1. Laboratory of Dr. Dominic Smiraglia Roswell Park Cancer Institute
Targeting the methionine salvage pathway as a metabolic point of leverage in novel therapeutic approaches for prostate cancer.
Irianna M. Torres
Laboratory of Dr. Dominic Smiraglia
Roswell Park Cancer Institute

2. Why study prostate cancer?
Among american men

3. Prostate Cancer
The prostate gland sits underneath the bladder. It secretes an alkaline fluid that
comprises 30% of seminal fluid. This fluid nourishes and protects sperm.
There is a high
concentration of acetylated polyamines in prostatic fluid.
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4. Prostatic Intraepithelial neoplasia
Prostate Cancer is an androgen driven disease.
Prostatic Intraepithelial neoplasia
Androgen-independent prostate cancer is highly refractory to additional therapies and we want to prevent patients from ever reaching this point in their disease through the approach we are taking in developing novel therapeutics
Prostatic intraepithelial neoplasia, established precursor of prostate adenocarcinoma.
Metastatic or biochemical recurrent prostate cancer is treated with Androgen
Deprivation Therapy (ADT).
Overwhelming majority of men treated with ADT recur months after treatment.
Androgen-independent prostate cancer is highly refractory to additional therapies.

5. Prostatic epithelial cells secrete acetylated polyamines.
Prostatic epithelial cells secrete acetylated polyamines.
= Polyamines
Basal cells
Prostate Cell
Luminal Cell
Lumen
Prostate cells are unique in that they secrete a large amount of acetylated polyamines into the lumen. We pros posed that biosynthetic flux increases to replenish intracellular stores of polyamines. We wanted to know what biosynthetic pathways would be involved in polyamine biosynthesis and what are the biological consequences of this increased secretion of acetylated polyamines.
What biosynthetic pathways are connected to polyamine biosynthesis?

6. Polyamine biosynthesis is interconnected with various pathways.
N1-acetylspermine
N1-acetylspermidine
SSAT
Polyamine
Biosynthesis
dcSAM
dcSAM
MTAP
SAM
one carbon units that are carried throughout these biosynthetic pathways to generate important substrates for polyamine biosynthesis and because of this interconnection there are biological consequences that occur as a result of increased secretion of acetylated polyamines and increased biosynthetic flux. So we’ll begin at the top, and we can follow this one carbon unit through the cycle to the generation of polyamines putrescine, spermine and spermidine. These polyamines are acetylated by SSAT and these acetylated polyamines are secreted into the lumen of the prostate cell. we can follow this one carbon unit again as it travels through the methionine cycle and produce s-adenosylmethionine known as SAM which is a substrate that is used for polyamine biosynthesis and methylation processes. And if we follow this last one carbon unit it travels through one carbon metabolism pathway which we can see generates substrates needed for the methionine cycle but also importantly results in the substrate deoxythymidine triphosphate dTTP which is important for DNA synthesis.
What are the biological consequences of this secretion?

7. Intracellular POLYAMINES
EXPORT
SSAT activity leads to export of acetylated polyamines.
Acetylated polyamines
The increase in biosynthetic flux to replenish
intracellular polyamines decreases SAM and dTTP
pools leading to stress.
SSAT
Intracellular POLYAMINES
dcSAM
SAM
Methionine
Cycle
So this is a more simplified version of these pathways. So ultimately what we can see is that this increased secretion of acetylated polyamines has biological consequences such as decreases in SAM pools which effects methylation process and decrease in dTTP which effects DNA synthesis.
This stress is increased even more in prostate cancer cells that secrete higher amounts of polyamines.
One- Carbon
Metabolism
dTTP

8. Intracellular POLYAMINES
EXPORT
The Methionine Salvage Pathway
recycles MTA, the one carbon unit, back to the
methionine cycle for polyamine biosynthesis.
Acetylated polyamines
SSAT
Intracellular POLYAMINES
Methionine Salvage Pathway
MTA
MTAP
dcSAM
SAM
Methionine
Cycle
So another important aspect of this pathway I want to point out is the methionine salvage pathway in green. polyamine biosynthesis generates a byproduct 5’-methylthioadenosine or MTA. The sole function of the enzyme MTAP is to recycle MTA to SAM. However if this pathway were to be inhibited, this would place even more stress onto SAM pools, the methionine cycle, dTTP and one carbon metabolism because this pathway would no longer be able to mitigate the deficiency of intracellular polyamines.
One- Carbon
Metabolism
dTTP

9. Can we leverage this stress towards a therapeutic approach?

10. Hypothesis
We hypothesize that adding more stress to an already strained system, by targeting two different aspects of these metabolic pathways, will result in the development of novel therapeutic strategies to complement existing therapies, with the goal of enhancing the extent and/or duration of clinical benefit.

11. Increase metabolic stress using BENSpm .
N1-acetylspermine
N1-acetylspermidine
SSAT
BENSpm
Polyamine Biosynthesis
dcSAM
dcSAM
MTAP
MTDIA
SAM
The manner in which we want to carry out this 2-sided approach is by using two pharmaceuticals. BENSpm is a polyamine analogue that upregulates the enzyme SSAT that forms acetylated polyamines. By upregulating SSAT we hope to see an increase in export of acetylated polyamines. Our second pharmacologic, MTDIA, is going to inhibit the cells ability to mitigate this deficiency by inhibiting the enzyme MTAP and ultimately inhibiting the methionine salvage pathway from recycling MTA back to the methionine cycle to generate SAM which is used for polyamine biosynthesis.
Increase metabolic stress using BENSpm .
Block mitigation of that stress with MTDIA.

12. Can we demonstrate an increase in SSAT activity with BENSpm?
So conceptually we thought BENSpm would be a good pharmaceutical for uprgulating SSAT and increasing exportation of polyamines within the cell. Now can we demonstrate this experimentally?

13. Treatment with BENSpm for 96 hours in the androgen sensitive LNCaP cell line causes upregulation of SSAT in (+) and (-) DHT.
SSAT
B-actin
(-) DHT
(+) DHT
BENSpm (uM)
It also does so at the activity level. The data is not shown here.
Hayley Affronti.

14. Is the methionine salvage pathway important?
Conceptually it seems that the methionine salvage pathway is significant, can we validate this concept?

15. MTAP knockdown inhibits tumor growth in LNCaP xenograft
We desgined an experiment to answer this question. We knocked down MTAP in LNCaP cells which we then injected 1 million of these cells subcutaneously in nude skid mice which are immunocompromised and observed tumor volume over a period of 78 days. We used an sh scramble as our control. We can see on this graph that MTAP knockdown inhibits tumor growth in this LNCaP xenograft and validates the importance of the methionine salvage pathway in cellular proliferation.
P < t-test
Can we block tumor growth in LNCaP xenograft pharmacologically with MTDIA?
Gaia Bistulfi, PhD.

16. MTDIA, a pharmacological MTAP inhibitor, blocks growth of tumor in LNCaP xenografts.
n=20 per group
So now that our concept is validated and we can see that MTAP inhibition leads to profound block in tumor growth, we wanted to see if we would still see a profound block in tumor growth when using a pharmaceutical that blocks MTAP. We subcutaneously injected nude skid mice with LNCaP cells and fed one group MTDIA drug in their water, and fed the control group just vehicle without drug. We yet again observed a profound inhibitory effect on the growth of the tumor.
Genetic and pharmacological interference of the methionine salvage pathway profoundly blocks xenograft growth.
Gaia Bistulfi, PhD.

17. Could SSAT overexpression increase the sensitivity of prostate cells to MTDIA treatment?
Could there be a combinational effect here of using these two drugs together?

18. SSAT Protein Expression in LNG- 53 Cells +/- TET
B-Actin
LNG-53 cells -TET
0hr hr 48hr 72hr 96hr 120hr
We have now demonstrated that the drug BENSpm does in fact upregulate SSAT and MTDIA, the MTAP enzyme inhibitor, causes a profound block in tumor growth which validates the signifcance of the methionine salvage pathway. We asked ourselves” could SSAt overexpression increase the sensitivity of prostate cells to MTDIA treatment. As a critical proof of this principle we stably transfected LNCaP cells with a construct that overexpresses SSAT in the absence of tetracycline. This cell line is called LNG-53. If we look at this western blot we see that LNG -53 cells grown in the absence of TET at various timepoints, overexpress SSAT at 48, 72, 96 and 120 hours. Then we treated these cells that overexpress SSAT with various concentrations of MTDIA and saw that the IC 50 was in fact lowered. Cell lines with increased SSAT activity have an increased sensitivity to MTDIA treatment. This genetic manipulation suggests there might be synergy. Our question is do we see synergy with the use of BENSpm and MTDIA.
LNG-53 cells, which overexpress SSAT, have an increased sensitivity to MTDIA treatment. This
genetic manipulation suggests there may be synergy.

19. Results show synergy when LNCaP cells are treated with BENSpm and MTDIA.
Upregulation of SSAT with 2.5 uM BENSpm decreases the MTDIA IC50.
Combination Indexes show treatment with 0.1 to 5.0 mM MTDIA and 1.0 to 10.0 uM BENSpm is
synergistic (CI < 1).
We treated LNCaP cells with various concentrations of MTDIA alone as a control and subsequently treated LNCaP cells with various concentration of MTDIA and BENSpm. This graph shows treatment with 2.5 uM BENSpm and MTDIA at various concentrations and we do in fact see a lower MTDIA IC 50 as compared to the control group suggesting there is synergy. After calculating the combination indexes of these experiments we did in fact see synergy, values less then 1.
Hayley Affronti.

20. Conclusion
Genetic and pharmacological interference of the methionine salvage pathway profoundly blocks xenograft growth.
Overexpression of SSAT significantly increases sensitivity to MTAP inhibition, providing critical proof of principal.
Treatment with BENSpm in the androgen sensitive LNCaP cell line causes upregulation of SSAT in (+) and (-) DHT.
Resulting in increased sensitivity to MTAP inhibition.
Combination Index demonstrated treatment with MTDIA and BENSpm is synergistic.

21. Future Directions
In the future, we hope to further study the effects of BENSpm and/or MTDIA treatment in vivo in androgen independent xenografts and recurrent prostate cancer models.
Our long-term goal is to take advantage of this in a clinical setting to enhance the extent and/or duration of clinical benefit of current therapies.

22. Acknowledgements
Roswell Park Cancer Institute Principle Investigator: Dr. Dominic Smiraglia Smiraglia Lab Hayley Affronti Vineet Dhiman Karina Miller Mouse Tumor Model Resource Dr. Barbara Foster Dr. Mike Moser Bryan Gillard Laboratory of Animal Resources at RPCI Dr. Sandra Sexton Dr. Leslie Curtin
Johns Hopkins University Dr. Robert A. Casero 20th Annual McNair Research Conference 2014 Dean’s Research Scholarship Prentice Family Foundation Western New York Prosperity Scholarship
Brian
Ellen
Barb
Marc
Dr. Casero Johns Hopkins