DNA Damage Induced by Novel Drug Cocktail Regulates Chemokine Production in Leukemia through Cytosolic DNA Sensing

Discussion/Conclusion

Introduction

Abstract

A group of researchers at UCLA optimized a combination

therapy (kept confidential) to treat acute lymphoblastic
leukemia (ALL). This cocktail induces permanent DNA
damage by targeting cells nucleotide metabolism and DNA
repair pathways. Two of the drugs deplete the DNA nucleotide
pool, preventing the cancer cell from replicating its genome
properly.[1] The replication stress response resulting from the
first two drugs is diminished by the third drugs inhibition of
ATR, a kinase responsible for monitoring DNA damage.[2] This
compound overrides the cell's natural response to halt cell
replication in the presence of lethal, non-repairable DNA
damage.[2] This combination induces permanent DNA damage
by replication stress from two drugs and by overriding cellular
checkpoints during cell replication through loss of ATR
function, ultimately leading the cell to apoptosis.

This research investigates the mechanism of action for chemokine production induced by a novel cancer drug cocktail to induce
DNA damage in precursor-B acute lymphoblastic leukemia (ALL). Preliminary data supports a model in which DNA damage caused
by this drug cocktail leads to DNA leakage into the cytoplasm and the activation of innate immune DNA sensing pathways,
modulating the production of inflammatory cytokines, chemokines, and interferons. Using an ELISA array and siRNA knockdowns
for STING and MyD88 (DNA sensing common adaptor proteins), this project establishes the dependence on these common adaptor
proteins for production of chemokines in vitro and long term in vivo: treatments in vitro exhibit an increased production of Eotaxin
and KC after 1 day of treatment; and Eotaxin, MIP-1, MIP-1, and SDF-1 after 4 days of treatment. Knockdowns for STING and
MyD88 in vitro results in decreased expression of MCP-1, MIP-1, MIG, Eotaxin, and TARC in both treated and non-treated groups.
Exploring the role of innate immunity in cancer models is crucial to understanding the dynamic interaction between the immune
system and tumors and may lead to novel methods to treat cancer.

Hypothesis
Pharmacologically induced nucleotide depletion will alter chemokine production through cytosolic DNA damage
sensing pathways.

The cell has various innate immune sensing mechanism to

detect pathogenic or self-damage markers. Extranuclear DNA,
an indication of DNA damage, can be detected by various
cytosolic DNA sensors.[3] This sensing induces the production
of inflammatory proteins, which can attract immune cells to
induce cell death in the abnormal cells.[4] Using an ELISA
array and siRNA knockdowns for STING and MyD88 (DNA
sensing common adaptor proteins), this project establishes
the correlation between drug treatment and chemokine
production through innate immune sensing pathways,
implicating the existence of additional tumor responses to
treatment.

Results
For Figures 3 and 4, the treated group was compared to the vehicle group as a
baseline. For Figure 5, the treated knockdowns were compared to the treated
vehicle group as a baseline to determine whether the STING or MyD88 was
necessary for chemokine production.

The in vitro treatments exhibits the direct effect of the drug on

chemokine expression of tumor. In the 1 day treatment, the treated
group had an increase of the MIP-1, Eotaxin, KC, and MDC. In the
4 day treatment, the treated group has an increase of RANTES, MIP1, MIP-1, SDF-1, and Eotaxin, KC. The discrepancy between the
in vitro treatment data may result from the difference in drug
exposure time.
The in vitro knockdowns demonstrated the dependence of the
production of the chemokines on both STING and MyD-88 proteins to
different degrees.
As for the in vivo model, the direct correlation between treatment and
these tumor regulated chemokine production established in vitro is
missing, suggesting that there are many other factors in play i.e.
presence of other chemokines produced by infiltrating lymphocytes
and leukocytes in the tumor microenvironment.[5]
Ultimately, this drug cocktail induces chemokine production through
cytosolic DNA sensing pathways. Whether this treatment results in a
heightened immune system and increased tumor death or represents
another case of cancer plasticity and resistant mechanism remains to
be investigated.

For Figure 6, the knockdowns (no treatment) were compared to the vehicle (no
treatment) to determine the effects of knockdown on normally produced
chemokine levels. For Figures 7 and 8, the xenografted treated mice were
compared to the xenografted non-treated mouse using the vehicle (no xenograft,
no treatment) mouse as a baseline.

Treatment

DNA Damage

Figure 2. DNA sensor pathway

Figure 9. Immunoediting Capabilities of Tumor [6]

Cell Death from

lethal DNA
damage

Leakage of nucleic
DNA into cytoplasm
Activation of DNA
sensor pathways
Production of
Chemokines
Immunemediated
cell death

Tumor
Evasion

Figure 1. Proposed multiple interactions from this combinationdrug therapy.

References

Materials and Methods

Animals
C57BL/6 mice were bred and housed under specific pathogen-free
conditions and were treated in accordance with the UCLA Animal
Research Committee protocol guidelines. One mouse was a vehicle
mouse (no xenograft, no treatment); one mouse was a control
(xenograft, no treatment). Two mice were experimental (xenograft
and treatment as described below).

Figure 3. In Vitro, 1 day post

treatment.

Figure 4. In Vitro, 4 days post

treatment.

Xenograft and Treatment

200,000 p185BCR-ABL Arf/ preB ALL cells were injected into mice via
tail vein. Mice in the cancer and treatment group were administered
the drug cocktail orally.
Cell Culture and Treatment
p185BCR-ABL Arf/ preB ALL cells in the treatment group were grown
in DMEM in the presence of the drugs for 4 days. p185 cells in the
vehicle group were grown in DMEM media for 4 days.

Acknowledgements
Figure 5. In Vitro knockdowns, no
treatment.

Figure 6. In Vitro treated

knockdowns.

Figure 7. In Vivo, 40 days post

treatment, Mouse 1.

Figure 8. In Vivo, 40 days post

treatment, Mouse 2.

Knockdowns and Treatment

60-80% confluent p185BCR-ABL Arf/ preB ALL in a 6 well plate were
transfected with small interfering RNA against STING and MyD88
(Life Technology) using Lipofectamine RNAiMAX Transfection
Reagent (Life Technology) for 72 hours. The culture was then treated
with the drug cocktail for a 24 hour period.
ELISA
Samples were analyzed using the Multi-Analyte ELISArray (Qiagen)
according to the protocol provided with the kit.

Multi-Analyte ELISArray

[1] Nathanson, D., Armijo, A., Tom, M., Li, Z., Dimitrova, E., Austin, W., ... Radu, C.
(2014). Co-targeting of convergent nucleotide biosynthetic pathways for leukemia
eradication. Journal of Experimental Medicine, 211(3), 473-486.
[2] Ruzankina, Y., Schoppy, D.W., Asare, A., Clark, C.E., Vonderheide, R.H., and
Brown, E.J. (2009). Tissue regenerative delays and synthetic lethality in adult mice
after combined deletion of Atr and Trp53. Nat. Genet. 41, 11441149.
[3] Paludan, S., & Bowie, A. (n.d.). Immune Sensing of DNA. Immunity, 38, 870-880.
[4] Ono, S., Nakamura, T., Miyazaki, D., Ohbayashi, M., Dawson, M., & Toda, M.
(2003). Chemokines: Roles in leukocyte development, trafficking, and effector
function. Journal of Allergy and Clinical Immunology, 1185-1199.
[5] Balkwill, F. (2003). Chemokine biology in cancer. Seminars in Immunology, 49-55.
[6] Chow, M., & Luster, A. (2014, December 1). Chemokines in Cancer. American
Association for Cancer Research, 1125-1131.

I gratefully acknowledge the following people for supporting my project:

Soumya Poddar and Dr. Caius Radu from UCLA Ahmanson Translational
Imaging Division for mentoring on this project.
Lindsey Gannon and Sonia Dobias from Qiagen for donating Multi-Analyte
ELISArray kits.
Dr. Malhotra for her continuous scientific enlightenment.
My parents for supporting me throughout these years.