Preclinical potentiators of statin-induced anticancer effects By Aleksandra Anna Pandyra A thesis submitted in conformity with the requirements for the Degree of Doctor of Philosophy Department of Medical Biophysics University of Toronto © copyright by Aleksandra A. Pandyra 2014 Thesis – Aleksandra Pandyra Preclinical potentiators of statin induced anticancer effects Aleksandra Anna Pandyra Doctor of Philosophy Department of Medical Biophysics University of Toronto 2014 Abstract Statins have been used for decades in the treatment of hypercholesterolemic patients to decrease the incidence of adverse cardiovascular events. Statins target the rate-limiting enzyme of the mevalonate (MVA) pathway, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). Statin-mediated HMGCR inhibition in hepatocytes leads to depletion of MVA derived sterols, including cholesterol, and a subsequent uptake of low-density lipoprotein (LDL) cholesterol from the plasma. Aside from the intended cholesterol-lowering properties, statins exert many pleiotropic effects including anti-tumour activity. The well characterized pharmacokinetic profiles, safety and the fact that many statins are off patent has ensured their prompt entry into clinic for evaluation of anti-cancer efficacy. We hypothesize that the therapeutic window of statins can be increased through (i) the use of a pharmacological screen to identify compound/s that synergize with statins to induce tumour- specific apoptosis and (ii) the use of a genome-wide small hairpin ribonucleic acid (shRNA) screen to identify novel pathways/targets whose knockdown enhances the vulnerability of tumour cells to statin-inhibition. Firstly we carried out a screen in a multiple myeloma (MM) ii Thesis – Aleksandra Pandyra cell line where we combined atorvastatin with other off-patent FDA approved drugs. We identified dipyridamole, a commonly used anti-platelet agent as synergizing with and potentiating statin-induced apoptosis in a variety of MM and acute meylogenous leukemia (AML) cell lines and primary patient samples. The efficacy of the combination was demonstrated in vivo. Secondly, through genome-wide shRNA screen, we identified several putative targets whose knockdown potentiated statin induced anti-cancer effects in the A549 lung cancer cell line. Amongst the most promising hits, sterol regulatory element binding transcription factor 2 (SREBF2) was validated in lung and breast cancer cell lines. The knockdown of SREBF2 was found to dramatically potentiate statin-induced apoptosis and block the upregulation sterol-responsive gene targets. Taken together, this research has uncovered a novel combination of clinically translatable drugs with strong preclinical efficacy in hematological malignancies. Furthermore we have identified several novel validated targets that sensitize the MVA pathway to statin-induced apoptosis. iii Thesis – Aleksandra Pandyra Acknowledgements First, I would like to thank my supervisor, Dr. Linda Penn, for the opportunity to work in her lab, in one of the best research centers in the world. I have learned so much from her and I will never forget my experience in this incredibly supportive lab. Along with Dr. Linda Penn’s unwavering support, I would like to thank the members of my supervisory committee, Drs Aaron Schimmer and Robert Kerbel for their scientific guidance. I felt like I could always go to them for advice and this has been instrumental in helping me through graduate school. Furthermore, I would like to thank my family for their never-ending support and love, most important Mom, Tat, and Magda and certainly every other member of the Polish gang, Kate, Aga, all the Piotr’s, Ciocia, Wujek, Grandma, Grandpa and the little ones, and the new ones (Jon and Allison). I would like to thank Mark, my best friend, who has always pushed me to be the best I can be in every aspect of my life and go through life screaming and fighting. And of course my other friends, I can’t even imagine going through life without knowing you: Tracy, Amanda, Sara, Pete, Cory, Manpreet, Carolyn, Paul B., Janice, Laura, Cynthia, Ric, Lisa, Trudy, Larry, Jamie, Kevin, Stepho, Tracey, Valery. A special thanks to Maria K., I wish I could have been a better friend. Moreover, I would like to thank all the collaborators, Drs. Jason Moffat, Corey Nislow, Mark Minden, Paul Boutros and Suzanne Trudel, who have all been instrumental in contributing to the work presented in this thesis. iv Thesis – Aleksandra Pandyra Also, I would like to thank Dr. Karl Lang for giving me the great opportunity to work in his oustanding, excellent and awesome lab, his everlasting support and guidance on present and future research endeavours. Finally I would like to thank Philipp Lang, for everything. There’s too much to say than a few sentences could cover. v Thesis – Aleksandra Pandyra Table of contents Abstract  ........................................................................................................................................................  ii   Acknowledgements  .................................................................................................................................  iv   Table  of  contents  ......................................................................................................................................  vi   List  of  figures  .............................................................................................................................................  ix   Table  of  tables  ...........................................................................................................................................  xi   Abbreviations  ...........................................................................................................................................  xii   Chapter  1  :  Introduction  ................................................................................................................  1   1.1  Treatment  of  Cancer  .........................................................................................................................  2   1.1.1  Cytotoxic  Chemotherapy  ............................................................................................................................  3   1.1.2  Targeted  Molecular  Therapies  .................................................................................................................  3   1.1.2  Drug  Repositioning  .......................................................................................................................................  6   1.2  Repositioning  Statins  as  anti-­‐cancer  agents  .............................................................................  7   1.2.1  Statins  in  the  cardiovascular  setting  .....................................................................................................  7   1.2.2  The  pharmacology  of  statins  ....................................................................................................................  8   1.2.3  The  mevalonate  pathway  and  cancer  ................................................................................................  11   1.2.4  Statins  as  preventative  anti-­‐cancer  agents  ......................................................................................  15   1.2.5  Statins  in  the  clinic  .....................................................................................................................................  17   1.3  Combination  therapy  ......................................................................................................................  21   1.4  Research  Objectives  and  thesis  outline  ....................................................................................  23   1.5  References  ..........................................................................................................................................  25   Chapter  2  :  Combining  statins  and  dipyridamole  effectively  targets  acute   myelogenous  leukemia  and  multiple  myeloma  .................................................................  35   2.1.  Abstract  ..............................................................................................................................................  36   2.2.  Introduction  ......................................................................................................................................  37   vi Thesis – Aleksandra Pandyra 2.3.  Results  ................................................................................................................................................  40   2.3.1  A  screen  of  pharmacologically  active  drugs  identifies  dipyridamole  as  a  potentiator  of   the  anticancer  effects  of  atorvastatin  ............................................................................................................  40   2.3.2.  The  combination  of  statins  and  dipyridamole  is  synergistically  antiproliferative  and   induces  apoptosis  in  AML  and  MM  cell  lines  .............................................................................................  44   2.3.  3.  The  combination  of  statins  and  dipyridamole  induces  apoptosis  in  primary  AML  and   MM  cells  .....................................................................................................................................................................  53   2.3.4.  The  combination  of  statins  and  dipyridamole  delays  tumour  growth  in  leukemia   xenografts  .................................................................................................................................................................  60   2.3.5.  Mediators  of  cAMP  signaling  in  combination  with  statins  induce  apoptosis  and  raise   intracellular  cAMP  levels  in  leukemia  cells  ................................................................................................  65   2.4.  Materials  and  Methods  ..................................................................................................................  74   2.5.  Discussion  ..........................................................................................................................................  79   2.6.  References  .........................................................................................................................................  84   Chapter  3  :  Genome  wide  shRNA  screen  reveals  novel  sensitizers  of  statin-­‐induced   apoptosis  .........................................................................................................................................  89   3.1  Abstract  ...............................................................................................................................................  90   3.2  Introduction  .......................................................................................................................................  91   3.3  Results  .................................................................................................................................................  94   3.2.1     A  genome-­‐wide  screen  identifies  shRNA  drop-­‐outs  after  fluvastatin  exposure  ........  94   3.2.2  Validation  of  shRNA  hits  in  the  A549  cells  uncovers  potentiators  of  statin-­‐induced   apoptosis  and  anti-­‐proliferative  effects.  ....................................................................................................  101   3.2.3  Concomitant  targeting  of  PI4KB  and  HMGCR  showed  anti-­‐proliferative  effects  in  lung   and  breast  cell  lines.  ...........................................................................................................................................  115   3.2.4      Down-­‐regulating  the  SREBF2  mediated  sterol-­‐feedback  loop  in  combination  with   statins  is  an  effective  anti-­‐cancer  strategy  to  induce  lung  and  breast  tumour  cell  kill.  ........  122   vii Thesis – Aleksandra Pandyra 3.2.5      Fluvastatin  delays  tumour  growth  in  MDAMB231  xenografts.  ..........................................  129   3.4  Methods  ............................................................................................................................................  131   3.5  Discussion  ........................................................................................................................................  135   3.6.  References:  .....................................................................................................................................  143   Chapter  4  :  Discussion  ...............................................................................................................  149   4.1  Statins  as  anti-­‐cancer  agents  –  How  translational  is  the  cell  culture  work?  .............  150   4.2  Potentiating  statin-­‐induced  apoptosis  –  combinatorial  approaches  ..........................  154   4.2.1  Statins  and  rationally  crafted  combinations  .................................................................................  154   4.2.2  Statins  and  dipyridamole  ......................................................................................................................  156   4.2.3  Limitations  and  future  directions  of  this  work  ...........................................................................  158   4.3  Targeting  the  MVA  pathway  –  unbiased  knockdown  approaches  ................................  161   4.3.1  The  MVA  pathway’s  other  targets  .....................................................................................................  161   4.3.2  Limitations  and  future  directions  of  this  work  ...........................................................................  162   4.4  Concluding  remarks  and  anticipated  impact  ......................................................................  164   4.5  References:  ......................................................................................................................................  165   Publications  (2008-­‐2013):  ................................................................................................................  170   viii Thesis – Aleksandra Pandyra List of figures Figure 1-1: The MVA pathway. ............................................................................................... 12   Figure 2-1: A drug screen reveals dipyridamole as a potentiator of the anti-proliferative effects of atorvastatin. ...................................................................................................... 43   Figure 2-2: The statin-dipyridamole combination is synergistic in AML and MM cell lines. 46   Figure 2-3: Dipyridamole potentiates the anti-proliferative effects of fluvastatin and the combination is synergistic. ............................................................................................... 49   Figure 2-4: The statin-dipyridamole combination induces apoptosis in AML and MM cell lines. ................................................................................................................................. 52   Figure 2-5: The statin-dipyridamole combination induces apoptosis in the OCI-AML3 cell line. ................................................................................................................................... 54   Figure 2-6: The statin-dipyridamole combination induces apoptosis in primary AML and MM patient cells. ...................................................................................................................... 59   Figure 2-7: Dipyridamole plasma concentrations are higher following i.p. administration than p.o. .................................................................................................................................... 61   Figure 2-8: The statin-dipyridamole combination delays tumour growth in leukemia xenografts. ........................................................................................................................ 64   Figure 2-9: The relative sensitivity to doxorubicin in 8226 and 8226 parental cells is not DOX significantly altered in response to dipyridamole exposure ............................................. 67   Figure 2-10: Cilostazol, a PDE3 inhibitor, potentiates the anti-proliferative activity of statins. .......................................................................................................................................... 70   Figure 2-11: Modulators of cAMP induce apoptosis and increase intracellular cAMP levels in AML cells. ........................................................................................................................ 73   ix Thesis – Aleksandra Pandyra Figure 3-1: A genome wide dropout screen uncovers putative shRNAs that potentiate fluvastatin-induced cell death. .......................................................................................... 97   Figure 3-2: HMGCR inhibition by statins triggers a restorative feedback loop in hepatocytes ........................................................................................................................................ 103   Figure 3-3: Line plots of the two best hairpins for each hit chosen for validation show the dropout over time. .......................................................................................................... 105   Figure 3-4: Generation of A549 sublines stably expressing shRNA constructs demonstrates knockdown of protein or mRNA target. ......................................................................... 109   Figure 3-5: shRNAs targeting gene hits identified in the screen potentiate fluvastatin-induced apoptosis and anti-proliferative effects. ......................................................................... 114   Figure 3-6: Transient knockdown of PI4KB sensitizes lung and breast cancer cells to the anti- proliferative effects of fluvastatin. ................................................................................. 118   Figure 3-7: The combination of fluvastatin and a pharmacological inhibitor of PI4KB is anti- proliferative and induces apoptosis in lung and breast cancer cells. .............................. 121   Figure 3-8: Stable knockdown of SREBF2 sensitizes breast cancer MCF7 and MDAMB231 cells to the pro-apoptotic and anti-proliferative effects of fluvastatin. .......................... 125   Figure 3-9: Knockdown of SREBF2 abrogates the upregulation of HMGCR and HMGCS1 upon fluvastatin exposure. .............................................................................................. 128   Figure 3-10: Fluvastatin delays tumour growth in MDAMB231 xenografts. ........................ 130   Figure 3-11: Targeting the MVA pathway in tumour cells. ................................................... 140   x