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29 MAY 2014
I VOLUME 123, NUMBER 22
l l l RED CELLS, IRON, & ERYTHROPOIESIS
Comment on An et al, page 3466
Of
mice and men…
----------------------------------------------------------------------------------------------------James Palis
UNIVERSITY OF ROCHESTER MEDICAL CENTER
In this issue of Blood, An et al provide human and murine transcriptome data
revealing significant stage- and species-specific differences in gene expression
during erythroblast maturation.1
T
he process of erythroblast maturation is
characterized by a progressive decrease in
cell size, nuclear condensation and ultimate
extrusion (in mammals), and the loss of
cytoplasmic basophilia due, in part, to the
accumulation of hemoglobin. As shown in the
figure, these striking morphological features
have been used by hematologists in the clinic
to classify erythroblasts in disease states and by
investigators in the laboratory to study erythroid
maturation both during development and across
species. The overarching similarities of this
morphological process both in humans and in
mice have implied the existence of similar
molecular mechanisms regulating erythropoiesis
in these organisms and has helped to justify the
use of mouse models to study gene function and
erythroid cell maturation.
Over the last decade, global gene expression
studies have been carried out to better
understand the processes associated with the
synthesis of red cells. These have included,
among several, the characterization of genes
expressed in cultured human erythroblasts
found in Hembase2 and the Human Erythroblast
Maturation database,3 in a cultured murine
Human and murine erythroblasts undergo comparable progressive stages of maturation characterized by changes in
cell size, nuclear condensation, and loss of cytoplasmic basophilia. These features are used to classify the cells as
proerythroblasts (ProE), basophilic erythroblasts (BasoE), polychromatophilic erythroblasts (PolyE), and orthochromatic erythroblasts (OrthoE). All erythroblast images were kindly provided by Dr Xiuli An, New York Blood Center, with
mouse erythroblast images adapted from Liu et al.8 All images were color corrected and cropped by J.P.
BLOOD, 29 MAY 2014 x VOLUME 123, NUMBER 22
erythroid cell line following expression of the
transcriptional regulator Gata1 in G1EDb,4 and
in primary murine erythroblasts directly isolated
from embryos,5,6 as well as from fetal liver and
adult bone marrow in erythronDB.6
Although many of these studies have relied
on flow cytometry to isolate pure populations
of staged erythroblasts, the erythroid lineage,
unlike its lymphoid and myeloid cousins in the
marrow, has lacked a plethora of cell surface
markers to help distinguish progressive stages
of maturation. Expression of glycophorin A
(CD235/Ter119) and the transferrin receptor
(CD71) has been widely used to analyze
erythroblast progression by flow cytometry.
Recent refinements by the authors have
brought band 3 and a4-integrin to the flow
cytometry-based isolation of staged human
erythroblasts7 and CD44 expression to the
isolation of their murine counterparts.8
Using these novel flow cytometric
approaches, An et al analyzed the global gene
expression of cultured human erythroblasts
and primary murine erythroblasts, as they
mature (see figure) from proerythroblast
to orthochromatic erythroblast stages.
Importantly, they find that each specific stage
of erythroblast maturation is characterized by
a unique transcriptome, which complements
the well-recognized morphological differences
evident in these populations. These findings
also support the concept that erythroid
maturation is characterized by cell divisions
that result in 2 daughter cells that differ
markedly from their parent cell. Clearly
erythroblasts can walk (proliferate) and
chew gum (differentiate) at the same time.
The authors also compare the human and
murine erythroid transcriptomes and find both
broad similarities, as well as marked differences,
eg, in the expression of genes associated with the
mitogen-activated protein kinase pathway and
the E3 ubiquitin ligase family of genes. These
dissimilarities add to previously identified
differences between mouse and human
erythropoiesis, most notably the lack of a fetal
3367
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form of hemoglobin in the mouse. A similar
conclusion regarding the marked differences in
gene expression between human and mouse
erythropoiesis has also been reached by an
independent analysis of previously published
gene expression data of human and mouse
erythroblasts isolated by flow cytometry.9
These marked differences in mouse and
human erythroid transcriptomes are surprising
given the morphological similarities that occur in
both species to ultimately form a biconcave disc
containing mostly hemoglobin. There certainly
can be different pathways that achieve the same
end result. However, a caveat of both studies1,9
is that the mouse erythroblasts were isolated
directly from the bone marrow, whereas the
human erythroblasts were derived from
prolonged in vitro culture of neonatal or adult
CD34-positive cells that mature as a cohort.
Because mammalian erythroblasts normally
mature attached to macrophage cells within
erythroblastic islands, it remains to be
determined how many of the species-specific
differences in gene expression might be due to
the in vitro microenvironment of the cultured
human erythroblasts.
Most studies of global erythroid gene
expression have relied on chip-based
technologies. A significant strength of this study
is the use of RNA-Seq (RNA Sequencing) to
generate the gene expression data. For example,
this approach has now permitted the global
analysis of ribosomal genes, which surprisingly
make up a significant proportion of the most
abundantly expressed genes present in
human proerythroblasts and basophilic
erythroblasts. In addition, this approach offers
the possibility of analyzing the expression of
splice variants, including the identification of
novel erythroid-specific variants, as well as
noncoding RNAs during erythroid maturation.
Such analyses of these databases are eagerly
awaited and should certainly lead to new insights
regarding the regulation of human erythropoiesis,
as well as that of its closest model organism.
Conflict-of-interest disclosure: The author declares
no competing financial interests. n
REFERENCES
1. An X, Schulz VP, Li J, et al. Global transcriptome
analyses of human and murine terminal erythroid
differentiation. Blood. 2014;123(22):3466-3477.
2. Miller JL. A genome-based approach for the study of
erythroid biology and disease. Blood Cells Mol Dis. 2004;
32(3):341-343.
3. Merryweather-Clarke AT, Atzberger A, Soneji S, et al.
Global gene expression analysis of human erythroid
progenitors. Blood. 2011;117(13):e96-e108.
3368
4. Welch JJ, Watts JA, Vakoc CR, et al. Global regulation
of erythroid gene expression by transcription factor
GATA-1. Blood. 2004;104(10):3136-3147.
5. Isern J, He Z, Fraser ST, et al. Single-lineage
transcriptome analysis reveals key regulatory pathways in
primitive erythroid progenitors in the mouse embryo.
Blood. 2011;117(18):4924-4934.
6. Kingsley PD, Greenfest-Allen E, Frame JM, et al.
Ontogeny of erythroid gene expression. Blood. 2013;
121(6):e5-e13.
7. Hu J, Liu J, Xue F, et al. Isolation and functional
characterization of human erythroblasts at distinct
stages: implications for understanding of normal and
disordered erythropoiesis in vivo. Blood. 2013;121(16):
3246-3253.
8. Liu J, Zhang J, Ginzburg Y, et al. Quantitative analysis
of murine terminal erythroid differentiation in vivo: novel
method to study normal and disordered erythropoiesis.
Blood. 2013;121(8):e43-e49.
9. Pishesha N, Thiru P, Shi J, Eng JC, Sankaran VG,
Lodish HF. Transcriptional divergence and conservation
of human and mouse erythropoiesis. Proc Natl Acad Sci
USA. 2014;111(11):4103-4108.
© 2014 by The American Society of Hematology
l l l CLINICAL TRIALS & OBSERVATIONS
Comment on Flinn et al, page 3406; and Kahl et al, page 3398; and Brown et al,
page 3390
CLL
and NHL: the end of chemotherapy?
----------------------------------------------------------------------------------------------------Bruce D. Cheson
GEORGETOWN UNIVERSITY HOSPITAL
“The times they are a changin’”—Bob Dylan
In this issue of Blood, Flinn et al, Kahl et al, and Brown et al provide further
encouragement that the possibility of a chemotherapy-free world is, indeed,
a rapidly approaching reality in indolent non-Hodgkin lymphomas (NHLs),
mantle cell lymphoma (MCL), and chronic lymphocytic leukemia (CLL).1-3
T
he therapeutic holy grail for lymphoid
malignancies has long been an effective
chemotherapy-free strategy to avoid the
scourge of the nonspecific toxic drugs that
patients have been subjected to for decades,
such as anthracyclines, alkylating agents, vinca
alkyloids, and purine analogs. Initial forays into
the nonchemotherapy realm included singleagent rituximab,4 doublets of monoclonal
antibodies,5,6 or rituximab combined with the
immunomodulatory agent lenalidomide.7
Each of these well-tolerated regimens
produced high response rates with durable
remissions. Nevertheless, the diseases remain
incurable. Recent interest has focused on
a series of agents that inhibit intracellular
pathways that promote the proliferation and
survival of malignant lymphocytes. Several of
these pathways are activated through B-cell
receptor signaling (see figure). One of the first
such drugs was fostamatinib disodium, a spleen
tyrosine kinase inhibitor that exhibited only
modest activity and is not being actively
pursued in lymphoid malignancies.8
More recently, idelalisib (formerly known
as CAL-101 and GS-1101) has induced
considerable interest. This potent, highly
selective, orally bioavailable small molecule
inhibits the d isoform of phosphatidylinositol
3-kinase (PI3K), a pathway that is overactive
in a number of lymphoid malignancies as well
as solid tumors. PI3K exists in 4 different
isoforms: p110a, -b, -g, and -d, the last
being most relevant to B lymphocytes.
As described in each of the 3 articles,
PI3K-mediated phosphorylation activates
the serine/threonine kinase AKT and,
subsequently, mammalian target of
rapamycin (mTOR). Overexpression of
PI3K/AKT appears to contribute to the
pathogenesis of various lymphoid
malignancies, including indolent NHL,
MCL, and CLL. Inhibiting PI3K results
in cell death through apoptosis.
Rarely have we encountered enthusiasm
for drugs such as that for idelalisib,
which is based on phase 1 data. Flinn et al1
administered idelalisib to 64 heavily
pretreated patients with indolent NHL, more
than half of whom were refractory to previous
chemotherapy. The response rate was 47%,
but it was 69% in those treated at the now
accepted dose of $150 mg twice daily, with
a median progression-free survival (PFS) of
16.8 months at this dose level. These data have
been confirmed in a subsequent phase 2 trial.9
Kahl et al2 treated 40 patients with MCL, with
a response rate of 69% at a dose of $150 mg
BLOOD, 29 MAY 2014 x VOLUME 123, NUMBER 22
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2014 123: 3367-3368
doi:10.1182/blood-2014-04-565457
Of mice and men…
James Palis
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