, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 2 | Fred Hutchinson Cancer Research Center 
 
Gut Microbial Diversity Associated With Plant 
Food Metabolites 
February 16, 2015 
    JM Kocarnik 
While the phrase 'you are what you eat' has long been popular, researchers are getting ever more 
adept at explaining why certain diets may affect human health.  One major area of recent research 
involves the ‘microbiome’, measuring the makeup of microorganisms living on or in the human 
body.  Previous research indicates that these microbiota can have distinct impacts on human health, 
either positive or negative, through metabolic or other processes.  In a recent report in Cancer 
Epidemiology, Biomarkers, and Prevention, Drs. Meredith Hullar and Johanna Lampe in the Public 
Health Sciences Division found that urinary excretion of plant metabolites was associated with both 
the diversity and the composition of the gut microbial community, potentially impacting downstream 
health effects. 
Lignans are compounds found in the woody portions of plants, seed coats, and the bran layer of 
grains.  Epidemiologic evidence suggests that increased consumption of lignans is associated with 
lower risk of breast and colon cancer.  These lignans are metabolized by the gut microbiota into the 
enterolignans enterodiol and enterolactone, bioactive chemicals that are thought to possess a 
variety of activities related to human health.  Importantly, several biochemical steps are needed to 
transform plant lignans into enterolignans, and these steps require an assorted group of gut bacteria 
for the entire process to occur.  This suggests that the complexity and diversity of the gut microbial 
community is essential for maximizing this conversion. 
To evaluate and characterize this relationship, the authors measured enterolignan levels and 
microbial diversity in a group of 115 women.  Enterolignan levels were measured from urine 
samples, and the gut microbial community was measured from stool samples.  To determine the 
makeup of the microbial community in each sample, the authors sequenced the 16s rRNA gene and 
then aligned the sequences into operational taxonomic units.  This taxonomic classification allowed 
the authors to determine the diversity of the microbial community, both within (alpha diversity) and 
between (beta diversity) individuals.  In order to account for differences in diet, the women also 
recorded everything they ate or drank for three days.  
 
 
February 16, 2015 SCIENCE SPOTLIGHT 
 
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Overall, the authors found that the global microbial community differed by tertile of enterolactone, but 
not enterodiol, and that this diversity increased with higher enterolactone excretion.   They also 
found that four bacterial genera were associated with the high enterolactone 
excreters: Moryella, Streptobacillus, Fastidiosipilla, and Acetanaerobacterium.  This diversity 
difference was also positively associated with fiber intake, which aligns with prior evidence that diet 
can influence the human gut microbiome.  This association between dietary patterns high in fiber 
and increased microbial diversity may also suggest additional health benefits, in terms of resilience 
to infection, environmental factors, or obesity. 
While this cross-sectional study will require additional follow-up, it suggests that the environmental 
exposures from dietary intake (such as lignans from plant food consumption) can be altered by the 
metabolic capacity of the gut microbiome (which may be represented by its diversity), which may 
have downstream health effects (such as alterations in cancer risk).  This may be particularly 
important for the catabolism of dietary fibers, which carry wide biochemical and structural 
forms.  Without these helpful microorganisms, humans might otherwise not be able to process these 
fibers into their bioactive forms, missing out on all their associated health benefits.  The authors 
hope that future studies will further our understanding of this complicated relationship. 
Other PHS researchers contributing to this project were Mr. Samuel Lancaster, Mr. Wade Copeland, 
and Dr. Timothy Randolph. 
 
 
Hullar MA, Lancaster SM, Li F, Tseng E, Beer K, Atkinson C, Wahala K, Copeland WK, Randolph 
TW, Newton KM, Lampe JW. 2014. Enterolignan producing phenotypes are associated with 
increased gut microbial diversity and altered composition in premenopausal women in the United 
States. Cancer Epidemiol Biomarkers Prev. pii: cebp.0262.2014. 
 
 
 
 
 
February 16, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 2 | Fred Hutchinson Cancer Research Center 
 
 
 
 
Image provided by Dr. Meredith Hullar 
The composition of the gut microbial community is significantly 
different between high and low enterolactone (ENL) excreters 
(MRPP; P<0.0005). Bars are the amount of urinary ENL (ng/ mg 
creatinine), color-coded by tertile. (Low = green, Medium = black, 
and High = blue). 
 


Gut Microbial Diversity Associated With Plant Food Metabolites

April 20, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 4 | Fred Hutchinson Cancer Research Center 
 
Following the Fate of a Naïve B Cell 
April 20, 2015 
    L Pattacini 
After antigen recognition by its receptor, the naïve B cell migrates to the border of the T cell zone 
where it receives signals to proliferate and originates several types of effector cells, such as antibody 
secreting plasma cells, germinal center (GC) cells and GC-independent memory cells. The question 
of whether a single naïve B cell has the potential to give rise to all three of these effector cells or is 
already committed to a specific path is central for vaccine development. 
In an article published on Science, Dr. Justin Taylor from the Vaccine and Infectious Disease 
Division (VIDD) at Fred Hutch follows the fascinating path of a single naïve B cell after antigen 
recognition. The results showed immense heterogeneity in the progeny of different B cells. In 44% of 
cases, a single cell originated a single effector cell subset (plasma cells, GC cells, GC-independent 
memory cells or activated precursor cells). Only 4 out of 74 clonal population contained all the 
subsets and the rest 2-3 subsets. Not surprisingly, large clonal populations were more likely to give 
rise to multiple subsets. Surprisingly however, no relationship was observed between the size of the 
clonal population and the capacity to proliferate. Dr. Taylor and his colleagues hypothesized that the 
size of the responding population was related to the ability of a clonal population to resist apoptosis. 
Indeed, single B cells resistant to apoptosis by genetic knockout of the pro-apoptotic mediator Bim 
carried the potential to generate a bigger population of effector cells and were more likely to 
generate multiple subsets. 
Previous research had suggested that another factor influencing the fate of a naïve B cell is the 
affinity of the receptor for the antigen. To test this hypothesis, the response of transgenic B cells to 
high or medium affinity antigen were compared. Stimulation with high affinity antigen resulted in the 
preferential production of plasma cells and GC cells, and fewer GC-independent memory cells as 
compared to the medium affinity stimulated clones. This role that receptor affinity for antigen plays in 
effector cell differentiation is similar to recent findings with T cells. 
This study represents a milestone in B cell and vaccine research. As Dr. Taylor puts it, "The 
implication of our work is that not every B cell stimulated by a vaccine is going to respond the way 
that we want it to.  What we need to do is figure out how to ensure that the most useful B cells resist 
apoptosis and differentiate down the pathway that will yield a long-lived protective response." 
 
April 20, 2015 SCIENCE SPOTLIGHT 
 
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Taylor JJ, Pape KA, Steach HR, Jenkins MK. 2015. Humoral immunity. Apoptosis and antigen 
affinity limit effector cell differentiation of a single naive B cell. Science, 347(6223), 784-787. 
 
 
Image provided by Dr. Justin Taylor 
Schematic representation of the optimal B cell response to 
vaccination, on the left, and actual response, on the right. 
 


Following the Fate of a Naïve B Cell

Zentner, Gabriel E.

January 19, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 1 | Fred Hutchinson Cancer Research Center 
 
A Day at the BEACH for Synapse Formation 
January 19, 2015 
      GE Zentner 
The number of synaptic connections in the human brain is estimated to be greater than the number 
of stars in the Milky Way.  Disruptions in this galaxy of neuronal connections lead to diverse 
neurological disorders including autism, epilepsy, and schizophrenia, and understanding the basic 
building blocks of the brain thus has profound implications for combating these conditions.  The brain 
contains two types of synapses, chemical and electrical, but the mechanisms underlying the 
formation of the latter are poorly understood.  To gain insight into the formation of electrical 
synapses, postdoctoral fellow Dr. Adam Miller and colleagues in the laboratory of Dr. Cecilia Moens 
(Basic Sciences Division) undertook a zebrafish genetic screen.  The authors found that the 
gene neurobeachin (nbea) is required for the formation of both chemical and electrical synapses and 
is necessary in the "information receiving" postsynaptic neuron for these processes.  These results 
may also have implications for understanding neurological disorders as mutations in human Nbea 
are associated with autism. 
To identify genes necessary for electrical synapse formation, the authors focused on the Mauthner 
neural circuit (M), which is involved in the startle response and contains a relatively simple circuitry 
of chemical and electrical synapses. M makes both electrical and chemical synapses with unique 
targets in the hindbrain and spinal cord. Mutations in the zebrafish genome were induced using a 
chemical mutagen and electrical synapse formation was assessed in mutant embryos 3 days post-
fertilization (dpf) by staining for a marker called Cx36. The authors identified a mutation termed 
disconnect4 (dis4) that resulted in markedly decreased Cx36 staining in M, as well as in other 
prominent electrical synapses in the brain, suggesting a general role for the dis4-encoded protein in 
electrical synapse formation. 
To map the dis4 mutation, the authors made use of a high-throughput RNA sequencing (RNA-seq) 
approach that they previously developed (Miller et al., 2013).  This analysis led to the identification of 
a nonsense mutation in nbea that would presumably lead to degradation of its mRNA.  Indeed, dis4 
mutant embryos displayed reduced expression of Nbea.  Independent mutant alleles in Nbea 
generated via TALEN enzymes resulted in identical synapse defects as seen in dis4 mutants, 
confirming that dis4 is an allele of nbea and that Nbea is required for electrical synapse formation. 
 
January 19, 2015 SCIENCE SPOTLIGHT 
 
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Nbea is a large protein and is a member of the BEACH (Beige and Chediak-Higashi) protein family, 
which has been implicated in cellular cargo transport.  Nbea has also been previously shown to be 
required for efficient chemical synaptic transmission. The authors found that nbea mutants had 
defects in electrical synapse function. Moreover, the mutants could perform normal escape 
behaviors in response to startling stimuli, but at a reduced frequency compared to wild-type 
animals.  
Synapses require two neurons, one presynaptic or "information sending", the other postsynaptic or 
information receiving. Further analysis revealed that Nbea is required in the postsynaptic neuron at 
both electrical and chemical synapses.  The authors also analyzed the effect of removing nbea on 
the structural formation of the M dendrites, which are the specialized neuronal postsynaptic 
compartment. Neurons are extremely polarized cells, generally containing many branchlike dendrites 
and a single. In the nbea mutants, there is greatly reduced dendritic branching and a loss of fine 
dendritic processes (see figure), indicating that Nbea is needed in M for the maintenance of dendritic 
complexity. 
The results presented in this study reveal a widespread and unexpected role for Nbea in synapse 
formation.  "Because of the very different biochemical properties of electrical and chemical synapses 
it was surprising that Nbea controlled the formation of both types. And this points to the critical open 
question, what is the molecular function of Nbea in synapse formation? Currently we don't know the 
details, however based on its localization and inferring from the function of related proteins, Nbea 
likely acts in the cellular "postal" system (endomembrane compartments) to control the trafficking of 
synaptic components within the postsynaptic neuron," said Dr. Miller 
 
Miller AC, Voelker LH, Shah AN, Moens CB. 2014. Neurobeachin Is Required Postsynaptically for 
Electrical and Chemical Synapse Formation. Curr Biol 25(1):16-28. 
See also: Miller AC, Obholzer ND, Shah AN, Megason SG, Moens CB. 2013. RNA-seq-based 
mapping and candidate identification of mutations from forward genetic screens. Genome 
Res 23(4):679-686. 
 
 
 
January 19, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 1 | Fred Hutchinson Cancer Research Center 
 
 
 
 
Image provided by Dr. Adam Miller 
(A) In completely wild-type embryos, there is high dendritic 
complexity as indicated by the orange and cyan projections. (B) In 
wild-type embryos with a nbea mutation in the Mauthner circuit, 
dendritic complexity is lost. 
 


A Day at the BEACH for Synapse Formation

Neves, Alexandre

January 19, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 1 | Fred Hutchinson Cancer Research Center 
 
A WEE1 Pain In the (Head and) Neck 
January 19, 2015 
     A Neves 
As with many other types of cancer, patients with head and neck squamous cell carcinoma 
(HNSCC) that have not responded to surgery, radiation or chemotherapy, have limited treatment 
options after the disease has spread. A Fred Hutch study published in the journal Clinical Cancer 
Research, led by Russell Moser and Dr. Chang Xu, in the Kemp (Human Biology and Public Health 
Sciences Divisions) and Méndez (Clinical Research Division) labs, respectively, with significant 
contribution by the lab of Dr. Carla Grandori (Human Biology Division), sought to identify new 
therapeutic targets for this disease. 
The tumor suppressor p53 is the most commonly mutated gene in head and neck cancer. Because 
mutations in p53 are common in HNSCC and are associated with metastasis,  resistance to therapy 
and poor patient survival, identifying vulnerabilities of p53 mutant HSNCC is of great clinical 
interest. "Tumors that harbor p53 mutations are more lethal to the patient, yet there are no tailored 
therapies for these tumors," said Dr. Eduardo Méndez.  
To explore such vulnerabilities of HSNCC cells, the investigators performed a short interfering RNA 
(siRNA) screen using 5 HSNCC cell lines derived either from primary or recurrent/metastatic tumors 
that targeted the entire complement of human protein kinases (713 in total, collectively referred to as 
the kinome), and assayed for cell viability. Due to the heterogeneity of human cell lines, the authors 
also carried out a similar kinome siRNA screen using 5 low-passage, murine squamous cell 
carcinoma cell lines (MSCC), which were obtained from mice harboring mutations in p53 pathway 
genes (Atm, Prkdc, p19Arf and Trp53 itself), that were subjected to chemical carcinogens to induce 
squamous cell carcinoma (SCC). 38 out of the 508 kinases shared between human and mouse cells 
were selected for further studies based on several criteria, such as effects of siRNA knockdown on 
viability of both human and mouse SCC, p53 mutational status and metastatic potential. Kinases for 
which inhibition impaired viability of human foreskin fibroblasts were excluded, leaving 28 kinases for 
follow-up studies. Secondary screening of additional HSNCC lines with independent siRNAs using 
viability and programmed cell death as endpoints confirmed a set of 10 kinases as essential for 
cancer cell survival. 
To corroborate the siRNA data and to identify compounds for in vivo testing, the researchers tes ted 
small molecule inhibitors targeting 6 kinases. AZD1775, a small molecule inhibitor of the WEE1  
January 19, 2015 SCIENCE SPOTLIGHT 
 
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kinase, had the broadest and most potent effect. Since WEE1 is a kinase that regulates the G2/M 
transition of the cell cycle, the authors hypothesized that p53-deficient SCC cells were particularly 
dependent on the G2/M checkpoint. To test this, they performed flow cytometry experiments that 
demonstrated that inhibition of WEE1 by AZD1775 resulted in unscheduled entry into the cell cycle 
and programmed cell death. Finally, to ascertain the therapeutic potential for WEE1 inhibition in a 
preclinical model, the investigators carried out a four arm, double blind study using a HSNCC cell 
line with high metastatic potential, which demonstrated that AZD1775 potentiates the efficacy of 
cisplatin, the standard chemotherapeutic agent for HSNCC. "We identified a number of potential 
kinase targets and focused on the G2/M cell cycle regulator WEE1.  Inhibition of WEE1 with a small 
molecule inhibitor AZD1775 was effective against tumors in mice.  These studies directly led to a 
phase I clinical trial in head and neck cancer patients", summarized Dr. Méndez. 
Overall, this study demonstrated that a collaborative effort can rapidly drive a basic research project 
to a clinical trial and demonstrated the feasibility of exploiting vulnerabilities of cancers cells in the 
clinic. This approach could lead to better therapies not only for head and neck cancers, but also for 
other, difficult-to -treat cancers. 
 
Moser R, Xu C, Kao M, Annis J, Lerma LA, Schaupp CM, Gurley KE, Jang IS, Biktasova A, 
Yarbrough WG, Margolin AA, Grandori C, Kemp CJ, Mendez E. 2014. Functional kinomics identifies 
candidate therapeutic targets in head and neck cancer. Clin Cancer Res, 20(16), 4274-88. PMCID: 
PMC4135446 
January 19, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 1 | Fred Hutchinson Cancer Research Center 
 
 
Image provided by Mr. Russell Moser. 
Identification of survival kinases in squamous cell carcinoma (SCC) 
using comparative functional kinomics. Top panel: drug sensitivity of 
p53 wild-type, p53 wild-type positive for human papilloma virus and 
p53 mutant SCC cells to chemical inhibition of G2/M kinases. 
Bottom left: differential cell viability in RNA interference validation 
screens. Bottom right: Cartesian plot of Z-scores from interspecies 
kinome identified kinases with highest shared impact on cell 
viability. 
 


A WEE1 Pain In the (Head and) Neck

Zentner, Gabriel

May 18, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 5 | Fred Hutchinson Cancer Research Center 
 
A Genetic Basis for Human Centromeres 
May 18, 2015 
     GE Zentner 
Accurate chromosome segregation relies upon the kinetochore, a protein complex that attaches to 
specific sites on chromatin known as centromeres.  In humans, centromeres lie within arrays of 
repeated a-satellite sequences, making analysis of their function, identity, and evolution 
difficult.  Most annotated a-satellite arrays are higher-order repeats (HORs), which comprise tandem 
copies of an a-satellite array, itself composed of multiple tandem ~170-base pair (bp) units with 
varying degrees of divergence from one another.  For instance, the X chromosome DXZ1 HOR is 
comprised of tandem copies of a 12-repeat array, wherein each repeat unit is on average only 77% 
identical to its neighbors.  As a-satellite DNA is estimated to comprise 2-3% of the human genome, 
or ~500,000 a-satellite repeats per haploid genome, ~20,000 a-satellites are expected on each 
chromosome.  However, the assembled a-satellite sequences represent nearly 100 times less a-
satellite sequence than expected. 
To identify functional centromeres, data analyst Jorja Henikoff, postdoctoral fellow Jitendra Thakur, 
and graduate student Sivakanthan Kasinathan in the laboratory of Dr. Steven Henikoff (Basic 
Sciences Division) identified DNA sequences associated with kinetochore proteins and introduced 
an unbiased computational approach to characterize functional centromeric 
sequences.  "Conventional sequence assembly methods are unable to map the most homogeneous 
repetitive sequences, leaving gaps in the ~170-bp a-satellite repeat arrays, 'black holes' that each of 
our centromeres is embedded in," said Dr. Henikoff.  "We realized that by isolating the sequences 
bound to centromere-specific chromatin proteins we could identify centromere sequences by 
function. We found that two dimeric alpha satellite units dominate the most homogeneous, and 
therefore the youngest, a-satellite repeats bound by centromere-specific chromatin proteins. 
Remarkably, these dimeric units showed precise positioning of chromatin particles, which protect 
100 base pairs centered over each of the units of the dimer, whereas a-satellite units that have been 
mapped to the edges the gaps showed little if any precise positioning." 
To enrich for presumably functional centromeric sequences, the authors performed chromatin 
immunoprecipitation and high-throughput sequencing (ChIP-seq) for the kinetochore proteins CENP-
A and CENP-C.  Because highly repetitive sequences are difficult to map unambiguously, most 
genome assembly methods skip over them, resulting in missing sequences in the reference  
 
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genome.  As a-satellite arrays are not assembled into the human genome, the authors first aligned 
the DNA associated with these kinetochore proteins to bacterial artificial chromosomes (BACs) 
known to contain a-satellite arrays.  The most homogenous, and therefore youngest, a-satellite 
sequences displayed the most robust enrichment of CENP-A and CENP-C, and decreased 
occupancy by these proteins was associated with increased degeneracy (evolutionary age) of a-
satellite repeats.  The authors thus hypothesized that young, homogenous sequences were 
functional centromere sequences. 
The authors next used a phylogenetic approach to identify the most abundant sequences recovered 
in the CENP-A ChIP-seq to further define functional centromeric sequences.  One group of these 
sequences matched a-satellite arrays from chromosomes 1, 5, and 19, and another group matched 
sequences from chromosomes 13, 14, 21, and 22.  The authors then generated a consensus 
sequence for each group: Cen1-like and Cen13-like.  Several sequences also matched unplaced 
clones from the human genome assembly.  These abundant sequences were composed of two a-
satellite repeats flanking a CENP-B box, the binding site of the only identified mammalian sequence-
specific centromere-binding protein. 
To analyze the effect of a-satellite dimers on chromatin organization, the authors aligned their 
CENP-A and CENP-C ChIP-seq data to the Cen1-like and Cen13-like dimeric consensus 
sequences.  This revealed a striking pattern of CENP-A enrichment, wherein two ~100 bp 'pillars' of 
CENP-A flank a 'pedestal' containing the CENP-B box.  In contrast, alignment of these ChIP-seq 
data to previously the most abundant consensus sequence from old, degenerate HORs revealed a 
different arrangement, with a single poorly positioned particle adjacent to a CENP-B pedestal.  From 
this, the authors concluded that young, but not old, a-satellite arrays precisely position CENP-A 
nucleosomes. 
  
This work strongly supports the idea that young, homogeneous a-satellite arrays are functional 
centromeric sequences and also challenges an established idea about how human centromeres are 
defined.  "Our finding that functional human centromeres are dominated by specific sequences with 
a unique chromatin conformation argues that human centromeres are defined by DNA sequence, in 
contrast to the common perception that they are epigenetically defined," said Dr. Henikoff. 
Henikoff JG, Thakur J, Kasinathan S, Henikoff S. 2015. A unique chromatin complex occupies young 
a-satellite arrays of human centromeres. Sci Adv 1(1):e1400234. 
 
May 18, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 5 | Fred Hutchinson Cancer Research Center 
 
 
 
 
Image from the publication 
(Left) At young a-satellite dimers, such as the Cen1-like consensus, 
two CENP-A containing nucleosomes are precisely positioned, 
protecting ~100 bp of DNA on either side of a CENP-B pedestal. 
(Right) At old a-satellite dimers, such as the D11Z1 HOR, only a 
single poorly positioned CENP-A-containing nucleosome is found 
adjacent to a CENP-B pedestal. 
 


A Genetic Basis for Human Centromeres

July 20, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 7 | Fred Hutchinson Cancer Research Center 
 
A Bird's Eye View of Advanced Prostate Cancer 
July 20, 2015 
     A Neves 
 
A multi-institution integrative sequencing 
analysis of DNA and RNA from 150 metastatic, 
castration resistant prostate cancers reveals 
both known and novel cancer pathways that are 
clinically actionable, an important step towards 
precision medicine. 
 
Image adapted from the publication. 
 
 
 
An estimated 220,000 American men are diagnosed with prostate cancer each year, with 30,000 
expected to die of it this year. While androgen deprivation therapy (ADT) is initially effective against 
metastatic disease, most men develop tumors that ultimately become resistant to ADT, including the 
most recent ADT regimen (abiraterone acetate and enzalutamide). This condition is known as 
metastatic castration-resistant prostate cancer (mCRPC). While many studies have cataloged 
somatic alterations in primary prostate cancers, genomic studies of mCRPC have been limited to 
either autopsy samples or preclinical models. To address the gap in our understanding of mCRPC, 
an international and multi-institutional consortium that included Fred Hutch investigators and 
benefitted from key contributions from Dr. Pete Nelson’s Laboratory (Human Biology, Clinical 
Research and Public Health Sciences Divisions), sequenced the DNA and RNA from biopsies of 
bone or soft tissue metastases prospectively from 150 mCRPC affected individuals. This work was 
recently published in the journal Cell. 
This integrative analysis represented an entry point of ongoing clinical trials on combination therapy 
in mCRPC. The biopsies were obtained from lymph nodes (42%), bone (28.7%), liver (12.7%) as 
well as other soft tissues (16.7%). Compared to primary prostate tumors, mCRPCs were enriched 
for mutations in the Androgen Receptor (AR) and the tumor suppressor TP53. Based on both 
biological and statistical criteria, mCRPCs could be subdivided into discrete molecular subtypes. 
These subtypes were grouped based on knowledge of biological pathways and alteration clustering 
July 20, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 7 | Fred Hutchinson Cancer Research Center 
 
and included, among others, the PI3K pathway, WNT pathway, DNA repair pathway and the cell 
cycle machinery. Accordingly, genes that were most frequently altered were AR (62.7%), genes 
encoding ETS family transcription factors (56.7%), TP53 (53.3%) and PTEN (40.7%). Importantly, 
the metastases of most mCRPC cases harbored alterations deemed to be clinically actionable, 
meaning that they have either diagnostic or prognostic significance or that they predict therapeutic 
response. 
The AR signaling pathway was found to be altered in 73% (107/150) of cases, either by mutation in 
AR itself, or in regulators of AR signaling such as the NCOR1/2, SPOP and ZBTB16 genes. 
Because most mCRPCs have AR pathway mutations, they are likely to be dependent on AR for 
viability. As such, future studies may be able to link specific AR mutations to clinical outcomes. 
Additionally, while mutations in the PI3K pathway are well described in cancer, this study uncovered 
novel alterations in the catalytic subunit PIK3CB, suggesting that patients with PIK3CB alterations 
may benefit from PIK3CB-specific inhibitors.  
Additionally, 19/150 (12.7%) cases harbored alterations in BRCA2, that encodes a DNA repair 
protein best known for its role in inherited breast and ovarian cancer. This prompted further 
examination of the DNA repair pathway, which revealed alterations in other well-known DNA repair 
genes, such as BRCA1 an ATM. Interestingly, previous work has demonstrated that cancers with 
DNA repair mutations might be particularly sensitive to poly ADP ribose polymerase (PARP) 
inhibitors. Overall, this study demonstrates that prospective genomics of advanced metastatic 
prostate cancer is not only feasible, but also enabled discovery of novel mutations that had not been 
observed in multiple prior studies of primary prostate tumors. Finally, this work suggests that 
prospective genomics of metastatic disease in treated individuals could potentially be applied to 
other types of cancer. "We were quite excited to find that a majority of metastatic castration resistant 
prostate cancers (mCRPC) harbor actionable mutations.  The findings suggest there is potential 
clinical utility of genetic testing to guide optimal therapy in the setting of mCRPC.  For example, a 
large number of cases had either somatic or germline DNA repair pathway alterations which may be 
predictive of favorable response to platinum or PARP inhibitor therapy," said Dr. Colin Pritchard, a 
UW investigator involved in the study. 
Robinson D, Van Allen EM, Wu Y-M, Schultz N, Lonigro RJ, Mosquera J-M, Montgomery B, Taplin 
M-E, Pritchard CC, Attard G, Beltran H, Abida W, Bradley RK, Vinson J, Cao X, Vats P, Kunju LP, 
Hussain M, Feng FY, Tomlins SA, Cooney KA, Smith DC, Brennan C, Siddiqui J, Mehra R, Chen Y, 
Rathkopf DE, Morris MJ, Solomon SB, Durack JC, Reuter VE, Gopalan A, Gao J, Loda M, Lis RT, 
Bowden M, Balk SP, Gaviola G, Sougnez C, Gupta M, Yu EY, Mostaghel EA, Cheng HH, Mulcahy 
July 20, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 7 | Fred Hutchinson Cancer Research Center 
 
H, True LD, Plymate SR, Dvinge H, Ferraldeschi R, Flohr P, Miranda S, Zafeiriou Z, Tunariu N, 
Mateo J, Perez-Lopez R, Demichelis F, Robinson BD, Schiffman M, Nanus DM, Tagawa ST, 
Sigaras A, Eng KW, Elemento O, Sboner A, Heath EI, Scher HI, Pienta KJ, Kantoff P, de Bono JS, 
Rubin MA, Nelson PS, Garraway LA, Sawyers CL, Chinnaiyan AM. 2015. Integrative clinical 
genomics of advanced prostate cancer. Cell, 161(5),1215-28. 
 
 


A Bird's Eye View of Advanced Prostate Cancer

August 17, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 8 | Fred Hutchinson Cancer Research Center 
 
ERG finally has something to YAP about in 
prostate cancer 
Aug. 17, 2015  
 
     A Neves 
 
 
 
 
 
A large prostate tumor expressing keratin 8 
(red) in a male transgenic mouse 
overexpressing ERG in prostate epithelial 
cells. A small fragment of normal prostate 
gland expressing keratin 5 in basal cells 
(green) and keratin 8 in luminal cells (red) is 
present at the bottom right corner of the 
image. Cell nuclei are in blue. 
 
Image provided by Dr. Valeri Vasioukhin 
 
 
 
 
 
ERG is a transcription factor of the ETS (erythroblastosis virus E26) family that is rearranged and 
overproduced in approximately 50% of all human prostate tumors. ERG overproduction appears to 
be important because knockdown of ERG in a human prostate cancer cell line (VCaP) that 
overexpresses ERG has been shown to decrease cell invasion, a hallmark of metastasis. 
Conversely, overexpression of ERG in non-cancerous prostate epithelial cells (RWPE-1) results in a 
marked increase in cell invasion. However, whether overexpression of ERG is sufficient for prostate 
cancer development and the potential molecular mechanisms of ERG in prostate cancer have not 
been determined. A new Fred Hutch study from Dr. Valeri Vasioukhin’s Laboratory (Human Biology 
August 17, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 8 | Fred Hutchinson Cancer Research Center 
 
Division), in collaboration with Dr. Pete Nelson’s Laboratory (Human Biology, Clinical Research, and 
Public Health Sciences Divisions), tackled this question by providing a detailed characterization of a 
mouse model wherein ERG is overexpressed in the prostate epithelium at levels comparable to 
those observed in ERG-rearranged human prostate cancers. This study was recently published in 
Cancer Cell. 
The authors first generated a mouse model that expressed clinically relevant levels of ERG 
specifically in the prostate epithelium. They found that these mice were short-lived, and that half of 
them developed late-onset prostate tumors. This finding demonstrated that overproduction of ERG in 
prostate epithelial cells in vivo is sufficient for prostate cancer development, indicating that 
rearrangement and overproduction of ERG plays a causal role in prostate cancer initiation. The 
researchers then analyzed expression of gene targets of major signaling pathways known to 
regulate cancer development, and found that even before tumor development, the ERG-
overexpressing prostate epithelial cells exhibited high activity of YAP1 (Yes-associated protein 1). 
Because YAP1 is a transcriptional regulator and a key effector of the Hippo signaling tumor 
suppressor pathway, the investigators used RNA sequencing to compare the transcriptional profiles 
of RWPE-1 cells overexpressing either ERG, a constitutively active form of YAP1 (YAP1S127A), or 
both ERG and YAP1S127A. Gene set enrichment analysis of the RNA sequencing data revealed 
strong overlap between the ERG and YAP1 transcriptional programs.  
Next, the researchers investigated the functional relevance of YAP1, and its close relative TAZ, 
using a 3D prostate organoid culture system. These experiments revealed that knockdown of either 
YAP1 or TAZ blunted both the proliferation and the invasive properties of human RWPE-1 cells that 
overexpressed ERG. To address the mechanism by which ERG promotes high YAP1 activity, the 
authors performed chromatin immunoprecipitaiton followed by high throughput DNA sequencing 
(ChIP-seq) to identify DNA sequences bound by ERG and TEAD4, a YAP1 binding partner. ChIP-
seq experiments in both VCaP and RWPE-1 cells revealed that ERG and TEAD4 were co-bound at 
many Hippo pathway genes, and that ERG increased both histone acetylation and gene activation at 
these sites. Importantly, 44% (52 of 116) of primary human prostate cancers analyzed co-expressed 
ERG and YAP1, and expression of nuclear YAP1 was linked to tumor recurrence after treatment.  
To evaluate the role of YAP1 in ERG-driven tumors in vivo, the investigators administered an FDA-
approved inhibitor of YAP1 (Verteporfin) to pre-established human orthotopic xenografts tumors, and 
found that Verteporfin treatment suppressed the growth of ERG-positive, but not ERG-negative, 
prostate tumor xenografts. Finally, the authors addressed whether activation of YAP1 was sufficient 
for tumor formation by driving expression of YAP1S127A in the prostate epithelium, which resulted in 
August 17, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 8 | Fred Hutchinson Cancer Research Center 
 
late-onset prostate tumors that were strikingly similar to ERG-driven tumors, both in histology and 
transcriptional profile. 
Said Dr. Vasioukhin, "ERG is overexpressed in ~50% of human prostate cancers, but the role and 
mechanisms of ERG in prostate cancer remained poorly understood. This study demonstrated that 
ERG expression is sufficient to cause prostate cancer development in aged individuals and revealed 
a connection between ERG and the Hippo signal transduction pathway, which can explain the 
molecular mechanisms of ERG in prostate cancer initiation." In conclusion, this important study 
showed that overexpression of either ERG or YAP1 results in late-onset prostate tumors and it 
revealed the mechanism responsible for oncogenic function of ERG. Furthermore, since YAP1 
activity is required for ERG-driven tumors both in vitro and in vivo, it suggests that the YAP1 
antagonist Verteporfin may provide novel therapeutic opportunities. 
Nguyen LT, Tretiakova MS, Silvis MR, Lucas J, Klezovitch O, Coleman I, Bolouri H, Kutyavin VI, 
Morrissey C, True LD, Nelson PS, Vasioukhin V. 2015. ERG activates the YAP1 transcriptional 
program and induces the development of age-related prostate tumors. Cancer Cell, 27(6), 797-808. 
Funding for this study was provided in part by the National Cancer Institute and the Prostate Cancer 
Foundation. 
 
 


ERG finally has something to YAP about in prostate cancer

Frost, Sophia

March 16, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 3 | Fred Hutchinson Cancer Research Center 
 
A For Alphas: Radioimmunotherapy Scores High 
March 16, 2015 
     SHL Frost 
Although the advancement of radioimmunotherapy (RIT) has benefitted patients with non-Hodgkin 
lymphoma to a certain degree, additional improvements are needed to further reduce the relapse 
rates. With RIT, malignant cells are selectively killed using tumor-targeting antibodies labeled with 
radioactive isotopes. This technique is especially well-suited for treatment of B cell lymphomas, due 
to their exquisite radiation-sensitivity. While initial responses to therapy often are encouraging, for 
most patients with advanced stage indolent (slow-growing) non-Hodgkin lymphoma and mantle cell 
lymphoma the benefit is unfortunately merely temporary. The reason is spelled MRD, or "minimal 
residual disease", referring to microscopic clusters of treatment-resistant tumor cells that require 
upgraded therapy for complete eradication. For Fred Hutch's Dr. Damian Green, that upgrade 
comprises alpha particle emitting isotopes, or more precisely, the alpha emitter astatine-211. In a 
recent publication in Blood, Dr. Green and colleagues from the Press Lab in the Clinical Research 
Division demonstrated that complete eradication of disease could be achieved in 70% of mice with 
disseminated lymphoma after treatment with astatine-211-labeled antibodies directed against the 
CD20 antigen. Contrastingly, no animals survived in matching control groups. 
This therapeutic success relied on the physical properties of alpha particle radiation. In contrast to 
the more commonly used beta emitting radionuclides, alpha emitters release particles with very high 
energy combined with extremely short range. In general, alpha radiation emitted from astatine-211 
only travels about 50 to 70 µm in tissue, making it ideal for treating microscopic tumors while 
minimizing the exposure to surrounding healthy tissue. Targeted alpha therapy studies have long 
been hampered by production issues and a lack of efficient radiochemical labeling methods, but 
substantial progress has been made in recent years. "Recently, we have been able to generate 
sufficient quantities of short path-length alpha emitting radionuclides to support clinical applications; 
in addition, our collaborators at the University of Washington have discovered a way to provide 
critical stability to alpha particle-labeled monoclonal antibodies," Dr. Green explained. Furthermore, 
he added that in comparison to first-generation RIT beta emitters like yttrium-90 and iodine-131, 
"alpha emitters have greater than 500 times more energy per unit length, and as few as 1 to 5 alpha 
particle emissions can be sufficient to cause irreparable DNA damage and induce cell death." 
In addition to the MRD model, the Fred Hutch research team explored targeted alpha therapy of 
mice with solid lymphoma tumors (approximately 100 mm
3
 at the start of therapy). In this disease 
March 16, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 3 | Fred Hutchinson Cancer Research Center 
 
setting they saw a radically different outcome; none of the animals were cured, although survival 
time after astatine-211-RIT was prolonged 2–3-fold in comparison with controls. Striking as this was, 
Dr. Green was not all that surprised. "The results confirmed our hypothesis that alpha emitting 
radionuclides would be most effective in micrometastatic or minimal residual disease settings." In 
fact, the outcome validated predictions made through mathematical models that took the features of 
alpha particle radiation into consideration. Depending on the distribution of radiolabeled antibodies 
within the tumor mass, bulky tumors may be more efficiently obliterated using beta radiation; this is 
due to the longer in vivo range of beta particles (up to about 10 mm), which may make up for a 
certain degree of suboptimal targeting. 
"Based on our findings, the best application of targeted alpha emitter therapy is in the clinical setting 
of micrometastatic or minimal residual disease," concluded Dr. Green, who now hopes to translate 
these preclinical results into clinical trials with astatine-211 targeted to B cell lymphomas using anti-
CD20 antibodies. In addition, the findings will support ongoing investigations regarding the use of 
alpha-RIT for treatment of multiple myeloma and other cancers of the blood and bone marrow. 
Green DJ, Shadman M, Jones JC, Frayo SL, Kenoyer AL, Hylarides MD, Hamlin DK, Wilbur DS, 
Balkin ER, Lin Y, Miller BW, Frost SHL, Gopal AK, Orozco JJ, Gooley TA, Laird KL, Till BG, Back T, 
Sandmaier BM, Pagel JM, Press OW. 2015. Astatine-211 conjugated to an anti-CD20 monoclonal 
antibody eradicates disseminated B-cell lymphoma in a mouse model. Blood. pii: blood-2014-11-
612770. [Epub ahead of print] 
 
 Image provided by Dr. Damian Green 
Kaplan-Meier survival curves 
demonstrating the efficacy of 
astatine-211-
radioimmunotherapy in a 
minimal residual disease model. 
Disease progression was 
monitored through 
bioluminescence imaging of 
luciferase-transduced Granta 
cells. Mice received 15 µCi of 
astatine-211 via either the anti-
CD20 antibody 1F5 or a non-
binding control antibody 
(HB8181), or no therapy (n = 10 
mice per group). 


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