Discussion

MYC is indisputably a genetic heavy weight. Studies in both Burkitt’s lymphoma and the Drosophila genome have shown that the MYC/ MAX heterodimer occupies approximately 15% of gene promoters in cells (Li et al., 2003; Orian et al., 2003), affecting processes including angiogenesis, apoptosis, differentiation, protein synthesis, and many others (Adhikary & Eilers, 2005; Dang, 1999; van Riggelen et al., 2010). Furthermore, overexpression of MYC has been linked to upwards of 70% of cancers (Dang, 2012) with estimated 100,000 annual U.S. cancer deaths directly caused due to deregulation of MYC activity (Boxer, 2001). Most importantly, we know that inactivation of MYC can lead to tumor regression and apoptosis without adversely affecting normal tissue (Arvanitis & Felsher, 2006; Soucek et al., 2008). It seems abundantly clear that identifying a small molecule disrupting the MYC/MAX heterodimer or modulating c- MYC function in vivo should be very high on the list of anyone who wants to “cure” cancer.

The fact that MYC has been considered “undruggable” for so long due to its lack of typical binding pockets is a golden opportunity— whoever creates a viable treatment targeting the protein will surely achieve greatness. It is through the Koehler Lab, and the novel small molecule MYC and MAX modulators being developed in-house, that I have had the opportunity to contribute to this cause. When considering my own thesis I was inspired by the papers written by Muellner et al. and Stratikopoulos et al. examining inhibitor resistance in the PI3K/AKT/mTOR pathway. Muellner et al. had demonstrated that resistance to PI3K inhibitors was dependent on c- MYC induction, which was reversible through using RNA interference (RNAi) of MYC mRNA (Muellner et al., 2011). Stratikopoulus et al. examined PI3K inhibitor resistance using a metastatic breast cancer mouse model driven primarily by either PIK3CA overexpression or loss of PTEN, finding it was resistant to both a pan-class 1 PI3K inhibitor GDC-0941 and BET inhibitor JQ1, but not both together (Stratikopoulos et al., 2015). Looking at these results I had to ask: what about a combinatorial study using the PI3K inhibitor GDC-0941 in conjunction with the Koehler Lab’s MYC modulators instead of the BET inhibitor JQ1? KI-MS1-001 and KI- MS2-008 would affect MYC/MAX’s transcriptional ability while JQ1 would affect the ability for MYC to even be transcribed, so I thought there might perhaps be interesting things to be learned from comparing these two approaches.

As the first step in pursuit of my thesis, I wanted to examine whether the novel small molecule modulators behaved as heterodi- mer inhibitors. This was an ongoing question in the Koehler Lab at the time, so that we better understood the mechanism of action of these modulators to put their effects in the cell lines in context. I began this step by growing E. coli bacteria overexpressing SBP-tagged MYC protein and working to purify the protein from the rest of the lysate, performing a western blot to confirm MYC was being produced and was purified properly. However, as mentioned earlier, the lightest band is seen from the FT-TEV lane while the darkest band was from the Beads lane, the opposite of what I would have wanted (Figure 2a). Although I was able to purify MYC from the rest of the lysate and the SBP-tag as determined from the presence of the FT-TEV band, an ideal purification would have had a much stronger band for MYC in that lane, and weaker bands in both of the other lanes. Especially the Beads lane made from boiling the leftover Streptavidin agarose beads in Laemmli running buffer, as the only thing remaining on these beads should have been the SBP-TEV tag and not MYC.

One explanation why the western blots were against expectations could be because the AcTEV Protease was older than believed and degrading, and thus wouldn’t be able to catalyze the cleavage reaction as well explaining the little MYC in the FT-TEV. Another reason could be because I changed the protocol after the first attempt to purify came out very poorly from the optimal incubation conditions of 3 hours in a 37° C water bath to an overnight incubation at 4 ° C. Furthermore, I didn’t think to wash the beads after removing the FT-TEV and perhaps there was a lot of cleaved MYC that was just stuck in the beads considering MYC is a very sticky protein. Repeating these purification experiments with new AcTEV Protease would be appropriate, but I decided at Dr. Caballero’s recommendation to simply continue to use the SBP- MYC in further experiments as ultimately the SBP tag is small and would not interfere with the MYC/MAX binding interaction. In addition, it is clear that there was overflow in the lanes of the western blot due to my error in loading them, and a repeated effort should load less sample. Any future attempt to purify SBP-MYC would take these issues into consideration.

To examine whether KI-MS1-001 or KI-MS2-008 inhibited the MYC/MAX heterodimer I first performed co-immunoprecipitation experiments between purified SBP-MYC and pure MAX protein in the presence and absence of the drug compound 10074-G5 to establish data for a positive control (Figure 2b). I expected that increasing concentration of the positive control would lead to decreasing concentration of MAX binding to the SBP-MYC, but this turned out to be harder to prove than anyone had reasonably considered. Generally, an increasing concentration of 10074-G5 caused a progressively lesser concentration of MAX to bind to the SBP-MYC (Figure 3). Unfortunately, there was an extremely small amount of MAX binding to the SBP-MYC in the control sample to which no 10074-G5 was added (Figure 3). Considering that the control within this blot clearly didn’t work, I performed three more co-immunoprecipitations with 10074-G5 and two with PY9, another positive control inhibitor—and yet, all of these blots were very blotchy so I was still unable to either replicate what was seen in Figure 2b or contradict it.

There are a couple of reasons that could explain why these western blots were unsuccessful. One reason could be that 100 μL of streptavidin agarose beads was insufficient to incubate with, although this amount had been standard in the lab. Another problem could have been that incubating the five samples for 1.5 hours at room temperature, prior to adding MAX and after, was not long enough for meaningful binding. A third reason for these difficulties could be due to the binding buffer solution used in incubation steps being a buffer solution from an EMSA kit, potentially causing issues in a co-immunoprecipitation; the choice to use this buffer solution had been made because the original plan was to later perform an EMSA testing whether the positive control inhibitor, KI-MS1-001, or KI-MS2-008 affected MYC/MAX binding to DNA, and I had been advised to prepare these Western blots using similar conditions to what would come later. This preparation proved completely unnecessary as I did not end up having time to perform an EMSA. Either way, I feel that these positive control data are inconclusive as I was ultimately unable to properly replicate results in readable gels.

Following the co-immunoprecipitation with the positive control inhibitors, the plan was to test whether the novel small molecule modulators inhibit the MYC/MAX heterodimer by incubating a solution of purified MAX and SBP-MYC with increasing concentrations of both KI- MS1-001 and KI-MS2-008. Unfortunately, I was also unable to perform this due to time constraints. Therefore, I have instead included in this study co-immunoprecipitation experiments that my second mentor Andrew Chen performed with KI-MS2-008 to use as a comparison to my experiment (Figure 2c). As expected, no bands were seen at all for the flow through or wash lanes, and there was only a light band in the control lane (Figure 2c). I was a bit surprised to see bands for MYC and MAX coming from the control sample incubated with normal IgG rabbit antibody, but this is most likely due to non-specific binding as MYC is known to be a sticky protein (Figure 2c). Most importantly, KI-MS2-008 did not seem to significantly impact the amount of MAX pulled down by MYC which suggests that it in fact does not interfere with in vitro MYC/MAX binding (Figure 2c). In the years following these experiments the Koehler Lab has also compiled more evidence to support this case.

I recognize that a couple key conditions are different between the two protocol designs and should be addressed, but I do not think any of these distinctions would disqualify the data from being a useful comparison: firstly, my experiment was performed using SBP-MYC purified from E. coli lysate while Andrew Chen’s was performed using normal MYC expressed in the Burkitt’s lymphoma cell line P493-6; secondly, my experiment used streptavidin agarose beads while Andrew Chen’s used magnetic Dynabeads; and thirdly, I incubated using EMSA kit binding buffer and Andrew Chen incubated using the Dynabead kit binding buffer. Given time, I would have liked to perform the ideal co-immunoprecipitation and EMSA experiments with both KI-MS1-001 and KI-MS2-008 under similar conditions to the positive control experiment.
Following this first goal, the second goal of my thesis was to characterize the independent IC50s of the four drug compounds in cell viability assays. Given limited time, I chose cell lines different along multiple axes to amplify information gained i.e. the cell lines that overexpressed the PI3K pathway were in different cancer types, breast (MDA-MB-468) and brain (U87MG), and the third cell line was a control with no overexpression of the PI3K pathway (HCC1599). I reasoned that without hard independent data in these three cell lines with these four drugs (pan-class I PI3K inhibitor GDC-0941, BET inhibitor JQ1, and the two MYC and MAX modu- lators KI-MS1-001 and KI-MS2-008), it would be unclear later on whether data produced through a characterization of a synergistic drug combination was actually causing synergistic effects or merely additive ones. Due to a desire to gain more data points from which to better visualize a sinusoidal curve and calculate IC50s of the drug compounds, I chose to perform two-fold serial dilutions from 40 μM to .156 μM and then further to 0.00122 μM.

Interestingly, all of the drug compounds seemed to be capable of reducing cell viability in the HCC15599 cell line: JQ1 (Figures 4a and S4a) and GDC-0941 (Figures 4b and S4b) consistently had IC50s less than 1 μM, while KI-MS1-001 (Figures 4c and S4c) and KI-MS2-008 (Figures 4d and S4d) had higher IC50s, between 4 μM and 14 μM. This was against expectations, as the HCC1599 cell line was expressly chosen because it has neither loss of PTEN nor a PIK3CA mutation. Instead, the cell line is known to be negative for expression of Her2- Neu, p53, estrogen receptor (ER), progesterone receptor (PR). Perhaps the results here, that drugs prospectively meant for the PI3K pathway and MYC/MAX modulators affect the viability of triple negative breast cancer, are the most unexpected gains from my thesis and could be further pursued by the Koehler Lab outside of this study. In the U87MG cell line, it did not seem like any of the drug compounds were particularly effective at all, which combined with the HCC1599 data possibly suggests that there is more of an importance placed on the tissue than the specific mutations than I expected (Figures 5 and S5). Finally, the data from the MDA-MB-468 cell line showed internal contradictions, with all drug compounds having IC50s around 1 μM in the second replicate (Figures 6), but KI-MS1-001 and KI-MS2-008 jumping up in the third replicate (Figures S6c and S6d). One commonly used MYC/ MAX heterodimer inhibitor compound that was discovered in the same study as the 10074-G5 used in this thesis is 10058-F4 (Yin et al., 2003). This compound initially was recorded by Yin et al. as having an IC50 of 49 μM in HL60 cells, but improved synthetic chemistry efforts from the same lab still only led an increases in potency to approximately IC50 = 20 μM (Wang et al., 2007; Yin et al., 2003). I would want to repeat the cell viability assays a few more times in MDA-MB-468 to look for more consistency in IC50 values due to the internal contradictions present between the replicates, but considering the data from the Wang et al. paper I think KI-MS1- 001 and KI-MS2-008 show promise.

During this stage I took care to do both three biological and technical replicates in order to be most thorough, and naturally there were plenty of opportunities for error when collecting data. The potential for human error whenever I was plating drugs, or media, or cells into the hundreds and hundreds of wells over the course of the months is a particularly large problem. A lot of the variation between technical replicates could probably be explained to a deficiency in technique, especially when I was first performing the assays. Performing these experiments with a robotic system would be a good way to avoid this source of error. Additionally, the western blotting to see if there were dose-dependent decreas- ing concentrations of the MYC and p-AKT proteins as a result of increasing concentrations of the drug compounds also did not work well. This is perhaps due to the HCC1599 and MDA-MB-468 cell lines not producing enough MYC considering that they are not known to overexpress that protein to begin with. In the future it would be prudent to first determine exactly how concentrated the total protein in the lysate from these cell lines must be to get a detectable band in western blotting.

The third aim had been to bring the thesis study to a close by characterizing the IC50s of the synergistic combinations of the PI3K/BET inhibitors and PI3K/MYC inhibitors, finally testing the hypothesis that the synergistic effects of the PI3K and MYC inhibi- tors would have a stronger impact on the cell lines and produce a lower IC50 value than the effects of the PI3K and BET inhibitors. As should be clear at this point, I unfortunately wasn’t actually able to perform either a combinatorial or synergistic study of the PI3K inhibitor and MYC/MAX modulators. Therefore, the future directions are clear. Stratikopoulos et al. had demonstrated the BET inhibitors blocked PI3K inhibition resistance caused by exposure to GDC-0941, and PI3K/BET inhibition blocked AKT reactivation (Stratikopoulos et al., 2015). They showed this through in vitro proliferation assays performed while holding one of the compounds at a constant 1 μM and adding to it a serial dilution from 8 μM to .125 μM of the other compound. The Koehler Lab plans on finish- ing my line of questioning by performing cell viability assays using these cancer cell lines; however instead of holding one compound at a constant 1 μM the plan is to vary the concentrations of both compounds along a two-fold serial dilution from 40 μM to .156 μM, in order to more precisely define where the best synergy is.

Through these future studies, we aim to present the novel MYC and MAX modulator compounds KI-MS1-001 and KI-MS2-008 as viable options to put into the cancer researcher’s toolkit. As dysregu- lated transcription factors become increasingly attractive targets for cancer research, developing a small molecule modulator of MYC or MAX function in vivo will only increase in importance as well (Darnell, 2002). In particular, evidence demonstrating elevated lev- els of MYC increases tumor recurrence in PIK3CAH1047R-driven mammary cancers show that MYC is an important oncoprotein that cannot be overlooked even when attempting to study a seemingly unrelated cancer driver (Liu et al., 2011). The clinical significance of MYC cannot be underestimated, especially in consideration of the work done by Stratikopoulus et al. and Liu et al. in showing that a single PI3K inhibitor isn’t enough to use as a treatment for a cancer of the PI3K/AKT/mTOR pathway. Even if the planned synergistic study here does not show that the modulators in their current chemical forms produce better results than the combination of JQ1 and GDC-0941, this wouldn’t be a reason to give up on this line of reasoning. These compounds are continually being improved and optimized by the Koehler Lab with the belief that the failures of today will inform the successes of tomorrow. The main goal is not an immediately translatable treatment, but a solid foundation from which further research and treatments can be built upon.