‫تقرير نهائي لمشروع بحث‬
Research project final report /
Rapport final du projet de recherche
1
2014 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2014
Programme de subvention à la recherche scientifique au Liban – 2014
‫مستند إداري‬
Administrative Document
--------
Administrative information / ‫المعلومات اإلداري‬
:‫المرجع‬
Project Title - )‫عنوان المشروع (عربي وأجنبي‬
Understanding the molecular mechanisms of action of targeted therapies on the
eradication of leukemia stem cells in HTLV-I associated adult T-cell
leukemia/lymphoma
.‫فهم ﺂلية العﻣﻝ للعالجات الموجهة لسرطان الدم والغدد اللمفاوية ﻋﺑﺭ الﻗﺿاﺀ ﻋلﻰ الﺨﻻﻴا الجﺫﻋﻴة السرطاﻧﻴة‬
Caractérisation moléculaire du mécanisme d’action des thérapies
spécifiques ciblant les cellules souches leucémiques de la leucémie T de
l’adulte associée au rétrovirus HTLV-1
Principal Investigator - ‫الباحث الرئيسي‬
Ali Bazarbachi, MD, PhD
Professor of Medicine,
Hematology and Oncology
Associate Dean for Basic
Research, Faculty of
Medicine
Director, Bone Marrow
Transplantation program
American University of
Beirut
bazarbac@aub.edu.lb
‫العنوان‬
Dr Ali Bazarbachi
Address
‫العنوان االلكتروني‬
Name &
surname
AUB
‫المؤسسة‬
Institution
Professor of Medicine
‫الوظيفة‬
e-mail
01 350000 ext 5344
‫رقم الهاتف‬
Telephone
‫االسم والشهرة‬
Post
2
2014 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2014
Programme de subvention à la recherche scientifique au Liban – 2014
.1
Co-investigators - ‫الباحثون المشاركون‬
‫العنوان االلكتروني‬
e-mail
he21@aub.edu.lb
‫المؤسسة‬
Institution
AUB
‫االسم والشهرة‬
Name and surname
Dr Hiba El Hajj
me00@aub.edu.lb
AUB
Dr Marwan El Sabban
rn03@aub.edu.lb
AUB
Dr Rihab Nasr
Duration and starting date of the research / ‫المدة التعاقدي للمشروع وتاريخ بدء البحث‬
2 years
Duration (year) / ‫المدة التعاقدية للمشروع‬
September 2012
Starting date of the research /‫وتاريخ بدء البحث‬
Scientific Information / ‫العلمي‬
‫ المعلومات‬.2
ّ
Objectives - ‫الهدف‬
(mandatory field to fill 5-8 lines) – )‫ أسطر‬8-5 : ‫( معلومات إلزامية‬
Specific aim 1: Role of PML in the “stemness” of ATL cells
Specific aim 2: Biochemical mechanisms of Tax degradation
Specific aim 3: Effect of Tax degradation on p53 reactivation
Specific aim 4: Role of Tax in the maintenance of the leukemic phenotype
Achievements - ‫أالنجازات المحقق‬
(mandatory field to fill 5-8 pages) – )8-5 : ‫( معلومات إلزامية‬
In the first year of this proposal, we showed that the combination of
arsenic, interferon-alpha, and zidovudine restores an “immunocompetent-like”
micro-environment in patients with adult T-cell leukemia lymphoma (ATL). HTLV-I
associated ATL carries a dismal prognosis due to chemo-resistance and immunocompromised micro-environment. The combination of zidovudine and interferonalpha (IFN) significantly improved survival in ATL. Promising results were
reported by adding arsenic trioxide to zidovudine and IFN. We assessed
3
2014 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2014
Programme de subvention à la recherche scientifique au Liban – 2014
Th1/Th2/Treg cytokine gene expression profiles in 16 ATL patients before and 30
days after treatment with arsenic/IFN/zidovudine, in comparison with HTLV-I
healthy carriers and sero-negative blood donors. ATL patients at diagnosis
displayed a Treg/Th2 cytokine profile with significantly elevated transcript levels of
Foxp3, interleukin-10 (IL-10), and IL-4 and had a reduced Th1 profile evidenced
by decreased transcript levels of interferon-γ (IFN-γ) and IL-2. Most patients
(15/16) responded, with CD4+CD25+ cells significantly decreasing after therapy,
paralleled by decreases in Foxp3 transcript. Importantly, arsenic/IFN/zidovudine
therapy sharply diminished IL-10 transcript and serum levels concomittant with
decrease in IL-4 and increases in IFN-γ and IL-2 mRNA, whether or not values
were adjusted to the percentage of CD4+CD25+ cells. Overall, the observed shift
from a Treg/Th2 phenotype before treatment toward a Th1 phenotype after
treatment with arsenic/IFN/zidovudine may play an important role in restoring an
immuno-competent micro-environment, which enhances the eradication of ATL
cells and the prevention of opportunistic infections. The attached manuscript
(Kchour et al. 2013) reporting these results is published in Retrovirology (impact
Factor 6.47).
During the second year of this proposal, we showed in another model of
virus associated lymphoproliferative disorder, namely primary effusion lymphoma
(PEL) associated with Kaposi Sarcoma Herpes Virus (KSHV) that the
combination of arsenic and IFN inhibits expression of KSHV latent transcripts and
synergistically improves survival of mice with primary effusion lymphomas. These
results were published in PLOS ONE (El Hajj et al. 2013; impact factor 3.73,
manuscript attached).
Although HTLV-1 and HTLV-2 share similar genetic organization, they
have major differences in their pathogenesis and disease manifestation. HTLV-1
is capable of transforming T lymphocytes in infected patients resulting in ATL
whereas HTLV-2 is not clearly associated with lymphoproliferative diseases.
Numerous studies have provided accumulating evidence on the involvement of
the viral transactivators Tax-1 versus Tax-2 in T cell transformation. Tax-1 is a
potent transcriptional activator of both viral and cellular genes. Tax-1 posttranslational modifications and specifically ubiquitylation and SUMOylation have
been implicated in nuclear factor-kappaB (NF-κB) activation and may contribute
to its transformation capacity. Although Tax-2 has similar protein structure
compared to Tax-1, the two proteins display differences both in their proteinprotein interaction and activation of signal transduction pathways. Recent studies
on Tax-2 have suggested ubiquitylation and SUMOylation independent
4
2014 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2014
Programme de subvention à la recherche scientifique au Liban – 2014
mechanisms of NF-κB activation. In the attached manuscript, we summarized
structural and functional differences between Tax-1 and Tax-2. Specifically, we
addressed their subcellular localization, nuclear trafficking and their effect on
cellular regulatory proteins. A special attention was given to Tax-1/Tax-2 posttranslational modification such as ubiquitylation, SUMOylation, phosphorylation,
acetylation, NF-κB activation, and protein-protein interactions involved in
oncogenecity both in vivo and in vitro. These results were published in Frontiers
Microbiology (Shirinian et al. 2013; manuscript attached).
Finally, we showed that ATL-derived cells are addicted to continuous Tax
expression, implying that Tax degradation underlies clinical responses in mice or
patients. Several studies previously demonstrated that arsenic and IFN enforce
PML nuclear body (NB) formation and partner protein recruitment. Upon
treatment with arsenic/IFN, we demonstrated that Tax is recruited onto NBs and
undergoes PML-dependent hyper-sumoylation by SUMO2/3, but not SUMO1.
These results suggest that arsenic/IFN is a targeted therapy in ATL and that Tax
degradation is mechanistically similar to that of PML/RARA in acute
promyelocytic leukemia (APL). The manuscript describing these findings
(Dassouki et al.) is in preparation.
Perspectives - ‫آفاق البحث‬
(mandatory field to fill 5-8 lines) – )‫ أسطر‬8-5 : ‫( معلومات إلزامية‬
Despite the fact that Tax transgenics develop a typical ATL, that Tax is
the actual driver of the disease and is required until leukemic stage has remained
controversial (Romanelli et al., 2013). Here, using sh-RNA-mediated Tax
silencing, we demonstrate that ATL-derived cells are strictly dependent on
continued Tax expression for their survival, exemplifying complete addiction to the
driving oncoprotein. Therefore, arsenic/IFN-initiated Tax degradation is most likely
responsible for the apoptosis of ATL cell lines and clinical remissions in mice and
patients, as recently demonstrated for PML/RARA in APL (Ablain et al., 2014; de
The and Chen, 2010; El Hajj et al., 2010; Kchour et al., 2009). Since Tax
degradation by the arsenic/IFN combination drives clinical response, unravelling
its biochemical mechanism is important. PML NBs function as in situ partner
sumoylation and degradation machineries (Lallemand-Breitenbach and de The,
2010; Sahin et al., 2014). IFN not only induces PML expression and NB
biogenesis, but also dramatically enhances SUMO availability. That steady-state
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2014 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2014
Programme de subvention à la recherche scientifique au Liban – 2014
Tax/PML interactions are massively increased upon proteasome inhibition reflects
basal Tax turnover within NBs. Tax is recruited on PML NBs upon treatment with
AS/IFN, most likely through its four SUMO interaction motifs, which serve as NBtargeting signals (Sahin et al., 2014). In ATL derived cells, we showed that Tax
recruitment results in a PML-dependent increase in SUMO2/3 conjugation. Why
Tax is preferentially conjugated by SUMO2/3 is unclear and deserves further
exploration. Thus, the basis of clinical efficacy of arsenic (oncoprotein
degradation by NB-associated proteolysis) appears identical in ATL and APL (de
The and Chen, 2010). In addition to oncogene degradation, PML NBs have a key
role in enforcing p53 activation and a senescence-like process, which is ultimately
responsible for APL cure (Ablain et al., 2014). Interestingly, p53 mutations were
associated to therapy resistance in ATL patients treated with IFN (Datta et al.,
2006), supporting the idea that, as in APL (Ablain et al., 2014), therapy must first
degrade the driving oncogene, but then also activate this PML/p53 senescence
axis to eradicate the disease.
More broadly, our results suggest that PML NBs, whose formation is
fostered by the IFN/arsenic combination, can act as degradation centres for
pathogenic proteins. Indeed, apart from PML/RARA and Tax, aggregation of
neurotoxic poly-glutamine containing proteins was show to be responsive to PML
or SUMO and the clinical disease to be IFN-reversible (Chort et al., 2013). Thus,
enforcing NB-formation, hypersumoylation and degradation of partner proteins
may have clinical benefit in other malignancies than ATL or APL.
REFERENCES
Ablain, J., K. Rice, H. Soilihi, A. de Reynies, S. Minucci, and H. de The. 2014.
Activation of a promyelocytic leukemia-tumor protein 53 axis underlies acute
promyelocytic leukemia cure. Nat Med.doi: 10.1038/nm.3441
Chort A, Alves S, Marinello M, Dufresnois B, Dornbierer JG, Tesson C, Latouche
M, Baker DP, Barkats M, El Hachimi KH, Ruberg M, Janer A, Stevanin G, Brice A,
Sittler A. 2013. Interferon β induces clearance of mutant ataxin 7 and improves
locomotion in SCA7 knock-in mice. Brain. 136:1732-45.
Datta, A., M. Bellon, U. Sinha-Datta, A. Bazarbachi, Y. Lepelletier, D. Canioni,
T.A. Waldmann, O. Hermine, and C. Nicot. 2006. Persistent inhibition of
telomerase reprograms adult T-cell leukemia to p53-dependent senescence.
Blood. 108:1021-1029.
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2014 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2014
Programme de subvention à la recherche scientifique au Liban – 2014
de The, H., and Z. Chen. 2010. Acute promyelocytic leukaemia: novel insights
into the mechanisms of cure. Nat Rev Cancer. 10:775-783.
El Hajj, H., M. El-Sabban, H. Hasegawa, G. Zaatari, J. Ablain, S.T. Saab, A.
Janin, R. Mahfouz, R. Nasr, Y. Kfoury, C. Nicot, O. Hermine, W. Hall, H. de The,
and A. Bazarbachi. 2010. Therapy-induced selective loss of leukemia-initiating
activity in murine adult T cell leukemia. J Exp Med. 207:2785-2792.
Kchour, G., M. Tarhini, M.M. Kooshyar, H. El Hajj, E. Wattel, M. Mahmoudi, H.
Hatoum, H. Rahimi, M. Maleki, H. Rafatpanah, S.A. Rezaee, M.T. Yazdi, A.
Shirdel, H. de The, O. Hermine, R. Farid, and A. Bazarbachi. 2009. Phase 2 study
of the efficacy and safety of the combination of arsenic trioxide, interferon alpha,
and zidovudine in newly diagnosed chronic adult T-cell leukemia/lymphoma
(ATL). Blood. 113:6528-6532.
Lallemand-Breitenbach, V., and H. de The. 2010. PML nuclear bodies. Cold
Spring Harb Perspect Biol. 2:a000661.
Romanelli, M.G., E. Diani, E. Bergamo, C. Casoli, V. Ciminale, F. Bex, and U.
Bertazzoni. 2013. Highlights on distinctive structural and functional properties of
HTLV Tax proteins. Frontiers in microbiology. 4:271.
Sahin, U., O.Ferhi, M. Jeanne, S. Benhenda, C. Berthier, F. Jollivet, M. NiwaKawakita, O. Faklaris, N. Setterblad, H. de The, and V. Lallemand-Breitenbach.
2014. Oxidative stress-induced assembly of PML nuclear bodies controls
sumoylation of partner proteins. J Cell Biol. in press.
Publications & Communications - ‫المنشورات والمساهمات في المؤتمرات‬
As per attached:
Kchour et al., 2013
El Hajj et al. 2013
Shirinian et al. 2013
Abstract - ‫موجز عن نتائج البحث‬
7
2014 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2014
Programme de subvention à la recherche scientifique au Liban – 2014
(mandatory field to fill 5-8 lines) – )‫ أسطر‬8-5 : ‫( معلومات إلزامية‬
We showed that the combination of arsenic, interferon-alpha (IFN), and zidovudine
restores an “immunocompetent-like” micro-environment in patients with adult T-cell
leukemia lymphoma (ATL). (Kchour et al. 2013). We validated the efficacy of the
arsenic/IFN combination in another model of virus associated lymphoproliferative
disorder, namely primary effusion lymphoma (PEL) associated with Kaposi Sarcoma
Herpes Virus (KSHV) (El Hajj et al. 2013). Finally, we showed that ATL-derived cells
are addicted to continuous Tax expression, implying that arsenic/IFN-induced Tax
degradation underlies clinical responses in mice or patients. We decorticated the
biochemical pathways of Tax degradation implicating PML-dependent hyper-sumoylation
of Tax. These results suggest that arsenic/IFN is a targeted therapy in ATL and that Tax
degradation is mechanistically similar to that of PML/RARA in acute promyelocytic
leukemia (APL).
‫توقيع الباحث‬
8
2014 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2014
Programme de subvention à la recherche scientifique au Liban – 2014