This document discusses a proposed study to target cancer stem cells in leukemia using tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-based immunotoxins. The objectives are to identify and characterize cancer stem cells from leukemia cell lines and patients, construct recombinant TRAIL-based chimeric proteins to selectively target these cancer stem cells, and test the efficacy and selectivity of the proteins. Cancer stem cells are resistant to chemotherapy and responsible for leukemia recurrence. TRAIL selectively induces apoptosis in cancer cells and has advantages over chemotherapy. The study aims to develop dual receptor targeted immunotoxins linking TRAIL to the interleukin-2 receptor to more effectively treat leukemia by eliminating drug-resistant cancer stem cells.

1. Identification and targeting of leukemic
stem cells using Tumor necrosis factor
(TNF)-related apoptosis-inducing ligand
(TRAIL)
TITLE OF THE PROJECT
Madhumathi J
Department of Biotechnology
Indian Institute of Technology Madras
Chennai 600036
madhurachel@gmail.com

2.  To identify and characterize cancer stem cells from leukemic cell lines and
patient samples.
To construct recombinant TRAIL based chimeric proteins to target cancer
stem cells.
To test the efficacy and selectivity of the chimeric proteins in normal cells,
lymphoma/leukemia cell lines, the isolated leukemic stem cells and patients.
OBJECTIVES
AIM
Targeting chemo-resistant cancer stem cells using TRAIL
based immunotoxins in leukemia

3. Cancer Stem Cells
Guzman ML, Jordan CT. Cancer Control. 2004
1. Chen, et al., Nature, 2012
2. Driessens,et al., Nature, 2012
3. Schepers, et al., Science, 2012
In 2012, Three independent studies provided first
evidence that CSCs do exist
Richard J. Gilbertson,
Resolving the stem-cell debate, Forum, Nature, 2012
Cancer stem cells (CSC’s) - cells in the tumor
growth with a tumor initiating potential.
Tumors were derived from mutated stem cells, the so-
called cancer stem cells (Martinez-Climent et al.,
2010).
CSC model: human cancers originate from tissue stem or
progenitor cells, and only a small fraction of these cells
(CSCs) have the capacity to proliferate indefinitely
Background and reasons for the proposed study

4.  Highly resistant to chemo- and radio-therapy
 Inhibition of apoptosis- long-term survival is primarily by deregulation of apoptosis.
 Self-renewal capability
 Quiescence
Characteristics of CSC’s
Resistance of CSC’s
CSC’s are typically resistant to cancer drugs and are
responsible for recurrence of all cancers.
1. Evasion of apoptosis is one of the major mechanisms of
immortality in human cancers (Hanahan and Weinberg,
2000).
2.Chemoresistance – due to efflux of anticancer drugs
through multidrug resistance transporter 1 (MDR1)

5. Guzman ML, Jordan CT. Cancer Control. 2004
Tannishtha Reya et al, Nature, 414, 2001
Targeting Cancer Stem Cells
Targeting both CSCs and the dividing cells would be required for complete
tumor eradication
Immunotoxins targeting specific surface proteins of cancer stem cells
could be the solution

6. IMMUNOTOXINS
Immunotoxin - chimeric protein composed of a targeting moiety, (ligand of a
receptor or an antibody to surface antigen), linked to a protein toxin moiety-
(Mathew and Verma, 2009; Potala and Verma, 2008, 2009, 2010 ; Madhumathi and Verma, 2012).
Specific for antigens selectively expressed on tumors
1. Cytokines- IL-2, IFN, TNF
targeting respective Receptors (IL-2 for
CTCL, renal cell cancer, melanoma)
2. Monoclonal antibodies- for tumor
specific antigens-EpCAM
3. Immunoglobulins – single chain
variable fragmens (ScFv).
4. Growth factors – VEGF, GnRH
Target Molecules Toxin Molecules
1. Bacterial toxins- Diphtheria Toxin,
Pseudomonas Exotoxin, Shiga, cholera,
anthrax toxins
2. Fungal toxins- a-sarcin, restrictocin
3. Plant toxins- ricin, abrin, saporin (SAP),
pokeweed antiviral protein (PAP), gelonin
etc
3. Humanized toxins-
Pro-apoptotic proteins-TRAIL, DFF,
Bcl-2, FASL and Rnases.

7.  Chemoresistant side population (SP), displayed higher sensitivity to TRAIL
compared to the non-SP cells (Sussman et al., 2007).
 CSC’s express higher levels of TRAIL receptors (DR4 and DR5) while the
normal stem cells do not express (Sussman et al., 2007).
 TRAIL signaling could bypass the drug efflux mechanisms by triggering the
death receptor-induced apoptotic pathway (Fulda and Pervaiz ,2010).
 Normal Mesenchymal stem cells (MSC’s) are resistant to TRAIL since their
receptors are inactive (Szegezdi et al., 2009).
REVIEW OF LITERATURE

8. TRAIL
 Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)- Induces apoptosis by
Extrinsic Pathway
 TRAIL- member of TNF superfamily- 40 kDa type II transmembrane protein
 Expressed by normal immune cells upon stimulation- T cells, NK cells, Macrophages,
Dendritic cells, B cells, monocytes and in prostate and spleen tissues.
 Role in normal cells:
Tumor suppressor, suppresses metastasis, prevents auto-immunity
TRAIL receptors
Binds to-
Death receptor 4 (DR4/TRAIL-R1),
Death receptor 5 (DR5/TRAIL-R2),
Decoy receptors DcR1 and DcR2 and
Soluble receptor osteoprotegrin
Receptors- R1 and R2 (DR4/DR5)- expressed in cancer cells
not normal cells -Selectively kills cancer cells but not most
normal cells
(Yagita, 2001)
DR4/DR5 expressed
in cancer cells
Decoy receptors
expressed in
normal cells

9.  Selective toxicity for tumour cells- (Yagita et al., 2004, Koschny et al., 2007, Carlo-Stella et
al., 2007).
 No apparent systemic toxicity of rTRAIL in non-human primates
 Shows bystander effect (therapeutic effect toward neighboring tumor cells that
lack expression of the target antigen).
 Induces apoptosis in premalignant cells-(Lu et al., 2004)
 Increased ability to induce apoptosis when combined with chemotherapy
(Liu et al.,2001)
 scFv54–sTRAIL : colorectal & breast carcinomas
 scFv425–sTRAIL :Squamous cell carcinomas
 scFvCD7–sTRAIL: Leukemias
TRAIL based immunotoxins
Advantages of TRAIL
Mathew and Verma, 2009
Bremer et al., 2004 a, b, 2005
The TRAIL based fusion constructs reported for solid tumors:

10. 1. IL-2 R Over-expressed in Leukemia and lymphoma- only a small percentage
of T cells are ordinarily IL2R+. [Targeting IL-2α receptor alone that cannot mediate
normal biologic signaling has been reported to minimize the toxicities of IL2 therapy
(Potala and Verma, 2010, Smith, 2006)].
2. Widely used for selective targeting of cancer cells (Foss et al., 1998). The majority
of clinical studies using diphtheria-based fusion protein toxin constructs
have been with the IL-2 receptor targeting constructs (Kreitman, 2006, Potala and Verma,
2008, Madhumathi and Verma., 2012).
3. IL-2 Induces intrinsic expression of TRAIL by immune cells (Baetu and Hiscott,
2002).
4. Acts as an adjuvant- promotes immune cells like NK cells and T cells that
clears cancer cells.
-IL-2 receptor-targeted therapies were used in treatment of neoplasia, autoimmune
diseases and transplantation (Potala and Verma, 2008). Low dose recombinant IL2 has
been proved to activate antitumor immune response in advanced malignancies
(Onizuka et al., 1999).
Advantages of IL-2

11. •Presence of surface
markers like CD34+ /
CD38-
•Side population
Hoechst efflux assay
•BrdU pulse chase
assay
•Magnetic activated cell sorting
(MACS)
•Isolation of resistant side
population by growth in
presence of anti-cancer drugs
•Colony forming assay, western
blot, RT-PCR
Characterization
and Isolation of
CSC’s
Identification of CSC’s
in leukemic cell lines and
patient samples
Analysis of cancer stem
cells (CSC’s) in leukemia
Cloning TRAIL
constructs from cDNA
Expression and Purification
of proteins
Receptor binding studies in
cell lines
Cytotoxicity assays (MTT)
Apoptosis assays- Annexin V,
caspase etc
Comparison with
Chemotherapy drugs
Construction and characterization of
Immunotoxins
Cytotoxicity studies in isolated CSC’s using
immunotoxins
METHODOLOGY

12. TRAIL
G3S
Linker
IL-2α
TRAIL based Immunotoxin Constructs
TRAIL Peptide
IL-2α
G3S
Linker
WORK PLAN
1. Isolation of side population (SP) cells by culturing them in presence of anti-cancer drugs
(Methotrexate and 5-Fluoro Uracil) to select for the resistant cells.
2. Identification and characterization of leukemic stem cells in side population –surface markers –
CD34, CD96, ABCG2, CD123 etc by immunostaining, Real time PCR of transcription factors
and signaling molecules of CSC, dye efflux assay, label retention assays and colony forming
assays.
3. TRAIL, IL-2-TRAIL, IL-2-TRAILpeptide fusion constructs will be genetically engineered and
cloned in pRSET vector. The proteins will be expressed in E.coli (BL21 DE3), purified and
characterized.
4. The specificity and cytotoxicity of the recombinant immunotoxins will be analyzed in leukemic
cell lines and isolated leukemic stem cells- receptor studies to localize TRAIL and IL2 receptor,
Apoptosis assays- MTT assay, Annexin-V FITC, analysis of membrane potential, western
blotting using anti-caspases 8, 3, PARP.

13. EXPECTED OUTCOME
 Development of novel dual receptor targeted recombinant protein to treat
leukemias and lymphomas more specifically and effectively with a wider
therapeutic window.
 Elimination of drug resistant side population and cancer stem cells by TRAIL
will overcome the problem of chemotherapy resistance and thus prevent
recurrence of disease.

14. PRELIMINARY RESULTS
HL60
Hoechst Anti-DR5-FITC Overlay
MOLT-4
K562
KG-1
Localization of TRAIL receptors
MOLT-4
Control Methotrexate Treated
HL60
K562
KG-1
Efflux of dye by Side population (SP)

15. Expression of ABCG2
Ant-ABCG2-FITC Hoescht Merged
Percentage viability of leukemia cells with
different concentrations of Methotrexate
Treatment with cancer drugs for isolation of SP population
Percentage viability of leukemia cells with different
concentrations of 5- FluroUracil

16. EXPERTISE IN THE FIELD
Sirisha Potala, Rama S. Verma, Journal of Biotechnology 148
(2010) 147–155
Expression of IL2-Receptor
DT-IL2 Immunotoxin
Mathew, Zaineb, Verma, Apoptosis (2013) 18:882–895
GMCSF-DFF40 Immunotoxin
Potala & Verma, Mol Biol
Rep, 2010
DT-HN-1 Immunotoxin
Other related Publications
1. Potala, Sahoo and Verma, Drug Discovery Today, 13, 2008
2. Mathew & Verma, Cancer sci, 2009, 100, (8), 1359-1365
3. Swati Choudhary, Mrudula Mathew and Rama S. Verma, 2011, 16, 495-503
4. Madhumathi & Verma, Current Opinion in Microbiology 2012, 15:300–309
Potala & Verma, Appl
Biochem Biotechnol, 2009
DT-SCF Immunotoxin

18. TRAIL induced apoptosis pathway
Receptors are trimerised - death-inducing signalling complex (DISC) assembled - adaptor molecule, Fas-associated death
domain (FADD), translocates to the DISC - interacts with intracellular death domain (DD) of the receptors Via its second
functional domain, the death effector domain (DED) - FADD recruits pro-caspases 8 and 10 to the DISC where they are
autocatalytically activated - activation marks the start of a caspase-dependent signalling cascade.