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

Why study prostate cancer? Among american men

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. © Copyright 2014 life-saving-naturalcures-and-naturalremedies.com

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 18-24 months after treatment. Androgen-independent prostate cancer is highly refractory to additional therapies.

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?

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?

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

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

Can we leverage this stress towards a therapeutic approach?

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.

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.

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?

Treatment with BENSpm for 96 hours in the androgen sensitive LNCaP cell line causes upregulation of SSAT in (+) and (-) DHT. SSAT B-actin 0 0.1 1.0 2.5 5.0 10.0 (-) DHT (+) DHT BENSpm (uM) It also does so at the activity level. The data is not shown here. Hayley Affronti.

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

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 < 0.001 t-test Can we block tumor growth in LNCaP xenograft pharmacologically with MTDIA? Gaia Bistulfi, PhD.

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.

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?

SSAT Protein Expression in LNG- 53 Cells +/- TET B-Actin LNG-53 cells -TET 0hr 24hr 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.

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.

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.

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.

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