Muhammed Ors ’17
One of the most common aberrations in human cancers is the overexpression of MYC, a master regulator transcription factor which functions in a heterodimer with the MYC-associated protein X (MAX) (Arvanitis & Felsher, 2006). Previously, it was shown that transduction of MYC caused resistance to PI3K inhibitor GDC-0941 (Muellner et al., 2011), and that the drug JQ1 inhibited MYC expression-induced PI3K rescue through JQ1’s inhibitory effects on the protein BRD4, which functions as a MYC enhancer (Stratikopoulos et al., 2015).
Here we use novel small molecule modulators of MYC transcriptional activity, KI-MS1- 001 and KI-MS2-008, in a comparative study across three human cell lines against the aforementioned PI3K inhibitor GDC-0941 and BET inhibitor JQ1. Co-immunoprecipitation experiments between purified Streptavidin Binding Protein (SBP) tagged MYC, the SBP tag being used to purify the MYC from whole cell lysate, and pure MAX protein in the presence and absence of the compounds imply that the novel MAX modulator KI-MS2-008 does not disrupt the MYC/MAX heterodimer binding, but data also seemed inconclusive and more work needed to be done for a definitive answer. Cell viability assays in the breast lines HCC1599 and MDA-MB-468 confirms that KI-MS1-001 and KI-MS2-008 (in addition to JQ1 and GDC-0941) reduces the viability of these cell lines, more so than these modulators have in previous studies done by the Koehler lab in other lines, but the brain line U87MG showed non convergent IC50 values for all compounds. Experiments are currently being done to determine if the compounds lower MYC and MAX protein levels in a dose dependent fashion in these cell lines. While this work does not rigorously support the hypothesis that the novel modulators would perform better than the other compounds, it contributes preliminary data for any future work the Koehler Lab will do that could more thoroughly test this hypothesis and optimize the modulators.
MYC and Justification for its Therapeutic Potential
A transcription factor is a protein that can bind to DNA to help control the rate of transcription. Dysregulated transcription factors can lead to unregulated cell growth and gene expression and therefore is a key therapeutic target in cancer research (Darnell, 2002).
Transcription factors as a class of therapeutic targets that would be both unique enough that drugs could be designed specifically to target the transcription factor of interest, and broad enough that treatment techniques would be more than just a single-disease solution.
MYC is a master regulator transcription factor due to its pervasive necessity in transcriptional activity; MYC is part of the MYC/MAX/MAD family of basic helix-loop-helix leucine zipper (bHLHZip) domain transcription factors (Adhikary & Eilers, 2005). It functions in a heterodimer with the protein MAX, the two intrinsically disordered individual proteins undergoing a coupled folding and binding to DNA and a number of additional cofactors in order to then either activate or repress transcription (Adhikary & Eilers, 2005). As demonstrated both in Burkitt’s lymphoma and in a study of the transcriptional network of Drosophila genome, the
MYC/MAX heterodimer occupies approximately 15% of gene promoters in cells, healthy and cancerous (Li et al., 2003; Orian et al., 2003). These occupied genes are incredibly diverse: MYC regulates cellular processes such as angiogenesis, apoptosis, differentiation, growth, metabolism, proliferation, protein synthesis, ribosomal biogenesis, and self-renewal (Adhikary & Eilers, 2005; Dang, 1999; van Riggelen, Yetil, & Felsher, 2010). In any one of these fields, MYC would be taken seriously as an important protein. As a result, the overexpression of MYC is one of the most common aberrations in human cancers, having been linked to upwards of 70% of cancers (Dang, 2012). Naturally, these aberrations range greatly from uncontrolled cell proliferation and immortalization to escape from immune surveillance and growth factor independence (Vita & Henriksson, 2006). Current estimates directly attribute 100,000 annual cancer deaths in the U.S. to the deregulation of MYC activity (Boxer, 2001).
One important concern when considering to target MYC as a cancer therapy is that for the same reason that inhibiting dysregulated MYC would be widely applicable and desirable, it could also cause dangerous toxicity to the healthy cells that rely on MYC activity for normal processes (Soucek et al., 2008). Fortunately, research has shown inactivation of MYC leads to tumor regression and apoptosis, and systemic effects of MYC inhibition are well tolerated and are completely reversible (Arvanitis & Felsher, 2006; Soucek et al., 2008). Furthermore, in osteogenic sarcoma even brief inactivation of MYC was sufficient, and reactivation of MYC did not restore the cancer (Jain et al., 2002). An important consideration for why systemic MYC inhibition may not be as harmful as once feared is because at any given time the normal cells in the body are quiescent and do not express much MYC anyway; side effects may not be any worse than side effects occurring in hematopoietic and gastrointestinal tissues – cells that are known to replicate often – due to current medicines (Prochownik & Vogt, 2010).
While effects of MYC inhibition appear to be tolerable for normal cells, this is decisively not the case for cancer cells where dysregulation of MYC is a big driver in their tumorigenesis. In these cases, cancers ranging from lymphoma, leukemia, osteosarcoma, and many more, inactivation of the MYC pathway alone can lead to sustained tumor regression through proliferative arrest, apoptosis, senescence, etc. (Felsher, 2010). This phenomenon is referred to as oncogene addiction: when a tumor heavily dependent on a single oncogenic pathway dies due to inhibition of that pathway. Therefore, it is clear that identifying a small molecule that disrupts the MYC/MAX heterodimer or otherwise modulates MYC function in vivo can have immense implications in drug design, cancer treatment, and survival rates. Unfortunately, MYC historically being considered “undruggable” due to lack of typical binding pockets has stunted research on it (Darnell, 2002). However, this moniker may be more outdated than accurate: research on a variant ETS transcription factor (ETV1) that undergoes undergoes chromosomal translocation in prostate cancers and Ewing sarcomas, among other conditions, using small-molecule microarray screens found a drug-like compound named BRD32048 that binds ETV1 directly and modulates its transcriptional activity (Pop et al., 2014). Simply considering oncogenic transcription factors “undruggable” is no longer good enough.
MYC Modulators Past and Present
With the goal of finding inhibitors of the MYC/MAX heterodimer, Yin, Giap, Lazo and Prochownik screened a chemical library of 10,000 low molecular-weight compounds using a yeast two-hybrid system in which disruption of the MYC/MAX heterodimer led to a decrease in beta-galactosidase activity (Yin, Giap, Lazo, & Prochownik, 2003). From this, seven compounds were found to be specific inhibitors of the MYC/MAX heterodimer in a control yeast strain (Yin et al., 2003). Compounds from this initial set have since been used as positive controls in inhibition experiments, although synthetic chemistry work has been done to try and increase their potency; one compound in particular, 10058-F4 (IC50 = 49 µM, in HL60 cells), can inhibit growth of fibroblasts, 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).
The Koehler Lab has been working to discover and develop new probes with better potency or selectivity over these existing MYC/MAX modulators. Previously, Bradner, McPherson, and Koehler developed small-molecule microarrays (SMMs), glass microscope slides onto which solid substrates have been arrayed with the purpose of identifying potential small molecule modulators for transcription factors (Bradner, McPherson, & Koehler, 2006). These SMMs were developed using a novel isocyanate-based covalent attachment technique which allows for the inclusion of compounds like bioactive small molecules, FDA-approved drugs, synthetic drug-like compounds, and natural products into screens—compounds which had not been intentionally synthesized to be arrayed onto a solid substrate, but because they had certain functional groups on them, like thiols, primary alcohols, and amines, the researchers were able to adhere them to the slides (Bradner et al., 2006). Following the attachment of compounds, SMMs are incubated with pure protein of interest or cellular lysate, with protein-small molecule interactions being detected through fluorescence (Bradner et al., 2006). Using this novel SMM development method, Clemons et al. performed a screen of more than 20,000 compounds against 100 transcription factor proteins, including MYC and MAX, and identified potential small molecule binders to MYC and MAX (Clemons et al., 2010). Koehler then performed another screen against pure MYC and pure MAX of more than 40,000 compounds, including ones previously identified in the Clemons et al. screen. It is from here that the prospective MYC/MAX modulators KI-MS1 and KI-MS2 emerged, and this is currently being written into another paper.
The goal of this unbiased-binding approach was to find novel direct probes of MYC independent of it being bound to MAX, for this could provide new information to the field as all known direct probes of MYC have been found through binding against the MYC/MAX heterodimer. The 313 positive hits discovered through the SMMs were subsequently evaluated in secondary reporter gene assays in HEK293T cells; 32 of these compounds specifically inhibited MYC-dependent transcription. Currently, the Koehler Lab is characterizing the mechanisms of action of the most promising of these hit compounds using cell-free and cell- based approaches, and further chemically optimize their potency and selectivity. An ultimate goal is to use these putative MYC modulators in translational studies involving actual cancers where dysregulated MYC has a large impact and thus break the ground around exploring therapeutic modulation of an “undruggable” oncogenic transcription factors like MYC.
MYC’s Role in the PI3K/AKT pathway
While inhibition of proteins and enzymes with roles in important kinase pathways provide attractive approaches to cancer treatment, sustaining this inhibition has proven to be a major challenge. One such pathway that develops inhibitor resistance is the PI3K/AKT/mTOR pathway, which is centered on phosphoinositide-3 kinase (PI3K) and is important in regulating processes involved in the cell cycle, proliferation, and longevity (Figure 1). Relevantly, MYC has been proven to be key player in it: during a study on the PI3K pathway using PIK3CAH1047R– initiated mammary tumors Liu et al. found that the tumors able to escape oncogene addiction had elevated levels of MYC, with knockdown of MYC reducing the incidence of recurrent tumors (Liu et al., 2011). Muellner et al. similarly observed that MYC mRNA translation and MYC transcriptional activity were amplified more often among human cell lines resistant to PI3K- mTOR inhibitors, and subsequently demonstrated that resistance to PI3K inhibitors was dependent on MYC induction (Muellner et al., 2011). Furthermore, RNA interference (RNAi) of MYC mRNA reversed the observed inhibitor resistance, showing its necessity and sufficiency for PI3K-mTOR resistance (Muellner et al., 2011).
Inspired by these results, Stratikopoulus et al. pursued a strategy to overcome PI3K inhibitor resistance using a metastatic breast cancer mouse model driven primarily by proteins PI3K and MYC. To make this model they crossed mice strains that expressed MYC mutations to strains that had either PIK3CA overexpression (H1047R mutation) or loss of PTEN expression (Ptenflox/flox) to create strains that overexpressed both MYC and PI3K, referred to as PI3K;MYC mice (Stratikopoulos et al., 2015). This model resisted a pan-class 1 PI3K inhibitor that hit both p110α and p110β subunits (GDC-0941) through feedback activation of tyrosine kinase receptors (RTKs), AKT, mTOR, and MYC. Another experiment showed their mouse model was also resistant to inhibitors of bromodomain and extra terminal domain (BET) proteins, which recognize acetylated-lysine residues in nucleosomal histones and facilitate recruitment of transcription proteins to chromatin. Stratikopoulos et al. chose to use the BET inhibitor JQ1 in order to target a member of the BET protein family BRD4, due to the fact that JQ1 had been previously shown to suppress MYC activity by downregulating MYC transcription and thereby causing potent anti-proliferative effects associated with cell cycle arrest and cellular senescence (Delmore et al., 2011; Stratikopoulos et al., 2015). Stratikopoulus et al. found that BET and PI3K inhibitors combined led to a sustained decrease of signaling of PI3K and proteins downstream of it in cell lines from the PI3K;MYC mice, causing cancer death and tumor regression both in vitro and in vivo (Stratikopoulos et al., 2015). They believe these results provide an appropriate step forward in pursuit of preventing the rescue of PI3K inhibition (Stratikopoulos et al., 2015).
Project and Hypothesis
This study seeks to capitalize on the preliminary data and approach communicated by Muellner et al. and Stratikopoulos et al. by performing an in vitro synergistic study of PI3K and BET inhibitors—but with the additional dimension of directly modulating MYC activity using the novel compounds of the Koehler Lab. The cell lines used include commercially available human cell lines corresponding to two breast cancers, HCC1599 and MDA-MB-468, and one brain cancer, U87MG. The latter two were chosen due to the loss of PTEN expression and/or mutation in PIK3CA which leads to overexpression of the PI3K pathway, and because both were present in the Stratikopoulos et al. study. HCC1559 will be a control line which does not have either loss of PTEN or a PIK3CA mutation. The compounds used are JQ1 and GDC-0941 from the previous study, and the Koehler Lab’s KI-MS1-001 and KI-MS2-008. The first aim is to determine if the MYC modulators disrupt the MYC/MAX heterodimer. The second aim is to characterize the independent IC50s of the four inhibitor compounds using cell viability assays, followed by western blotting to look for any dose-dependent decrease in MYC and p-AKT protein following incubation. The third aim, which was not completed during the thesis project period, involves determining the IC50s of the synergistic combinations of the PI3K/BET and PI3K/c- MYC inhibitors. The hypothesis is that the synergistic effects of the PI3K/MYC inhibitors will produce a lower IC50 than those of the PI3K/BET inhibitors.
Purification of SBP Tagged MYC
We grew bacteria expressing high amounts of SBP-MYC by using a frozen glycerol stock of E. coli bacteria which had previously been transformed by Dr. Caballero with a MYC vector pET28-6xHis-SBP-TEV (Addgene). After separating the SBP-MYC soluble lysate from the insoluble pellet, my next goal was to purify the SBP-MYC from the rest of the proteins in the cell lysate, and also the MYC protein from the SBP tag. I performed a Coomassie stain and a western blot to check the efficacy of the purification approach. The purified MYC would be found in the sample named FT-TEV on the blot, the supernatant separated from the streptavidin agarose beads after incubation with the TEV Reaction Mix (Figure 2a). A sample of the flow through after the overnight incubation was saved and named FT-Beads as a way to provide a comparison point to the purity of the isolated SBP-MYC (Figure 2a). The FT-Beads sample would be used to demonstrate just how much of the SBP-MYC is left unbound to the Streptavidin agarose beads after incubation, though admittedly this is perhaps not a positive or negative control exactly—I reasoned that it would provide an internal control of the efficacy of incubation conditions. Furthermore, a second internal control was devised to look at the efficacy of the protease reaction by simply adding Laemmli running buffer to the Streptavidin agarose beads following the TEV reaction, creating a sample that would prospectively show what was left attached to the beads and was unable to be isolated through the purification (Figure 2a).
The western blot showed MYC bands around 57 kDa for all three lanes, FT-Beads, FT- TEV, and Beads, which corresponds to the molecular weight of MYC (57-60 kDa) according to anti-MYC antibody manufacturer’s information, though the Koehler Lab has many times seen MYC run closer to 50 kDa. The lightest band is seen from the FT-TEV lane, while the darkest band was from the Beads lane (Figure 2a). From these results, the greatest concentration of MYC remained attached to the streptavidin agarose beads instead of being properly cleaved by the AcTEV Protease reaction. In order to produce cleaner and better quality gels to look at I performed these experiments twice, and the second run provided the images used for Figure 2a. However, the results did not change between these two runs and there was not enough time to try again. Considering that cleaving the SBP tag was not absolutely necessary to use the protein, I decided after talking to Dr. Caballero to change the original plan for the rest of the experiments and simply perform them with the SBP-MYC.
Novel Compounds Do Not Disrupt MYC/MAX heterodimer
The purpose of these experiments was to ask whether the modulators disrupt the MYC/MAX heterodimer binding. To that end, I chose to look at this question in vitro through co- immunoprecipitation assays between MYC and MAX in the presence and absence of modulator compounds, using the SBP-MYC lysate produced previously as my source of MYC. This first set of assays use the positive MYC/MAX heterodimer inhibitors 10074-G5 or KJ-Pyr-9 as the compounds modulating the interaction between SBP-MYC and MAX; the purpose of this set of assays was to behave as a large control for all of the co-immunoprecipitation assays that were to follow. Within this assay protocol, the negative control was a sample prepared without SBP- MYC, to determine if MAX would be pulled down by the streptavidin agarose beads in the absence of SBP-MYC. The positive control tubes were prepared with increasing concentrations of the MYC/MAX heterodimer inhibitors 10074-G5 (25 µM, 50 µM, and 100 µM) or KJ-Pyr-9 (10 µM, 15 µM, 20 µM). Additionally, all samples were kept at a final concentration of 1% DMSO to be consistent with conditions, as this is what the drug compound stock solutions had been diluted in.
Blots were visualized through chemiluminescence using the ChemiDoc MP Imaging System machine (Bio-Rad) and they were further analyzed using the Image Lab program (Bio- Rad) in order to ascertain the relative intensities of the visible bands (Figure 3). Bands for MYC were seen in all lanes but the lane where there was no SBP-MYC lysate added, as expected (Figure 2b). However, amongst the bands for MAX while there seemed to be a decrease in MAX protein present as 10074-G5 increases in concentration, I was surprised to see a band for MAX coming from the MYC-absent sample and no band from the sample incubated without the inhibitor compound (Figure 2b). The Image Lab program analysis of band intensity confirmed that while the MAX protein concentration was decreasing as 10074-G5 concentration was increasing from 25 µM to 100 µM, as evidenced by the decreasing ratio of MAX/MYC present in each lane, this was not the case when the concentration of 10074-G5 was 0 µM (Figure 3b).
Considering the unexpected developments encountered here, I performed technical replicates with the same lysate and biological replicates with another round of purified SBP-MYC lysate. However, I was unable to get data that would either replicate what was seen in Figure 2b or contradict it—all of the other gels were very blotchy with undecipherable bands. Therefore, I have chosen to include the clearest looking image in this thesis (Figure 2b).
Following co-immunoprecipitations done with incubation with the positive control compounds, the next step would be to use the small molecule modulators developed in lab, currently designated as KI-MS1-001 and KI-MS2-008. This would have been done to test the hypothesis that these too would inhibit the MYC/MAX heterodimer. Unfortunately, due to time constraints and concerns about moving onto other experiments for the thesis, I could not personally test this hypothesis. Luckily, my mentor Andrew Chen was already working on this question independently and allowed me to include his results here to serve as a comparison to the previous step (Figure 2c). Within this assay protocol there were two negative controls in a sense: a sample prepared with only DMSO, without either anti-MAX (C-17) antibody or KI-MS2-008, and secondly, for each condition tested three washes were also run as an internal control, as there should have been no proteins of interest visible from these wash steps. The incubation condition of anti-MAX (C-17) antibody without KI-MS2-008 would be the positive control, as the antibody should bind to MAX which would in turn pull down its heterodimeric partner MYC.
The blot was visualized through chemiluminescence using the ChemiDoc MP Imaging System machine (Bio-Rad). No bands were visible for the flow through (1% loaded) or wash (5% loaded) lanes, but there was a faint band for MYC immunoprecipitated from the control sample incubated with normal IgG rabbit antibody—likely due to non-specific binding (Figure 2c). Most importantly, KI-MS2-008 did not seem to significantly impact amount of MAX pulled down by MYC, suggesting it does not interfere with in vitro MYC-MAX binding (Figure 2c).
KI-MS1-001 and KI-MS2-008 Reduce Cancer Cell Viability
The human cancer cell lines used to perform the cell viability assays were two breast cancer cell lines, HCC1599 (suspension) and MDA-MB-468 (adherent), and one brain cell line, U87MG (adherent). U87MG and MDA-MB-468 both have loss of PTEN expression and/or mutations in PIK3CA which leads to overexpression of the PI3K pathway, and additionally both were present in the Stratikopoulos et al. study in a limited manner. HCC1599 has neither loss of PTEN nor a PIK3CA mutation and is meant to be a control line. These cell lines were chosen because they were all readily available from the MIT Koch Institute’s high throughput screening facility. The purpose in doing cell viability assays was to characterize the independent IC50s of the pan-class I PI3K inhibitor GDC-0941, the BET inhibitor JQ1, and the two MYC and MAX modulators KI-MS1-001 and KI-MS2-008.
In the first biological replicate all four drug compounds were serially diluted two-fold in DMSO from 40 µM to 0.156 µM (results not included in this manuscript). The second and third replicates were further serially diluted from 40 µM to 0.00122 µM due to a desire to better visualize a sinusoidal curve to calculate IC50s from. Cell lines incubated in compounds or DMSO and for both 3 days and 5 days, after which that plate would be visualized. These time points parallel those tested in the Stratikopoulos et al. paper. CellTiter-Glo was used to measure the amount of ATP present in the wells of the plates through luminescence, as these values are considered to be a standard indicator of metabolically active cells, and therefore a reasonable method to draw conclusions on cell viability (Riss, Moravec, & Niles, 2011).
Luminescence values were graphed, and IC50 values calculated, using Prism software (Graphpad). I performed three biological replicates and three technical replicates for each of the conditions tested. The technical replicates are accounted for through the error bars appearing on the graphs, and I have chosen to include both the second and third replicates as they each give interesting and partially conflicting information about the cell lines.
In the HCC1599 cell line all of the drug compounds seemed to be capable of reducing cell viability, with JQ1 (Figures 4a and S4a) and GDC-0941 (Figures 4b and S4b) consistently having 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 on Day 5. All drug compounds displayed lower IC50 values on Day 5 in comparison to Day 3, which would be expected; JQ1 seemed to be the most potent out of all four, going as low as 0.002 µM (Figure S4a). In the U87MG cell line, none of the drug compounds were particularly effective, with almost all of the IC50 values for the drugs being greater than 30 µM (Figures 5 and S5), and JQ1 even having an IC50 value that actually went up from 24 µM on Day 3 to 32 µM Day 5 (Figure 5a). Interestingly, in the third biological replicate while the Day 5 IC50 values for JQ1, GDC-0941, and KI-MS2- 008 (Figures S5a, S5b, S5d) could not be determined because the graphs did not converge, KI- MS1-001 alone had an IC50 value of 3.7 µM at Day 5 (Figure S5c). The data from the MDA- MB-468 cell line were especially interesting as KI-MS1-001 and KI-MS2-008 showed very different IC50s between the second and third biological replicates. All drug compounds had IC50s around 1 µM in the second replicate (Figure 6) but in the third replicate KI-MS1-001 went up to 5.5 µM (Figure S6c) and KI-MS2-008 jumped up to 30.5 µM (Figure S6d).
Following testing of cell viability under compound and DMSO conditions, HCC1599 and MDA-MB-468 were also prepared for western blotting against MYC, p-AKT, and GAPDH in order to test for potential dose-dependent differences in protein expression. I chose to incubate the cells for 24 hours because that was the time frame the Stratikopoulus et al. paper used and I wanted to be as comparable as possible. I chose to incubate with two-fold serial dilutions from 10 µM to 0.156 µM because the measured IC50s seemed to consistently fall within that range.
Total protein concentration was determined using a BCA assay in order to be able to prepare samples for the gel to have a consistent 30 µg concentration of protein, otherwise the comparison of bands would not be particularly meaningful. However, by the time the project ended I had been unable to produce any western blot data that showing the MYC protein present in the lysate.
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 heterodimer 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 modulators 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 decreasing 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 inhibitors 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 finishing 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 dysregulated 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 levels 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.
MATERIALS AND METHODS
Bacterial Expression of SBP tagged MYC
SBP-MYC was purified from E. coli bacteria previously transformed by Dr. Caballero with a MYC vector pET28-6xHis-SBP-TEV (Addgene). A frozen glycerol stock of this bacteria was thawed and incubated overnight at 37° C using 25 mg/mL Kanamycin (ThermoFisher) and 25 mg/mL Chloramphenicol (ThermoFisher) in Lysogeny Broth media (Boston Bio Products). The next day a sample of this was incubated in a 500 mL culture of media at the same temperature and same antibiotic concentration. When the OD600 of the bacteria was measured to be between 0.5-1, and thus in the exponential growth stage, bacterial growth was slowed on ice and expression of MYC with a streptavidin binding peptide (SBP) and Tobacco Etch Virus (TEV) tag attached was induced using 0.5 mM IPTG. After the bacteria culture was incubated overnight at room temperature the culture was centrifuged for 30 minutes at 3000 rpm at 4° C. The pelleted bacteria was resuspended in 20 mL of lysis buffer, a solution composed of 20 mL CelLytic B (Sigma-Aldrich), 0.8 mL Lysozyme (Sigma-Aldrich), 2.6 µL benzonase (Sigma- Aldrich), and 2 tablets of protease inhibitor cocktail (Roche). The lysate was incubated for 30 minutes at 37° C before being centrifuged for 15 minutes at 12,000 g at 4° C, discarding the pellet and storing the supernatant at -80° C for use in both purification and co- immunoprecipitation experiments.
Purification of SBP tagged MYC
300 µL of streptavidin agarose beads (ThermoScientific) were washed twice with cold PBS, centrifuging for 4 minutes at 200 g at room temperature. The streptavidin agarose beads were incubated overnight at 4° C with 10 mL of the previously collected lysate. After this incubation the flow through was stored, henceforth referred to as FT-Beads. The beads remaining in the tube were then washed twice again with cold PBS. To separate the MYC from the SBP-TEV tag and have pure MYC, AcTEV Protease (Invitrogem) was used to bind the TEV part and then cleave MYC from the SBP-TEV tag. The 300 µL of streptavidin agarose beads were incubated overnight at 4° C with a Reaction Mix solution composed of 30 µL of 20X TEV buffer, 6 µL of 0.1 M DTT, 4 µL of AcTEV Protease, and 600 µL of pure water. After incubation with the TEV Reaction Mix, the streptavidin agarose beads were centrifuged for 5 minutes at 200 g at 4° C, and the supernatant was then separately stored, henceforth referred to as FT-TEV. Finally, 300 µL of 2x Laemmli sample running buffer (Bio-Rad) was added to the beads and the tubes were boiled for ten minutes at 90° C, and centrifuged for 5 minutes at 12000 g at room temperature.
20 µL of the Laemmli buffer was added to 20 µL samples of the FT-Beads and FT-TEV and these were boiled for ten minutes at 90° C, centrifuged for 5 minutes at 12000 g at room temperature. These samples, in combination with a 40 µL sample of the boiled beads mentioned above, were run on 4-15% polyacrylamide gels (Bio-Rad) in 1X Tris/glycine/SDS buffer at 200 V for about 45 minutes. One gel was analyzed by Coomassie stain, and the other gel was transferred to a PVDF membrane (Bio-Rad) and analyzed by western blot using a 1:500 dilution of anti-MYC (9E10) mouse monoclonal antibody (Santa Cruz Biotech) and a 1:2000 dilution of anti-mouse monoclonal antibody (Santa Cruz Biotech). Blots were developed using a 1:1 solution of the two West Pico Stablizer/Enhancer solutions (ThermoScientific) and visualized through chemiluminescence using the ChemiDoc MP Imaging System machine (Bio-Rad).
Co-immunoprecipitation of SBP-MYC and MAX with 10074-G5
Co-immunoprecipitation assays were performed to look at the ability of SBP-MYC to bind to and pull down MAX in vitro while under the condition of being incubated with modulators of this interaction. This first set of assays use the positive MYC/MAX heterodimer inhibitors 10074- G5 (Selleck Chem) or PY9 (Sigma-Aldrich) as the compounds modulating the interaction between SBP-MYC and MAX. A binding buffer solution was produced that was composed of pure water, 10x Binding buffer (EMSA kit, ThermoScientific) diluted to a 1x final concentration, 50% glycerol diluted to a 2.5% final concentration, 100 mM MgCl2 diluted to a 5 mM final concentration, 1% NP-40 diluted to a 0.05% final concentration. This solution is henceforth referred to as simply binding buffer. 1 mL of Streptavidin agarose beads (ThermoScientific) were washed twice with cold PBS, centrifuging for 5 minutes at 200 g at 4° C. 100 µL of these washed beads were aliquoted into 4 tubes to be incubated with 100 µL of the SBP-MYC lysate previously frozen and 800 µL of the binding buffer. Another tube was prepared to be the negative control, containing only the beads and the binding buffer, no SBP-MYC. All 5 of these were incubated for 1.5 hours at room temperature and then centrifuged for 3 minutes at 300 g at 4° C, removing the supernatant. To all of these tubes 1 µL of pure His-tagged MAX protein (Abcam) and 889 µL of the binding buffer was added. The positive control tubes additionally had increasing amount of MYC/MAX heterodimer inhibitors 10074-G5 (25 µM, 50 µM, and 100 µM) or PY9 (10 µM, 15 µM, 20 µM) added to the solution, whereas the negative control and test tubes just had a final concentration of 1% DMSO, without additional drug. Again all 5 of these were incubated for 1.5 hours at room temperature and then centrifuged for 3 minutes at 300 g at 4° C, removing the supernatant. For the last time, the streptavidin agarose beads were washed twice with cold PBS.
100 µL of the Laemmli buffer was added to 100 µL samples of the beads and boiled for ten minutes at 90° C, centrifuged for 5 minutes at 12000 g at room temperature. 30 µL of each sample were run on 4-15% polyacrylamide gels (Bio-Rad) in 1X Tris/glycine/SDS buffer at 200 V for about 45 minutes. The gel was transferred to a PVDF membrane (Bio-Rad) and analyzed by western blot using a 1:500 dilution of anti-MYC (9E10) mouse monoclonal antibody (Santa Cruz Biotech), a 1:500 dilution of anti-MAX (C-124) rabbit monoclonal antibody (Santa Cruz Biotech), and 1:2000 dilutions of anti-mouse and anti-rabbit monoclonal antibodies (Santa Cruz Biotech). Blots were developed using a 1:1 solution of the two West Pico Stabilizer/Enhancer solutions (ThermoScientific) and visualized through chemiluminescence using the ChemiDoc MP Imaging System machine (Bio-Rad).
Co-immunoprecipitation of MYC and MAX from P493-6 cell lysates with KI-MS2-008
The protocols and experiments described in this section were all performed by my mentor, graduate student Andrew Chen. P493-6, a Burkitt’s lymphoma cell line which is engineered to express high levels of MYC in the absence of doxycycline, was cultured. 400 million P493-6 cells, as counted by the Cellometer Auto T4 Bright Field Cell Counter (Nexcelom Bioscience), were pooled and washed twice in cold PBS. Cells were resuspended in 10 mL of modified RIPA lysis buffer with 0.1% sodium deoxycholate, 1% NP-40, 0.004% buffer benzonase, 200 mM NaCl, 50 mM Tris, 1X protease inhibitor (Sigma-Aldrich), and 1X phosphatase inhibitor (Roche). After incubating on ice for 15 minutes, the cells were centrifuged for 10 minutes at 14000 g at 4° C.
Following this a Bradford assay was performed to determine the total protein concentration of the cell lysate (the supernatant from the previous step), and 4 mg of total protein was incubated for thirty minutes with either 2 µL DMSO or 20 µM KI-MS2-008 and topped off to a total volume of 1 mL with Ab Binding and Washing Buffer from the magnetic Dynabeads kit (ThermoScientific). Then the protein/compound mixtures were incubated overnight at 4° C with either 20 µL MAX (C-17) rabbit antibody (Santa Cruz Biotech) or 10 µL of normal rabbit IgG (Santa Cruz Biotech). 50 µL of the magnetic Dynabeads per tube were placed on a magnetic stand for a minute to settle the beads, and washed in 200 µL of the Ab Binding and Washing Buffer before transferring the protein/compound/antibody mixture prepared the night prior into the tubes. These new tubes were incubated for one hour at 4° C and then placed on the magnetic stand again to settle the beads, saving the flow through to run on the gel. This was washed three times using 200 µL of the Washing Buffer, saving the washes for the gel as well.
30 µL of 2X SDS buffer was added to Dynabead tubes and boiled for five minutes at 90° C, using the magnetic stand to settle the beads and separate the elution. 20 µL of each sample were run on 4-15% polyacrylamide gels (Bio-Rad) in 1X Tris/glycine/SDS buffer at 100 V for 10 minutes, followed by 200 V for about 35 minutes. The gel was transferred to a PVDF membrane (Bio-Rad) and analyzed by western blot using a 1:500 dilution of anti-MYC (9E10) mouse monoclonal antibody (Santa Cruz Biotech), a 1:500 dilution of anti-MAX (H-2) mouse monoclonal antibody (Santa Cruz Biotech), and 1:2000 dilutions of anti-mouse monoclonal antibodies (Santa Cruz Biotech). Blots were developed using a 1:1 solution of the two West Pico Stabilizer/Enhancer solutions (ThermoScientific) and visualized through chemiluminescence using the ChemiDoc MP Imaging System machine (Bio-Rad).
Cell Viability Assays
All cell lines were obtained from the MIT Koch Institute’s High Throughput Screening facility. The suspension human breast cancer cell line HCC1599 was cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium (ThermoFisher), the adherent human breast cancer cell line MDA-MB-468 was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (ThermoFisher), and the adherent human brain cancer cell line U87MG was cultured in Eagle’s Minimum Essential Medium (EMEM) (ATCC). All culture media was prepared with 10% Fetal Bovine Serum (FBS) (ATCC) and 1% Penicillin/Streptomycin (P/S) (Corning Incorporated).
Cell cultures were grown using an incubator kept at 37° C at 5% CO2 and cultures were split every 3 to 5 days, as recommended according to the manufacturer’s instructions. The compounds used are BET inhibitor JQ1 (Sigma-Aldrich) and PI3K inhibitor GDC-0941 (Selleck Chem), and KI-MS1-001 and KI-MS2-008 which are being produced internally at the Koehler Lab. All four were serially diluted two-fold in DMSO, from 40 µM to 0.156 µM and later from 40 µM to 0.00122 µM, while being kept at a final concentration of 0.4% DMSO.
50 µL of cells were plated in sterile 96 well plates (Corning Incorporated) at a density of 5,000 cells per well, two plates per condition. Suspension cells were directly treated with 50 µL of either JQ1, GDC-0941, KI-MS1-001, KI-MS2-008 or DMSO and placed into the incubator, while adherent cells were first incubated overnight before replacing the culture media and then treating them with compound or DMSO. Each cell line was incubated in compounds or DMSO for both 3 days and 5 days, time points after which a plate would be visualized. 100 µL of CellTiter-Glo reagent (Promega) was added to each well, and plates were incubated for 10 minutes at room temperature on a platform shaker. Luminescence was recorded using a Tecan Infinite 200 Pro spectrophotometer using an integration time of 0.5 second/well. Luminescence values were graphed, and IC50 values calculated, using Prism software (Graphpad).
Following testing of cell viability under compound and DMSO conditions, cells were also prepared for western blotting against MYC, p-AKT, and GAPDH. Using sterile 12 well plates (Greiner Bio-One) for the HCC1599 suspension cells and sterile 6 well plates (VWR) for the MDA-MB-468 adherent cells, 3 million cells from each cell line were incubated for 24 hours with two-fold serial dilutions, from 10 µM to 0.156 µM, of the four compounds at 0.4% DMSO. Suspension cells were directly treated with the compounds while adherent cells were first incubated overnight in culture media before being treated with compounds. Cells were pelleted and washed with cold PBS, centrifuging for 5 minutes at 200 g at 4° C. Then cells were resuspended in 60 µL of modified RIPA lysis buffer with 0.1% sodium deoxycholate, 1% NP-40, 0.004% buffer benzonase, 200 mM NaCl, 50 mM Tris, 1X protease inhibitor (Sigma-Aldrich), and 1X phosphatase inhibitor (Roche). Total protein concentration was determined using a BCA assay, and samples meant to run on the gel were either prepared to have a consistent 30 µg concentration of protein or 50 µg, depending on the total protein concentration from the cell line.
Enough 2X SDS buffer was added to samples for a 1:1 ratio of sample and running buffer, and samples were boiled for five minutes at 90° C, centrifuged for 5 minutes at 12000 g at room temperature. Samples were run on 4-15% polyacrylamide gels (Bio-Rad) in 1X Tris/glycine/SDS buffer at 200 V for about 45 minutes. The gel was transferred to a PVDF membrane (Bio-Rad) and analyzed by western blot using a 1:500 dilution of anti-MYC (9E10) mouse monoclonal antibody (Santa Cruz Biotech), a 1:500 dilution of anti-MAX (C-124) rabbit monoclonal antibody (Santa Cruz Biotech), a 1:1000 dilution of anti-p-AKT (T308) rabbit monoclonal antibody (Cell Signaling), a 1:1000 dilution of anti-GAPDH (14C10) rabbit monoclonal antibody (Cell Signaling), and 1:2000 dilutions of anti-mouse and anti-rabbit monoclonal antibodies (Santa Cruz Biotech). Blots were developed using a 1:1 solution of the two West Pico Stabilizer/Enhancer solutions (ThermoScientific) and visualized through chemiluminescence using the ChemiDoc MP Imaging System machine (Bio-Rad).
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