Bif application simon

Application for PhD Fellowship
Candidate: Carlos López Fernández de Castillejo
1. PhD Research Project: Dissecting the Leukaemic Stem Cell Niche
2. Abstract
Myeloproliferative neoplasms (MPN) are haematological malignancies originated by mutated
hematopoietic stem cells (HSC). They cannot be cured in most cases and are characterised by
overproduction of different blood cells. However, it remains unclear how a common mutation
can cause different clinical outcomes. Moreover, the drugs currently used in patients are only
capable of slowing down the disease, but cannot eradicate it. This suggests that other factors
critically contribute to MPN progression. Our research group has demonstrated specific
alterations of the microenvironment of HSC in MPN. We have demonstrated that mutated HSC
can only expand after damaging nerve terminals and mesenchymal stem cells (MSC) in the
bone marrow, overcoming the control that this environment can exert over mutated HSC
proliferation. We have thus shown a completely novel potential approach to treat these
diseases—e.g. preserving the environment, rather than targeting the mutated cell—. These
results have fructified in a clinical trial awarded as the winner of the Gateway/RTF-CCR/SAKK
Research Grant 2014, in collaboration with Prof. Radek Skoda (Basel University Hospital) and
multiple hospitals in Switzerland. The first specific aim of this project is to validate the efficacy of
this therapy in other MPN cases, including essential thrombocythemia and MPN with
aggravating additional mutations, which will be critical in order to maximize the number of
patients that could benefit from this potential therapy. On the other hand, MPN frequently
develops into acute myeloid lekaemia (AML), which is highly aggressive and exhibits poor
response to chemotherapy. Unlike MPN, our preliminary data suggest that survival of the
leukaemic HSC, responsible for tumour initiation, maintenance and relapse, could depend on
the microenvironment. As a second aim, we propose to investigate the contribution of the
hematopoietic microenvironment to the development of AML and its role in mutant HSC support.
If awarded, this Fellowship could increase our understanding of the biological processes
underlying AML and might lead to the design of more effective therapies for the treatment of
both chronic and acute myeloid leukaemia.

Carlos LF de Castillejo Dissecting the Leukaemic Stem Cell Niche
Abstract
Myeloproliferative neoplasms (MPN) are haematological malignancies originated by mutated
hematopoietic stem cells (HSC). They cannot be cured in most cases and are characterised by
overproduction of different blood cells. However, it remains unclear how a common mutation
can cause different clinical outcomes. Moreover, the drugs currently used in patients are only
capable of slowing down the disease, but cannot eradicate it. This suggests that other factors
critically contribute to MPN progression. Our research group has demonstrated specific
alterations of the microenvironment of HSC in MPN. We have demonstrated that mutated HSC
can only expand after damaging nerve terminals and mesenchymal stem cells (MSC) in the
bone marrow, overcoming the control that this environment can exert over mutated HSC
proliferation. We have thus shown a completely novel potential approach to treat these
diseases—e.g. preserving the environment, rather than targeting the mutated cell—. These
results have fructified in a clinical trial awarded as the winner of the Gateway/RTF-CCR/SAKK
Research Grant 2014, in collaboration with Prof. Radek Skoda (Basel University Hospital) and
multiple hospitals in Switzerland. The first specific aim of this project is to validate the efficacy of
this therapy in other MPN cases, including essential thrombocythemia and MPN with
aggravating additional mutations, which will be critical in order to maximize the number of
patients that could benefit from this potential therapy. On the other hand, MPN frequently
develops into acute myeloid lekaemia (AML), which is highly aggressive and exhibits poor
response to chemotherapy. Unlike MPN, our preliminary data suggest that survival of the
leukaemic HSC, responsible for tumour initiation, maintenance and relapse, could depend on
the microenvironment. As a second aim, we propose to investigate the contribution of the
hematopoietic microenvironment to the development of AML and its role in mutant HSC support.
If awarded, this Fellowship could increase our understanding of the biological processes
underlying AML and might lead to the design of more effective therapies for the treatment of
both chronic and acute myeloid leukaemia.
Introduction
Myeloproliferative neoplasms are a group of haematological disorders initiated by mutations
in the haematopoietic stem cell (HSC) compartment, and characterized by overproduction of
myeloid cells. The most common mutation found in patients who do not carry the Philadelphia
chromosome (formed by translocation of the genes BCR-ABL) is a point mutation in the Janus
Kinase 2 (JAK2) gene, which renders this protein constitutively activated. JAK2 is a tyrosine

kinase protein with important roles in cell survival and proliferation. The mutation JAK2-V617F is
found in 95% patients with polycythemia vera and 50% patients with essential thrombocythemia
and primary myelofibrosis1-4
. A fraction of patients with these diseases (3-10%) show mutations
in the gene encoding the receptor of thrombopoietin (MPL)5
. The majority of patients with
essential thrombocythemia and primary myelofibrosis who do not carry alterations in JAK2 or
MPL show mutations in the gene encoding calreticulin6,7
. Additionally, mutations in ASXL1,
CBL, IDH, IKZF1 and TET2 are also found in MPN patients and are associated with the
development of secondary acute myeloid leukemia (AML) and myelodysplastic syndromes,
among others5
. Acute leukaemias are characterized by high proliferation and survival of
leukaemic cells, together with a differentiation blockade. They can present chromosomal
translocations involving the MLL gene and can be phenotypically divided into acute myeloid
leukaemias (AML), acute lymphoid leukaemias, or mixed lineage leukaemias (MLL). In fact,
MLL translocations occur in the majority (>70%) of infant leukaemias, in 10% of adult AMLs,
and in most of therapy-related AMLs, and they are often characterized by a poor prognosis8
.
MLL fusions can transform normal HSCs into leukaemic stem cells (LSCs), which retain their
quiescence and self-renewal, are resistant to chemotherapy and are considered a major cause
of tumour relapse9
. Also, MPN treatments are currently symptomatic and can only delay disease
progression.
HSCs reside within a specific microenvironment or “niche”3
. The HSC niche provides the
necessary conditions for the regulation, survival, growth, and maintenance of HSCs. Although
the exact composition of the HSC niche is still object of discussion, our group successfully
identified an essential component of the HSC niche: a population of mesenchymal stem cells
(MSCs) that express the intermediate filament protein nestin (hereafter termed nestin+ MSCs).
Nestin+ MSCs localize in close association with HSCs and sympathetic nerve fibres, express
HSC-supporting molecules such as the chemokine CXCL12/SDF-1, and are required for homing
and maintenance of HSCs10,11
. In addition, we have shown that damage to the sympathetic
regulation of nestin+ MSCs is required for the manifestation of myeloproliferative neoplams12
.
These data have provided novel insights into diseases that were previously thought to be
independently driven by mutated HSCs. Sympathetic nerve fibres, supporting Schwann cells
and nestin+
MSCs were consistently reduced in the bone marrow of MPN patients and mice
carrying the JAK2-V617F mutation in HSCs. Unexpectedly, MSC reduction is not due to
differentiation into fibroblasts or osteoblasts, thereby contributing to the abnormal stromal
expansion. It is caused instead by bone marrow Schwann cell death triggered by interleukin-1β

produced by mutant haematopoietic progenitors, sympathetic neural damage and ensuing MSC
apoptosis that is not prevented by the JAK inhibitor ruxolitinib. Nestin+ cells activate the
Schwann cell program in MPN, which might be explained by the neural crest contribution to this
population that we have recently found13
. In turn, MSC loss worsens the disease. In vivo
depletion of nestin+
cells or their production of CXCL12 expanded mutant haematopoietic
progenitors and accelerated MPN progression. In contrast, administration of neuroprotective or
sympathomimetic drugs prevented mutant HSC expansion. Treatment with β3-adrenergic
agonists, which restored the sympathetic regulation of nestin+
MSC10,11
, prevented the loss of
these cells and blocked MPN progression by indirectly reducing the number of LSC. Our results
have demonstrated that damage to the niche, induced by genetically mutated HSC, critically
contributes to disease manifestation in MPN. These results have also identified niche-forming
MSC and their neural regulation as promising therapeutic targets in MPN12
. These results have
also fructified in a clinical trial awarded as the winner of the Gateway/RTF-CCR/SAKK Research
Grant 2014, in collaboration with multiple hospitals in Switzerland.
However, it is likely that a combined future therapy, targeting both the stem and niche cells, will
be ultimately required to eliminate LSCs, but this has not been achieved yet. For instance, JAK
inhibitors have little effect on allele burden, probably because they do not discriminate between
WT and mutant JAK2 and also due to the acquisition of pharmacological resistance14-16
. Further
research on the extrinsic mechanisms that regulate the proliferation and survival of leukemic
stem cell will prove to be a valuable tool for the eradication of LSCs.
We have recently uncovered novel pathways that regulate the proliferation and survival of
normal and leukaemic haematopoietic stem cells. Interestingly, men exhibit higher prevalence of
myeloid neoplasias as compared to women17,18
, but the reasons are unknown. Also, whether
sex hormones directly regulate normal and leukaemic haematopoietic stem cells has remained
largely unknown. Our recent data demonstrates differential expression and specific roles of
oestrogen receptors (ERs) in haematopoietic progenitors. ERα activation directly depleted
multipotent haematopoietic progenitors by apoptosis. In contrast, the selective ER modulator
tamoxifen induced proliferation of quiescent murine long-term HSCs, altered their self-renewal
signature and compromised haematopoietic reconstitution following myeloablation. These
effects persisted in two sets of haematological neoplasias: tamoxifen induced apoptosis of
murine and human MLL-AF9+
blasts, improved chemotherapy of MLL-AF9+
leukaemia and
reduced leukaemic burden in vivo; and tamoxifen alone blocked JAK2V617F
-induced
myeloproliferative neoplasm by restoring normal apoptosis levels in murine and human

malignant cells. These results uncover specific regulation of haematopoietic progenitors by
oestrogens and potential anti-leukaemic properties of selective ER modulators (Sánchez-
Aguilera A. et al. Cell Stem Cell, under review).
The first specific aim of this project is to validate the efficacy of this therapy in other MPN cases,
including essential thrombocythemia and MPN with aggravating additional mutations, which will
be critical in order to define suitable patients and maximize the number of patients that could
benefit from this potential therapy.
Analogous to HSCs, it is highly probable that AML LSCs require a specific niche for their
maintenance and proliferation. However, the composition of such niche and the extracellular
signals involved in the interaction between LSC and niche remain to be described. Furthermore,
it is not even clear whether the LSC niche is actually similar or different from the niche that
supports non-malignant HSCs. A thorough characterization of the components, the extracellular
signals, and the precise functions of the leukemic stem cell niche will be vital in our
understanding of the pathogenesis, progression, prognosis, and therapeutic treatment of
leukemia, and, more broadly, of myeloproliferative diseases. Our preliminary data suggest that
AML LSCs modify nestin+ MSC to favour their own survival but depend afterwards on survival
signals from these cells. The second specific aim will dissect these interactions, which might
offer new potential therapeutic targets in AML.
RESEARCH GOALS
The research line of my PhD project will focus on the study and characterization of the
leukaemic stem cell niche. The following are the two main research goals that I propose to
achieve by the end of my PhD.
1. New treatments of myeloproliferative neoplasms based on novel neuroendocrine
regulation of bone marrow stem cells.
We will study whether treatment with tamoxifen or β3-adrenergic agonists might have beneficial
effects in other types of MPN. For that purpose we will perform similar studies, as described
before12
, using a mouse model of essential trombocythemia (Vav1-cre;JAK2V617F
) and Mx1-
cre;JAK2V617F
mice with additional loss of function of Tet methylcytosine dioxygenase 2 (Tet2-
floxed), found in many MPN patients. We have obtained these models from our collaborator
Prof. Radek Skoda (Basel University Hospital). Mice will be chronically treated with these drugs
or vehicle upon disease manifestation, will be monitored and analyzed as previously

described12
. These results might increase the number of patients that could potentially benefit
from this promising novel therapy.
Our data suggest that JAK2V617F
HSCs are more sensitive than their normal counterparts to
tamoxifen’s effect, mainly based on the reduced mutant clone chimerism in competitive
transplantation experiments. We will perform additional competitive repopulation assays using
limiting dilutions of mutated cells, given their higher proliferation and survival. We will dissect the
molecular mechanism by which tamoxifen can abrogate the survival advantage of LSCs in
myeloproliferative neoplasms. Regulatory feedback loops between JAK2 and ER-α have been
described in breast cancer19
, but they seem to be cell type and context-dependent and the
underlying mechanisms are not clear yet. It is thought that increased STAT5 phosphorylation in
mutated JAK2V617F
HSCs contributes to reduced apoptosis levels in these cells. Thus, we will
determine whether tamoxifen treatment interferes with STAT5 phosporylation in JAK2V617F
HSCs. For that purpose, LSK cells will be isolated from tamoxifen/vehicle-treated mice and
Western Blot will be performed using commercial antibodies for STAT5, p-STAT5 and β-actin. If
reduced p-STAT5 levels are detected upon tamoxifen treatment, it is likely that ER-α affects the
expression of JAK2V617F
. We will then measure normal mouse Jak2 and mutant human
JAK2V617F
expression by qPCR in LSK cells from tamoxifen/vehicle-treated mice.
Caveat and alternative strategies: a target of JAK2, phospholipase 2A (PP2A), inactivates AKT
and ERK, and allows for normal apoptosis by inactivating anti-apoptotic proteins (such as Bcl-2,
Bcl-XL and Mcl-1) and by activating pro-apoptotic proteins (like BAD, caspase-4 and BIM).
PP2A is inactivated by the SET-SETBP1 complex, which is continuously targeted for
ubiquitinylation and proteasome degradation, allowing for normal apoptosis. If JAK-STAT
signalling does not show differences in tamoxifen-treated HSCs, we will study PP2A and SET-
SETBP1 activity.
2. Role of mesenchymal stem cells in acute myeloid leukaemic stem cell maintenance.
While bone marrow biopsies from different MPN patients showed reduced nestin+
cells12
, this
was not the case of samples from patients with AML (Fig. 1).

Figure 1. Nestin+
vascular cells are present in the bone marrow of patients with acute
myeloid leukaemia (AML). Representative immunohistochemistry of bone marrow biopsies of
three AML patients using antibodies against CD34 (red) to label leukaemic blasts and vascular
endothelial cells, and NESTIN (brown) to label NESTIN+ cells.
We have obtained from our collaborator Prof. Jürg Schwaller (Basel University Hospital,
Switzerland) an inducible mouse model of MLL-AF9 AML. Transplantation of mutant BM cells
into Nes-gfp mice also does not show a reduction in GFP+
cells, reproducing the clinical
observation (not shown). Also, while genetic depletion of nestin+
cells accelerated MPN
development12
, it reduces instead the number of MLL-AF9+
cells in the mouse AML model (Fig.
2a-b). These results clearly show striking differences in the role of the microenvironment in
acute and chronic myeloid leukaemia. We will perform a comparative study to unravel these
differences. Genetic depletion of nestin+
cells is able to reduce one week later AML burden,
suggesting that nestin+
cells are required for the survival of primitive leukaemic cells. It is also
well known that AML progenitors cannot survive alone in culture, suggesting that they depend
on microenvironmental cues for their maintenance. Intriguinly, we found that the leukaemic
population that shows the highest expansion in this AML model has the immunophenotype lin-
sca-1-
c-kitlow
(Fig. 2c). Among primitive leukaemic cells, this is also the population that is mostly
reduced after nestin+
cell depletion (Fig. 2d).
We will perform a combined transcriptomic and proteomic screening to identify survival factors
produced by nestin+
cells that are required for AML maintenance. Intriguinly, depletion of nestin+
cells in leukaemic mice does not affect normal haematopoietic progenitors but only their
leukaemic counterparts, as shown by competitive transplantation experiments (Fig. 2a-b).
These results suggest that AML cells might transform their microenvironment, and more
precisely nestin+
MSCs, to favour their own survival. We have generated Nes-gfp;Rosa26-
rtTA;MLL-AF9 mice and will induce the disease with doxycycline administration in drinking
water. We will sacrifice the mice upon disease manifestation in peripheral blood counts. BM
CD45-
CD31-
Ter119-
Nes-GFP+
cells will be isolated from control and leukaemic mice and RNA

will be extracted using the Arcturus Picopure RNA isolation kit (Life Technologies). RNA will be
amplified and prepared for RNA-Seq using the Ovation RNA-Seq System v2 (NuGEN) following
the manufacturer’s recommendations. The RNA sequencing library will be prepared with the
TruSeq RNA Sample Preparation v2 Kit (Illumina, San Diego, CA) to construct index-tagged
cDNA. The quality, quantity and the size distribution of the Illumina libraries will be determined
using the DNA-1000 Kit (Agilent Bioanalyzer). Libraries will be sequenced on the Genome
Analyzer IIx (Illumina) following the standard RNA sequencing protocol with the TruSeq SBS Kit
v5. Fastq files containing reads for each library will be extracted and demultiplexed using
Casava v1.8.2 pipeline. Sequencing adaptor contaminations will be removed from reads using
cutadapt software tool (MIT) and the resulting reads were mapped and quantified on the
transcriptome (NCBIM37 Ensembl gene-build 65) using RSEM v1.1734
.
We will perform RNAseq also in lin-
sca-1-
c-kitlow
cells isolated from the bone marrow of
leukemic mice with/without depletion of nestin+
cells. Comparison of the list of genes and gene
ontology pathways differentially expressed by Nes-GFP+
cells from control and leukaemic mice,
and by leukaemic haematopoietic progenitors in mice with/without nestin+
cell depletion, will
provide a first list of candidate survival signals that are induced by leukaemic cells in MSCs to
favour their own survival.
We will cross this list with proteins differentially expressed in the BM supernatant of AML mice
with/without depletion of nestin+
cells. Briefly, Nes-creERT2
;iDTA and control recipient mice will
be transplanted with Rosa26-rtTA;MLL-AF9 cells and treated after one week with doxycycline in
drinking water, and tamoxifen diet to deplete nestin+
cells. We will sacrifice the mice upon the
first signs of disease in peripheral blood counts. BM from femora and tibiae will be gently
flushed in minimal volume, and BM supernatant will be collected after centrifugation for
secretome proteomics analyses, as we previously described20
, with the exception of a previous
filtration with columns that deplete the supernatant from the most abundant proteins, to increase
the yield. We expect to detect qualitative differences in the BM in vivo secretome upon nestin+
cell depletion.

rtTA;MLL-AF9
CD45.1+
(106 cells) Nes-CreERT2;
R26lacZbpAfloxDTA
dox
WT
CD45.2+
(106 cells)
+
R26lacZbpAfloxDTA
(controls)
tamoxifen
elimination
of Nes+ cells
Nes+ cells intact
1 wk 4 wk
dox
tamoxifen
1 wk 4 wk
Leukemia development?
a
b c
d
N = 18 (combined data from 3 independent experiments) * p < 0.05
(gated on lin-)
LSKLK
ckitlow
LSKLK
ckitlow
BM CD45.1 LSK
0
20
40
60
80
BM CD45.1 ckit(low)
0
1000
2000
3000
4000
5000
CD45.1+ LSK cells (x103) CD45.1+ LK cells (x103) CD45.1+ ckitlow cells (x103)
p = 0.07
control NesCre;DTA control NesCre;DTA control NesCre;DTA
BM CD45.1 LK
0
100
200
300
400
BM CD45.2 LSK
0
20
40
60
80
100
BM CD45.2 LK
0
100
200
300
400
BM CD45.2 ckit(low)
0
100
200
300
400
control NesCre;DTA control NesCre;DTA control NesCre;DTA
CD45.2+ LSK cells (x103) CD45.2+ LK cells (x103) CD45.2+ ckitlow cells (x103)
Figure 2. Nestin+
cells maintain acute myeloid leukaemic progenitors. a, Experimental
paradigm used to analyse leukaemia development upon in vivo elimination of nestin+
cells. b,
Depletion of nestin+
cells selectively reduces primitive leukaemic cells. BM nucleated cells, MLL-
AF9+
/WT lin- or total nucleated cells in mice sacrificed upon leukocytosis. c-d, Leukaemic
myeloid progenitors show the highest expansion and are mostly reduced by nestin+
cell
depletion. MLL-AF9+
/WT haematopoietic progenitors (LSK; lin-
sca-1+
c-kit+
) and myeloid
progenitors (LK; lin-
sca-1-
c-kit+
and lin-
sca-1-
c-kitlow
). d, Representative FACS plots of
leukaemic (blue) and WT (orange) haematopoietic progenitors (right). Unpaired two-tailed t test.

Caveat and alternative strategy: AML is frequently characterised by a blast crisis, which means
that the BM is often fully infiltrated of leukaemic cells before a clear and rapid expansion of
myeloid cells can be observed in peripheral circulation. Therefore, the vast amount of proteins
secreted by leukaemic cells in the BM might mask the comparatively less represented BM
stroma-derived secretome. If necessary, we will perform similar proteomics analyses in a
xenograft model of human AML, which will allow us to discriminate, based on the species of
origin, leukaemic- (human) and BM stromal-derived (mouse) secreted proteins. BM samples will
be obtained under written consent from patients carrying the AML-AF9 fusion protein. CD34+
cells will be isolated immunomagnetically and injected intrafemorally into NSG mice sublethally
irradiated. Peripheral blood counts will be monitored every 2 weeks and mice will be sacrificed
upon first signs of disease in peripheral circulation. BM supernatant will be harvested and
secretome proteomics analyses will be performed as described above.
The combined transcriptomic and secretome proteomic analyses will provide a list of candidate
factors to test using a novel co-culture system. We have devised new culture conditions that
allow for the isolation and expansion of murine and human primitive MSC. We have devised a
simple way to isolate and culture them in novel conditions that preserve better their primitive
features, including their capacity to support haematopoietic stem cells20
. In these conditions,
MSCs are grown as mutipotent non-adherent spheres, hereafter termed mesenspheres. To
dissect the interactions of MSC with leukaemic cells, we have developed a coculture system in
which we can plate together leukaemic MLL-AF9 blasts and mesenspheres. The addition of
mesenspheres to the culture has striking anti-apoptotic effects (see Fig. 3), statistically and
significantly increasing the mean survival of the blasts, regardless of the addition of cytokines
normally used to grow these leukemic cells (67.15 ± 15.19% vs. 64.20 ± 19.26%). To
investigate whether this pro-survival effect could be mediated by cell-cell contact between the
leukaemic blast and the mesenspheres, we used transwells, which allow cells to share the
same culture medium while blocking physical contact. The pro-survival effect persisted in
transwell culture, as we have previously reported for normal HSCs20
(mean survival of blasts =
71.70 ± 2.40% with mesenspheres vs 37.35 ± 12.66 % without mesenspheres). Furthermore,
the use of conditioned media did not alter the beneficial effects of the mesenspheres, as the
differences in mean survival percentage were not significantly different between blasts cultured
in non-conditioned or conditioned media. Conditioned media was collected by saving the
extracellular fluid obtained from cocultured of blasts and mesenspheres. Conditioned media
remained frozen at -80ºC until needed. Combining the data obtained from the three

experimental strategies, it is clear that the mesenspheres are secreting an extracellular factor
out to the media that causes leukaemic blasts to survive better than when cultured without
mesenspheres. In addition, mesenspheres from each experiment were collected and saved for
RNA analysis by RNA-seq. The proteomics and transcriptomics analyses of extracellular fluid
obtained from cocultures together with the in vivo depletion analyses will provide us with a list of
candidates that we will test in our in vitro model.
References
1 James, C. et al. A unique clonal JAK2 mutation leading to constitutive signalling causes
polycythaemia vera. Nature 434, 1144-1148, doi:10.1038/nature03546 (2005).
2 Baxter, E. J. et al. Acquired mutation of the tyrosine kinase JAK2 in human
myeloproliferative disorders. Lancet 365, 1054-1061, doi:10.1016/S0140-6736(05)71142-
9 (2005).
3 Levine, R. L. et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera,
essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 7,
387-397, doi:10.1016/j.ccr.2005.03.023 (2005).
4 Kralovics, R. et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N
Engl J Med 352, 1779-1790, doi:10.1056/NEJMoa051113 (2005).
5 Tefferi, A. Novel mutations and their functional and clinical relevance in myeloproliferative
neoplasms: JAK2, MPL, TET2, ASXL1, CBL, IDH and IKZF1. Leukemia 24, 1128-1138,
doi:10.1038/leu.2010.69 (2010).
6 Klampfl, T. et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N
Engl J Med 369, 2379-2390, doi:10.1056/NEJMoa1311347 (2013).
7 Nangalia, J. et al. Somatic CALR Mutations in Myeloproliferative Neoplasms with
Nonmutated JAK2. New England Journal of Medicine 0, null,
doi:doi:10.1056/NEJMoa1312542.
8 Krivtsov, A. V. & Armstrong, S. A. MLL translocations, histone modifications and
leukaemia stem-cell development. Nat Rev Cancer 7, 823-833, doi:10.1038/nrc2253
(2007).
9 Cozzio, A. et al. Similar MLL-associated leukemias arising from self-renewing stem cells
and short-lived myeloid progenitors. Genes Dev 17, 3029-3035,
doi:10.1101/gad.1143403 (2003).
10 Mendez-Ferrer, S., Lucas, D., Battista, M. & Frenette, P. S. Haematopoietic stem cell
release is regulated by circadian oscillations. Nature 452, 442-447,
doi:10.1038/nature06685 (2008).
11 Mendez-Ferrer, S. et al. Mesenchymal and haematopoietic stem cells form a unique
bone marrow niche. Nature 466, 829-834, doi:10.1038/nature09262 (2010).
12 Arranz, L. et al. Neuropathy of haematopoietic stem cell niche is essential for
myeloproliferative neoplasms. Nature 512, 78-81, doi:10.1038/nature13383 (2014).
13 Isern, J. et al. The neural crest is a source of mesenchymal stem cells with specialized
hematopoietic stem-cell-niche function. eLife 3, doi:10.7554/eLife.03696 (2014).
14 Mullally, A. et al. Physiological Jak2V617F expression causes a lethal myeloproliferative
neoplasm with differential effects on hematopoietic stem and progenitor cells. Cancer
Cell 17, 584-596, doi:10.1016/j.ccr.2010.05.015 (2010).

15 Koppikar, P. et al. Efficacy of the JAK2 inhibitor INCB16562 in a murine model of
MPLW515L-induced thrombocytosis and myelofibrosis. Blood 115, 2919-2927,
doi:10.1182/blood-2009-04-218842 (2010).
16 Koppikar, P. et al. Heterodimeric JAK-STAT activation as a mechanism of persistence to
JAK2 inhibitor therapy. Nature 489, 155-159, doi:10.1038/nature11303 (2012).
17 Cancer Facts & Figures 2013, A.C. Society, ed. (Atlanta). (2013).
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Carlos López Fernández de Castillejo
3. Reasons for wishing to work with a particular group
The Stem Cell Niche Pathophysiology laboratory headed by Dr Simon Mendez
Ferrer provides me with the appropriate personal and professional environment for me
to develop my predoctoral research. The PI is a young and ambitious leader who has
made himself a name in the stem cell research world. During his postdoctoral stay in
Mount Sinai, New York, his research culminated in a series of articles, two of which
were published in Nature. Moreover, his discovery of Nestin+
mesenchymal cells as key
regulators of the haematopoietic stem cell niche has provided paradigm-shifting insights
into the regulation of stem cells. His efforts were rewarded by the Howards Hughes
Medical Institute with the prestigious grant Early Career Scientist. After he came back
to Spain to establish his lab at CNIC, Madrid, Dr Mendez Ferrer has continued to thrive
in the scientific world and the laboratory’s research continues to be published in the best
journals.
Besides having a renowned and highly competent Principal Investigator, the
laboratory is composed of four postdoctoral researchers and two graduate students.
Their experience in the field and their ambition will definitely be a huge positive factor
in pushing me to achieve the very best out of my own research projects.
Finally, the research center CNIC can be considered a top-class institution with
state-of-the-art facilities and equipment that will expose to an incredible variety of
opportunities in terms of the techniques that I will be able to put into practice for my
research. The skillset and tools that this center has to offer will without doubt serve me
for the rest of my career in biomedical research.

To whom it may concern:
I hereby confirm that Carlos López Fernández de Castillejo is enrolled in the
Advanced Biotechnology Master program offered by Universidade da Coruña.
Carlos López Fernández de Castillejo has successfully completed 60 ECTS
(European Credit Transfer System) in the program during the 2013-14 academic
year. The completion of the 60 ECTS grants him access to the PhD admission
process.
A Coruña, 30 September 2014
Signature:
Manuel Becerra Fernández
Coordinator of the Advanced Biotechnology Master

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AJISUEAII{Í-IAUO}TE

5. List of Publications:
The following is a list of publications in which I appear as author. All of them originate from my
work as an undergraduate student at Emory University. Even though the topics of research do
not correspond to the topic of my predoctoral research, I do believe that having several peer-
reviewed publications exemplifies my passion and determination to pursue a career in science.
1. Food plant-derived disease tolerance and resistance in a natural butterfly-plant
parasite interaction
Eleanore Sternberg, Thierry Lefèvre, James Li, Carlos Lopez Fernandez de Castillejo, Hui
Li, Mark Hunter & Jacobus de Roode . Evolution 66(11): 3367-76. [2012]
2. Behavioural resistance against a protozoan parasite in the monarch butterfly
Thierry Lefèvre, Allen Chiang, Mangala Kelavkar, Hui Li, James Li, Carlos Lopez
Fernandez de Castillejo, Lindsay Oliver, Yamini Potini, Mark Hunter & Jacobus de Roode.
Journal of Animal Ecology 81(1): 70-79. [2012]
3. Virulence evolution in response to anti-infection resistance: toxic food plants can
select for virulent parasites of monarch butterflies
Jacobus C. de Roode, Carlos Lopez Fernandez de Castillejo, Tyler Faits & Samuel Alizon
Journal of Evolutionary Biology 24(4): 712-722. [2011]

Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III
Melchor Fernandez Almagro 3, E-28029 Madrid, España · Tel. 91 453 12 00 | Fax. 91 453 12 40 · CIF. G82316753
Cardiovascular Development and Repair Department
Fundación Centro Nacional de Investigaciones
Cardiovasculares Carlos III (CNIC)
C/ Melchor Fernández Almagro, 3
28029 Madrid, Spain
Tel.: + 34 914531200
Fax: + 34 914531240
Boehringer Ingelheim Fonds
Stiftung für medizinische
Gurndlagenforschung
Re: Recommendation letter for Mr. Carlos López application
Madrid, September 30th
, 2014
Dear Members of the Board of Trustees:
I write this letter to enthusiastically express my strongest possible support for Mr. Carlos López
application for a PhD Fellowship from Boehringer Ingelheim Fonds.
Mr. López has an extraordinary academic record, personal and professional qualities and specific
lab expertise (despite the early stage of his career) that I believe make him an outstanding candidate
for this Fellowship. We would be extremely happy to accept him as a PhD student in our group so
that he can develop the experimental research project outlined in his proposal.
Mr. López completed his Bachelor in Biology in Emory University (Atlanta, GA, USA), where he
obtained top qualifications, receiving the Dean's List Award for academic excellence. He obtained
several competitive US fellowships that allowed him to participate in summer research programs
and train in the laboratory of Dr. Jacobus de Roode. He decided to return to Spain (where he was
born) for family reasons and is currently finishing his Diploma in Advanced Biotechnology in the
University of La Coruña (Spain), where he is originally from. He will finish his Diploma by La
Coruña University in the upcoming months and has already obtained sufficient credits to apply for a
PhD program in Spain.
We were fortunate that Mr. López decided to apply for a summer stay at the National
Cardiovascular Research Center (CNIC), where our group is based. He wisely chose our institute as
a Center of Excellence in Research (Severo Ochoa Award) and applied to my group based on his
interest in our research track. Over the past 10 years, and based on my PhD background in
neurobiology, we have been interested in the neural regulation of peripheral stem cell niches. We
showed that the brain regulates the bone marrow stem cell niche via the sympathetic nervous
system (Mendez-Ferrer S et al. Nature 2008) and that mesenchymal stem cells expressing the
intermediate filament protein nestin play a key role in regulating haematopoietic stem cells
(Mendez-Ferrer S et al. Nature 2010; Isern J et al. Cell Rep 2013). This association with the
peripheral nervous system is further emphasized by a common origin for peripheral neuroglia and
mesenchymal stem cells with specialized niche functions (Isern J et al. eLife 2014). We have
recently shown that this regulatory network is lost during myeloproliferative diseases, as a
consequence of the inflammatory insult caused in the bone marrow by the mutated haematopoietic
stem cells. However we have also demonstrated that rescue of this neuropathy can rescue the niche,
prevent the expansion of mutated cells and the development of fibrosis (Arranz et al, Nature 2014).
This work has been possible thanks to competitive international funding, such as the International
Early Career Scientist grant of the Howards Hughes Medical Institute. These results have fructified
in a clinical trial awarded as the winner of the Gateway/RTF-CCR/SAKK Research Grant 2014, in
collaboration with multiple hospitals in Switzerland.

Melchor Fernandez Almagro 3, E-28029 Madrid, España · Tel. 91 453 12 00 | Fax. 91 453 12 40 · CIF. G82316753
Cardiovascular Development and Repair Department
Fundación Centro Nacional de Investigaciones
Cardiovasculares Carlos III (CNIC)
C/ Melchor Fernández Almagro, 3
28029 Madrid, Spain
Tel.: + 34 914531200
Fax: + 34 914531240
Mr. López wants to build on our recent work to study the leukemic stem cell niche in the bone
marrow. Normal and leukaemic haematopoietic stem cells are regulated by a specialized
microenvironment (“niche”) which contributes to maintain them in a slowly proliferative state that
makes them more resistant to chemotherapy, a major cause of relapse. His proposal (which he has
written with very little help) builds on our new findings that challenge current dogmas in this field:
the identification of different mesenchymal stem cells with non-overlapping functions (skeletal and
hematopoietic stem cell maintenance); cell identity might be reversible in myeloproliferative
neoplasms, where the change of cell fate is pathogenic and can be reverted; our discovery that
mutated HSCs, considered to drive myeloproliferative neoplasms autonomously, can be controlled
by the niche, and are also sensitive to long-distance neuroendocrine signals that could be exploited
for the treatment of myeloproliferative diseases. A main aspect of his proposal builds on his
discovery—as a summer student in our lab—that leukemic stem cells change nestin+ mesenchymal
stem cells to favor their own survival. Mr. López has found that leukemic stem cells depend on
survival signals produced by nestin+ msenchymal stem cells. He has proposed a sophisticated
approach to discover the key survival signals that might represent novel therapeutic targets for the
treatment of these devastating incurable diseases.
Mr. López came highly recommended by his previous advisor and our interactions with him have
surpassed our best expectations. Mr. López is a brilliant student with a high degree of curiosity and
an outstanding commitment to science. He possesses the rare combination of perfectionism in
experimental research with ambition, dedication and productivity. He is a great team player but has
also a clear vision towards which he wants to move his scientific career, despite his early stage. I
would be delighted to accept him as a PhD student. If awarded, the Fellowship from Boehringer
Ingelheim Fonds would critically impact his career and the possibilities of developing this project.
Please do not hesitate if you need any further information for his evaluation.
With best wishes,
Simón Méndez Ferrer, PhD
(CNIC) Planta 3 Sur
Calle Melchor Fernández Almagro, 3
28029, Madrid
Tel 91 453 12 00, Ext. 3310
Fax 91 453 13 04
E-mail: smendez@cnic.es

Curriculum Vitae
PERSONAL INFORMATION
Title: Mr. Name: Carlos López Fernández de Castillejo Date of Birth: 28 / 12 / 1989 Gender: Male
Nationality: Spanish Phone: (+34) 619 32 17 94 DNI: 47401745X
Email: clopezf25@gmail.com
EDUCATION
University of La Coruña La Coruña, Spain
Master’s Programme in Advanced Biotechnology Class of 2014
Emory University Atlanta, GA (USA)
Bachelor of Science in Biology Class of 2012
GPA: 3.74 / 4.0
“Dean’s List” Award for Academic Excellence
RESEARCH EXPERIENCE
Research Laboratory at Spanish Cardiovascular Research Center (CNIC) Madrid
Predoctoral Research Student
September 2014 - Present
 Principal investigator: Dr Simón Méndez Ferrer
 Research focus: study of the pathophysiology of the hematopoietic stem cell niche; regulation of the stem
cell niche mediated by the mesenchymal stem cells.
Research Laboratory at Institute of Health Sciences La Coruña, Spain
Volunteer Research Student January 2014 - May 2014
 Principal investigator: Dr Alexander Mikhailov
 Research focus: study of arrhythmias and the transcription factors involved in the regulatory events
Occurring after myocardial infarction using the neonatal piglet as a model
Research Laboratory at Emory University Atlanta, GA (USA)
Research Assistant March 2008 – October 2010
 Principal investigator: Dr Jacobus de Roode, listed in the 2011 “Brilliant 10”
top scientist under 40 by the American magazine Popular Science
 Research focus: host-pathogen interactions of the parasitic protozoan Ophryocystis elektroscirrha
and its host, the monarch butterfly Danaus plexippus. The evolution of virulence in
host-pathogen interactions
 Assisted in the design, development, and analysis of various experiments

Field Research Experience Michoacán, Mexico
Field Research Assistant February 2009
 Principal investigator: Dr Jacobus de Roode
 Participated in field work for the collection and analysis of butterfly and protozoan samples
to use in future lab experiments and for evaluation of the ecology of the monarch butterfly
Research Laboratory at New York University New York, NY
Volunteer Research Student June-July 2010
 Principal investigator: Dr Wendy Suzuki
 Assisted with research projects on the organization of memory on the medial temporal lobe
using the Rhesus macaque as the system of study. I was mainly involved in the care, feeding,
and training of the macaques that were used for the experiments
TECHNIQUES
 RT-PCR, qPCR, primer design
 Eukaryotic and bacterial cell culture
 Confocal microscopy
 RNA and DNA extraction, purification
 Handling of mice
 Western blot
 Immunochemistry
 Flow cytometry
PUBLICATIONS
 Food plant-derived disease tolerance and resistance in a natural butterfly-plant parasite interaction
Eleanore Sternberg, Thierry Lefèvre, James Li, Carlos Lopez Fernandez de Castillejo, Hui Li, Mark Hunter & Jacobus
de Roode
Evolution 66(11): 3367-76. [2012]
 Behavioural resistance against a protozoan parasite in the monarch butterfly
Thierry Lefèvre, Allen Chiang, Mangala Kelavkar, Hui Li, James Li, Carlos Lopez Fernandez de Castillejo, Lindsay
Oliver, Yamini Potini, Mark Hunter & Jacobus de Roode
Journal of Animal Ecology 81(1): 70-79. [2012]
 Virulence evolution in response to anti-infection resistance: toxic food plants can select for virulent parasites of
monarch butterflies
Jacobus C. de Roode, Carlos Lopez Fernandez de Castillejo, Tyler Faits & Samuel Alizon
Journal of Evolutionary Biology 24(4): 712-722. [2011]
CONFERENCES AND POSTER PRESENTATIONS
 6th International Conference on Myeloproliferative Neoplasms [Estoril, October 2014]
- Organized by the European School of Haematology
- Topics include: The genomics and genetics of MPNs, Moilecular and cellular pathogenesis of MPNs,
Diagnosis , classification and molecular monitoring, JAK2 inhibitors and novel therapies in MPNs
 SIRE Emory Program Poster Presentation:
- “The effect of host plant on the longevity of the adult monarch butterfly” [Atlanta, 2009]

 SURE Emory Program Poster Presentation:
- “The interaction between the host plant and a protozoan parasite of the monarch butterfly” [Atlanta, 2009]
- Awarded 2nd
prize for best presentations in the area of Life Sciences
 Emory Undergraduate Research Symposium:
- “Testing the effect of host plant species on the virulence of a protozoan parasite” [Atlanta, 2010]
COURSES
 Integral Diagnosis in Haematology [Madrid, September 2014]
- Organized by the Ramon y Cajal Hospital
- Topics include: Myeloproliferative Neoplasms
AWARDS / GRANTS / SCHOLARSHIPS
 CNIC CICERONE 2014 Program Scholarship:
- Merit-based studentship to work in a research laboratory during the summer at the Spanish National
Cardiovascular Research Center
 Emory University SIRE Program (Scholarly Inquiry and Research at Emory):
- I was awarded an stipend to conduct research at my University during the 2008-09 academic year
in the lab of Dr. Jacobus de Roode
 Emory University SURE Program (Summer Undergraduate Research at Emory):
- Included a 3000$ grant to participate in a summer research program during 2009
 Banco Santander Master’s Program Scholarship:
- Merit-based 2500€ scholarship designed to finance post-graduate studies in Spain
during the 2013-14 academic year
 Phi Eta Sigma Fraternity Member:
- American honour society that acknowledges outstanding scholastic achievement at University
OTHER SKILLS
 Native level in written and oral English
 Native level in written and oral Spanish
 Proficient with Microsoft Office
 Advanced level in SPSS and ImageJ

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