utilizing sirna screens to identify potentiators of an akt ...

1. E. Randal Hofmann Utilizing siRNA Screens to Identify Potentiators of an AKT Inhibitor for Cancer Drug Target Discovery Today I going to talk about some of my work for drug target discovery in oncology while at GSK with respect the PI3k/Akt pathway. In particular, how we used an siRNA library to identify new drug targets that potentiate an Akt inhibitor.Today I going to talk about some of my work for drug target discovery in oncology while at GSK with respect the PI3k/Akt pathway. In particular, how we used an siRNA library to identify new drug targets that potentiate an Akt inhibitor.

2. I�ll begin with a little background on the PI3K/Akt pathway. Then discuss the rational for potentiators studies. Give a brief overview of the siRNA screen done in our core group at RTP and our analysis. Followed by how we confirmed the hits and finish with the findings and impact these are having on cancer research at GSK.I�ll begin with a little background on the PI3K/Akt pathway. Then discuss the rational for potentiators studies. Give a brief overview of the siRNA screen done in our core group at RTP and our analysis. Followed by how we confirmed the hits and finish with the findings and impact these are having on cancer research at GSK.

3. AKT Plays a Central Role in Tumorigenesis First discovered as the viral oncogene v-akt. Allows for survival of tumor cells under stress due to: Oncogene activation Anoikis Growth factor deprivation Hypoxia Chemo or radiation therapy Three closely related homologues (AKT1, AKT2, AKT3). S/T kinases belonging to the AGC (PKA, PKG and PKC) family. Hyperactivation of AKT is one of the most common molecular events in human malignancy. The discovery of the viral oncogene v-akt over 30 years ago was the first clue for a role of Akt in cell survival and growth. Akt allows for survival of tumor cells under stress due to oncogene activation, anoikis, growth factor deprivation, hypoxia and chemo or radiation therapy. Akt belongs to the AGC family of S/T kinases and there are 3 family members with non-overlapping functions in mouse development. Upon localization to the plasma membrane via it�s PH domain, Akt is phosphorylated in the activation loop at T308 and the carboxy-terminal tail at S473 which lead too it�s full activation. Hyper activation of Akt is one of the most frequent molecular events in human malignancy.The discovery of the viral oncogene v-akt over 30 years ago was the first clue for a role of Akt in cell survival and growth. Akt allows for survival of tumor cells under stress due to oncogene activation, anoikis, growth factor deprivation, hypoxia and chemo or radiation therapy. Akt belongs to the AGC family of S/T kinases and there are 3 family members with non-overlapping functions in mouse development. Upon localization to the plasma membrane via it�s PH domain, Akt is phosphorylated in the activation loop at T308 and the carboxy-terminal tail at S473 which lead too it�s full activation. Hyper activation of Akt is one of the most frequent molecular events in human malignancy.

4. The finding that Akt is constitutively activated in PTEN deficient fibroblasts and glioblastoma cells ~10 years ago placed Akt into the PI3K pathway. This was a landmark discovery because this pathway is commonly activated by RTKs such as IGF1R and EGFR and oncogenes such as Ras and Src. And as we know now, mutations in PI3K and PTEN are some of the most frequently observed mutations in cancer. Akt is able to drive oncogenesis through phosphorylation of its downstream substrates that regulate protein synthesis and cell growth, cell cycle, and evasion of both intrinsic and extrinsic apoptotic stimuli.The finding that Akt is constitutively activated in PTEN deficient fibroblasts and glioblastoma cells ~10 years ago placed Akt into the PI3K pathway. This was a landmark discovery because this pathway is commonly activated by RTKs such as IGF1R and EGFR and oncogenes such as Ras and Src. And as we know now, mutations in PI3K and PTEN are some of the most frequently observed mutations in cancer. Akt is able to drive oncogenesis through phosphorylation of its downstream substrates that regulate protein synthesis and cell growth, cell cycle, and evasion of both intrinsic and extrinsic apoptotic stimuli.

5. The rational for targeting AKt is further supported with evidence from the clinic where we find a high frequency of amplifications or mutations that activate Akt. More recently, activating mutations in the pleckstrin homology domain of Akt 1 has been found to occur at significant frequencies in breast, colorectal and ovarian cancers. AKT2 and AKT3 are also disregulated and their contributions to oncogenesis has been documented. AKT1&2 seem to be functionally redundant for survival in tumor cell lines as inhibition of both is more effective in cell killing than either alone.The rational for targeting AKt is further supported with evidence from the clinic where we find a high frequency of amplifications or mutations that activate Akt. More recently, activating mutations in the pleckstrin homology domain of Akt 1 has been found to occur at significant frequencies in breast, colorectal and ovarian cancers. AKT2 and AKT3 are also disregulated and their contributions to oncogenesis has been documented. AKT1&2 seem to be functionally redundant for survival in tumor cell lines as inhibition of both is more effective in cell killing than either alone.

6. Single agent response is more likely for those drugs whose target is the primary driver of the patients cancer � �oncogene addiction� When single agents fail in this setting it is likely that the target may still be important, it�s role is moderated by other events and so still retains utility as a combination therapy Tumor Models That Carry the Genetic Signature of the Native Malignancy are More Likely to Recapitulate Clinical Behavior The goal of targeted therapy is to inhibit the primary driver of the cancer cells. For example, in cancers where the primary driver is a growth factor receptor such as EGFR, inhibition with a single agent is likely to produce a response. This is your classic example of oncogene addiction. However, as we have seen in the case of EGFR inhibitors, single agent therapy fails when tumor cells acquire other drivers as not all patients with activating EGFR mutations respond to inhibitors. These cells are the ones that will likely require combination therapy. Knowing what these secondary drivers are would make attractive drug targets for combination therapy or even as single agents in cancers where they act as single drivers.The goal of targeted therapy is to inhibit the primary driver of the cancer cells. For example, in cancers where the primary driver is a growth factor receptor such as EGFR, inhibition with a single agent is likely to produce a response. This is your classic example of oncogene addiction. However, as we have seen in the case of EGFR inhibitors, single agent therapy fails when tumor cells acquire other drivers as not all patients with activating EGFR mutations respond to inhibitors. These cells are the ones that will likely require combination therapy. Knowing what these secondary drivers are would make attractive drug targets for combination therapy or even as single agents in cancers where they act as single drivers.

7. GSK690693: AKT Kinase Inhibitor A little over a year ago, GSK published preclinical data for the Akt inhibitor GSK690693, which went into the clinic in late 2007. This compound is a low nanomolar, ATP competitive inhibitor of all three Akts as well as other members of the AGC family. There are also some other kinases that are inhibited by 693, the significance of this inhibition is not known. 693 induces 50% growth inhibition in at least 6 cell lines at concentrations below 1 uM. However, most cell lines tested had gIC50s greater than 1 uM. What are the other drivers, or the mechanism of resistance in these cell lines?A little over a year ago, GSK published preclinical data for the Akt inhibitor GSK690693, which went into the clinic in late 2007. This compound is a low nanomolar, ATP competitive inhibitor of all three Akts as well as other members of the AGC family. There are also some other kinases that are inhibited by 693, the significance of this inhibition is not known. 693 induces 50% growth inhibition in at least 6 cell lines at concentrations below 1 uM. However, most cell lines tested had gIC50s greater than 1 uM. What are the other drivers, or the mechanism of resistance in these cell lines?

8. Tumor Cell Line Sensitivity to GSK690693 is not Defined by a Single Mechanism Sensitivity to inhibition of this pathway is not as simple as looking at the status of Akt activity. While sensitive cell lines do have higher Akt activity, as measured by phosphorylation at serine 473, many less sensitive cell lines also have hyperactivated Akt, often higher than those that are sensitive. Mutations in Kras and Braf likely play a role in resistance but, again, not all resistant cell lines have these mutations. So it is clearly more complex. Understanding the mechanisms, that is, knowing what proteins contribute to resistance to 693 could unveil new targets for cancer therapy. One approach to this question is to use functional genomics. Sensitivity to inhibition of this pathway is not as simple as looking at the status of Akt activity. While sensitive cell lines do have higher Akt activity, as measured by phosphorylation at serine 473, many less sensitive cell lines also have hyperactivated Akt, often higher than those that are sensitive. Mutations in Kras and Braf likely play a role in resistance but, again, not all resistant cell lines have these mutations. So it is clearly more complex. Understanding the mechanisms, that is, knowing what proteins contribute to resistance to 693 could unveil new targets for cancer therapy. One approach to this question is to use functional genomics.

9. Goals of a potentiation study: Identifying pathways or targets for combination therapy. Targets of existing compounds New targets for drug development Defining potentiator(s) to use for building dual specificity into a small molecule or biological. Goal will define the selection of cell lines, doses and scale (e.g. kinase, druggable genome, biopharm genome, or whole-genome screen). Screening siRNA Libraries in Combination with Compounds May Identify Targets for Combination Therapy of Cancer siRNA library screens are one way to identify other drivers or genes that are mediating survival in the presence of a single agent. What we have done is to use a focus set library of kinases and phosphatases to identify genes that when targeted by siRNAs, potentiate the effect of an Akt inhibitor. The goal of potentiation studies are to identify pathways or drug targets of existing compounds or for new drug development. A more ambitious goal would be to identify drug targets that could be used to drive development of back-up inhibitors for building dual specificity into a single small molecule or biological. The goal of a specific program will define the scale and design of the study. siRNA library screens are one way to identify other drivers or genes that are mediating survival in the presence of a single agent. What we have done is to use a focus set library of kinases and phosphatases to identify genes that when targeted by siRNAs, potentiate the effect of an Akt inhibitor. The goal of potentiation studies are to identify pathways or drug targets of existing compounds or for new drug development. A more ambitious goal would be to identify drug targets that could be used to drive development of back-up inhibitors for building dual specificity into a single small molecule or biological. The goal of a specific program will define the scale and design of the study.

10. siRNA Reagent Overview Typically used for in vitro studies where assay length is ~5-7 days Delivery into the cells must be efficient Commercially available lipid or peptide reagents � work best with transformed adherent cell lines Technologies are improving for primary cells and suspension cell lines Vendors utilize bioinformatic approaches or chemical modifications to select highly specific siRNA Dharmacon On Target Plus SMART Pools: 2�-O-methylation of the ribosal ring of key positions on the sense strand and in the �seed� region of the guide strand decreases thermo stability of off-target mechanisms Researchers have taken advantage of this process as a tool to silence expression of specific genes to understand their function. In mammalian cells, the first reagents and still the most commonly used reagents are synthesized siRNAs. These are typically used for in vitro studies where the assay length is about 5-7 days. The use of siRNA reagents requires efficient delivery into cells and there are many commercially available lipid and peptide based reagents. These usually work best in transformed and adherent cell lines, however, technologies are improving for delivery into primary and suspension cells. siRNAs have been designed by vendors using algorithms and more recently chemical modifications in an attempt to select highly potent and selective siRNAs. For example, Dharmacon sells reagents called�.Researchers have taken advantage of this process as a tool to silence expression of specific genes to understand their function. In mammalian cells, the first reagents and still the most commonly used reagents are synthesized siRNAs. These are typically used for in vitro studies where the assay length is about 5-7 days. The use of siRNA reagents requires efficient delivery into cells and there are many commercially available lipid and peptide based reagents. These usually work best in transformed and adherent cell lines, however, technologies are improving for delivery into primary and suspension cells. siRNAs have been designed by vendors using algorithms and more recently chemical modifications in an attempt to select highly potent and selective siRNAs. For example, Dharmacon sells reagents called�.

11. An siRNA Screening Approach to Identify GSK690693 Potentiators The potentiator screen was run by our core group in RTP. Briefly, using a kinase and phosphatase library, siRNAs targeting a single gene were arrayed in 96 well plates for transfection of the 7 resistant cell lines. Cells were then treated with either 693 or DMSO and proliferation data was captured 3 days later using the Cell Titer Glo assay from Promega. The data was analyzed for combinations of siRNAs and compound that were greater than additive and sensitized multiple cell lines to 693. In the end, we chose to follow-up on 26 candidates for confirmation studies hoping to find new targets and a better understanding of the pathways involved that may lead to biomarkers.The potentiator screen was run by our core group in RTP. Briefly, using a kinase and phosphatase library, siRNAs targeting a single gene were arrayed in 96 well plates for transfection of the 7 resistant cell lines. Cells were then treated with either 693 or DMSO and proliferation data was captured 3 days later using the Cell Titer Glo assay from Promega. The data was analyzed for combinations of siRNAs and compound that were greater than additive and sensitized multiple cell lines to 693. In the end, we chose to follow-up on 26 candidates for confirmation studies hoping to find new targets and a better understanding of the pathways involved that may lead to biomarkers.

12. This was done by retesting all 26 hits in all seven cell lines and capturing Taqman data. We used the following scheme to do this. This was done by retesting all 26 hits in all seven cell lines and capturing Taqman data. We used the following scheme to do this.

13. We repeated this two more times for most cell lines and included FACS to measure cell cycle and cell death effects. Potentiation was determined taking into account the drug effect and the siRNA effect. We repeated this two more times for most cell lines and included FACS to measure cell cycle and cell death effects. Potentiation was determined taking into account the drug effect and the siRNA effect.

14. Out of the 26 hits we followed up, 17 of them reproducibly potentiated 693 in at least one cell line. Pathway analysis of these genes and there function revealed two predominant mechanisms: inhibition of the MAP kinase pathway and sensitization to apoptosis. This suggests that these cells are depending on the MAPK pathway when Akt activity is blocked much like Akt activation protects cells when EGFR activity is blocked. And since Akt protects against apoptosis, one would expect that inhibition of Akt would lead to greater cell death when other survival genes were inhibited. Some outliers to these two mechanisms were genes that regulate metabolic pathways, including glycolysis.Out of the 26 hits we followed up, 17 of them reproducibly potentiated 693 in at least one cell line. Pathway analysis of these genes and there function revealed two predominant mechanisms: inhibition of the MAP kinase pathway and sensitization to apoptosis. This suggests that these cells are depending on the MAPK pathway when Akt activity is blocked much like Akt activation protects cells when EGFR activity is blocked. And since Akt protects against apoptosis, one would expect that inhibition of Akt would lead to greater cell death when other survival genes were inhibited. Some outliers to these two mechanisms were genes that regulate metabolic pathways, including glycolysis.

15. There are at least three scenarios that could explain the mechanism of potentiation for the hits identified in the screen. In the first scenario, both AKT and the potentiating kinase are in parallel pathways. Another scenario puts the cooperating kinase in the same pathway where it is effecting AKT signaling. The identity of a few of the hits suggest the third scenario is a real possibility as inhibition of off-target kinases could be mediating the effect. Although this is just preliminary results, one thing we wanted to begin to look at was the impact of these siRNAs on the AKT activation as this has been reported to be one mechanism of co-operation in a similar study. We were able to identify at least three hits that seem to impact the levels of AKT phosphorylation levels on their own suggesting they may act by sensitizing the cells to the inhibition of AKT. Interestingly, two of these hits also impact the levels of phospho ERK1/2 suggesting a second mechanism of co-operation.There are at least three scenarios that could explain the mechanism of potentiation for the hits identified in the screen. In the first scenario, both AKT and the potentiating kinase are in parallel pathways. Another scenario puts the cooperating kinase in the same pathway where it is effecting AKT signaling. The identity of a few of the hits suggest the third scenario is a real possibility as inhibition of off-target kinases could be mediating the effect. Although this is just preliminary results, one thing we wanted to begin to look at was the impact of these siRNAs on the AKT activation as this has been reported to be one mechanism of co-operation in a similar study. We were able to identify at least three hits that seem to impact the levels of AKT phosphorylation levels on their own suggesting they may act by sensitizing the cells to the inhibition of AKT. Interestingly, two of these hits also impact the levels of phospho ERK1/2 suggesting a second mechanism of co-operation.

16. To do this we simply deconvoluted the pool of siRNAs into their individual motifs. What we found was only one of the motifs had a dramatic effect on proliferation. When we looked at the protein levels of these cells, we found that all 4 motifs were knocking down the protein to various degrees. This kinase is one member of a family of six closely related kinases so we also looked at the protein levels of other family members that were expressed in this cell line. What we found was that the motif that was effective in inhibiting growth was also reducing the protein levels of 2 other kinases in this family. To do this we simply deconvoluted the pool of siRNAs into their individual motifs. What we found was only one of the motifs had a dramatic effect on proliferation. When we looked at the protein levels of these cells, we found that all 4 motifs were knocking down the protein to various degrees. This kinase is one member of a family of six closely related kinases so we also looked at the protein levels of other family members that were expressed in this cell line. What we found was that the motif that was effective in inhibiting growth was also reducing the protein levels of 2 other kinases in this family.

17. While siRNA screens that utilize biochemical assays are robust and have the advantage of a relatively simple work flow and reproducibility, they offer less insight into the cell biology. One of the most attractive features of the first genome wide RNAi screen was that they could screen for multiple phenotypes that could be visualized under a microscope. As you are all aware of now, there are multiple options now for doing relatively high through put screens with automated microscopy. These high content imagers allow us now to assay for a number of phenotypes such as�.and many of the vendors have improved the software for these instruments for rapid assay set up and on the fly analysis.While siRNA screens that utilize biochemical assays are robust and have the advantage of a relatively simple work flow and reproducibility, they offer less insight into the cell biology. One of the most attractive features of the first genome wide RNAi screen was that they could screen for multiple phenotypes that could be visualized under a microscope. As you are all aware of now, there are multiple options now for doing relatively high through put screens with automated microscopy. These high content imagers allow us now to assay for a number of phenotypes such as�.and many of the vendors have improved the software for these instruments for rapid assay set up and on the fly analysis.

18. At GSK, I was in charge of evaluating several of these instruments for our department and the one we found best for our needs was the MD image express. The hope was, before the re-structuring, was that we could use high content imaging in combination with siRNA libraries as a more powerful means to identify genes that affect the AKT pathway, for example, hear we used a FOXO-GFP reporter cell line during the demo of the instrument to evaluate nuclear translocation in response to inhibition of the pathway. At GSK, I was in charge of evaluating several of these instruments for our department and the one we found best for our needs was the MD image express. The hope was, before the re-structuring, was that we could use high content imaging in combination with siRNA libraries as a more powerful means to identify genes that affect the AKT pathway, for example, hear we used a FOXO-GFP reporter cell line during the demo of the instrument to evaluate nuclear translocation in response to inhibition of the pathway.

19. It is important however, to try different methods of analysis to determine which method best reflects the other relevant biology, in this case, cell killing as the different methods can give significantly different results.It is important however, to try different methods of analysis to determine which method best reflects the other relevant biology, in this case, cell killing as the different methods can give significantly different results.

20. Found that it was possible to identify reproducible potentiators of small molecules using siRNA screening. However, potentiation was modest at best. Indication of synergistic inhibition by targeting Akt and targets in the current GSK portfolio. Inhibitors of targets other than EGFR or MEK1/2 in the MAPK pathway may provide an improved therapeutic outcome in combination with GSK690693. Two of the RTKs have been added to the Biopharm portfolio of new targets. Summary of Potentiator Studies One of my goals for this project was to determine if these studies could identify reproducible potentiators. While we were able to demonstrate this, we did not find any homeruns, that is, a target that could dramatically shift the EC50 towards sensitivity. Our best potentiators are themselves very effective at inhibiting cellular proliferation when inhibited by siRNAs an therefore are potentially good stand alone targets that could provide a benefit in combination therapy. We did find that there may be an indication of synergistic inhibition with 693 with some of the compounds in the oncology portfolio and these studies are currently ongoing. We also found that there are other possible avenues to inhibiting the MAPK pathway that may provide an improved therapeutic outcome in combo with 693. It is clear that one will miss things with these types of studies and it is the follow up studies that require the most resource. One of my goals for this project was to determine if these studies could identify reproducible potentiators. While we were able to demonstrate this, we did not find any homeruns, that is, a target that could dramatically shift the EC50 towards sensitivity. Our best potentiators are themselves very effective at inhibiting cellular proliferation when inhibited by siRNAs an therefore are potentially good stand alone targets that could provide a benefit in combination therapy. We did find that there may be an indication of synergistic inhibition with 693 with some of the compounds in the oncology portfolio and these studies are currently ongoing. We also found that there are other possible avenues to inhibiting the MAPK pathway that may provide an improved therapeutic outcome in combo with 693. It is clear that one will miss things with these types of studies and it is the follow up studies that require the most resource.

21. Acknowledgements Cancer Research Heather Jackson Liz Docherty Tomas Vilimas Reannon Holland Debbie Jones Steve Blakemore Caretha Creasy Rakesh Kumar

22. Trasfection of �Clean� siRNA Motifs Fails to Inhibit Proliferation or Modify Dowstream Effectors So it was possible that there was redundancy amongst the family members so we used �clean� motifs of the kinases that seem to be targeted by the one motif in combination. Even knockdown of all three kinases didn�t result in growth inhibition. One possible explanation is that we are not sufficiently blocking signaling to putative downstream substrates. Or, that these kinases aren�t essential for survival in these conditions.So it was possible that there was redundancy amongst the family members so we used �clean� motifs of the kinases that seem to be targeted by the one motif in combination. Even knockdown of all three kinases didn�t result in growth inhibition. One possible explanation is that we are not sufficiently blocking signaling to putative downstream substrates. Or, that these kinases aren�t essential for survival in these conditions.