A subset of CB002 xanthine analogues bypass p53-signaling to restore a p53 transcriptome and target an S-phase cell cycle checkpoint in tumors with mutated-p53

Mutations in TP53 occur commonly in the majority of human tumors and confer aggressive tumor phenotypes including metastasis and therapy resistance. CB002 and structural-analogues restore p53 signaling in tumors with mutant-p53 but we find that unlike other xanthines such as caffeine, pentoxifylline, and theophylline, they do not deregulate the G2-checkpoint. Novel CB002-analogues induce pro-apoptotic Noxa protein in an ATF3/4-dependent manner, whereas caffeine, pentoxifylline, and theophylline do not. By contrast to caffeine, CB002-analogues target an S-phase checkpoint associated with increased p-RPA/RPA2, p-ATR, decreased Cyclin A, p-histone H3 expression and downregulation of essential proteins in DNA-synthesis and -repair. CB002-analogue #4 enhances cell death, and decreases Ki-67 in patient-derived tumor-organoids without toxicity to normal human cells. Preliminary in vivo studies demonstrate anti-tumor efficacy in mice. Thus, a novel class of anti-cancer drugs show activation of p53 pathway signaling in tumors with mutated p53, and target an S-phase checkpoint.


Introduction
Tumor suppressor p53 responds to cell stress signals from DNA damage, oncogene activation, oxidative stress and hypoxia. Upon activation by post-translational modifications and oligomerization, p53 signals cell cycle arrest, apoptosis or DNA repair, according to the extent of the cellular stress, thereby controlling cell fate and preventing tumorigenesis [1]. Thus, it is not surprising that the TP53 is the most commonly mutated gene (TCGA, 2020), including in ovarian, colorectal, esophageal, head & neck, lung and pancreatic cancers that are the most affected sporadic human cancer types [2]. TP53 is mutated in over 50% of human cancers and the other 50% involves a biological inactivation of its signalling pathway. Similar to other tumor suppressors, the mutated p53 protein results in loss-of-function but due to oligomerization can act in a dominant-negative fashion with regard to remaining wild-type p53 allele. Unlike other tumor suppressors, mutant p53 protein can also acquire a gain-of-function which contributes to aggressive tumor phenotypes including enhanced invasion, genomic instability and therapy resistance [3][4][5][6]. Consequently, patients whose tumors carry p53 mutations have a poor prognosis and decreased overall survival [7].
A common feature of cancer cells is genomic instability due to ineffective cell cycle checkpoint responses. Genomic instability is not necessarily due to defective checkpoints. The checkpoints may be intact but the repair may be deficient. Upon DNA damage, the normal cell cycle checkpoint response is to arrest the cell at the G1 phase. In cancer cells, the majority have an ineffective G1checkpoint due to p53 mutation but retain a functional G2-checkpoint and thus have the ability to undergo cell arrest at the G2 phase. Cancer cells depend on bypassing intra-S-phase and G2/M checkpoints for unrestrained cell proliferation. Stress signal transduction in the p53 pathway is initiated by activation of kinases ataxia-telangiectasia-mutated (ATM), ataxia telangiectasia and Rad3-related (ATR) and downstream checkpoint kinases Chk1/2 that serve as signaling sensors and mediators of p53 activation. It has been a long-standing dogma that ATM/Chk2 and ATR/Chk1 are independently activated but recent studies provide evidence of cross-talk between the kinases [8][9][10]. Chk1/2 are kinases that participate in cell cycle checkpoint control, with Chk1 being active in S-and G2-phase whereas Chk2 is active throughout the cell cycle [10][11][12].
Accumulation of genomic aberrations over time, renders cancer cells vulnerable to checkpoint targeting therapy. Since the discovery of checkpoint targets, small molecule inhibitors have been pursued in combination with ionizing radiation and chemotherapy agents in order to deregulate checkpoints thereby leading to cancer cell death. For example, combination of caffeine, a xanthine derivative, with irradiation or chemotherapy agents was found to deregulate the G2-checkpoint through ATM/ATR inhibition leading to therapy sensitization and enhanced cell death [13,14].
Nonetheless, translational cancer therapeutics studies were discontinued due to unachievable active concentrations in human plasma [15]. Thus, for the past two decades, the field has focused on the development of Chk1/2 inhibitors which are in clinical trials [16][17][18].
Another cancer therapeutic approach we and others have pursued involves restoration of p53 pathway signaling in tumors with mutant p53 or tumors that are null for p53. Despite efforts to restore the p53-pathway, to date there are no FDA-approved drugs that functionally restore the p53 in tumors with mutated p53. We previously reported a p53-pathway restoring compound CB002 whose mechanism of action was not fully elucidated. We showed that CB002 leads to apoptotic cell death mediated by p53 target Noxa, a pro-apoptotic protein [19]. Here we further evaluated more potent CB002-analogue compounds and uncovered a unique mechanism of action suggestive of a novel class of anti-cancer drugs. Based on their molecular structure as xanthine derivatives, the novel class of CB002-analogues, unlike caffeine and other established xanthine derivatives, do not deregulate the G2-checkpoint. By contrast, the novel CB002-analogue xanthines perturb S-phase and more importantly they restore the p53-pathway, a property not found with caffeine, pentoxifylline and theophylline. We sought to further characterize and define by transcriptomic and proteomic analysis the new class of small molecules with anti-tumor properties.

CB002 and structural analogues restore the p53 pathway independently of p53, while xanthines such as caffeine, pentoxifylline, and theophylline do not
We sought to identify more potent analogues of parental xanthine compound CB002. We tested CB002-analogues in the Chembridge library for the capability to induce the luciferase activity using a p53-regulated luciferase reporter stably expressed in the SW480 colorectal cancer cell line and also determined the IC50 values for the compounds by a Cell-Titer glow cytotoxicity assay ( Figure 1A-B, Figure S1). The majority of the CB002-analogues tested, with the exception of analogue #12, enhanced p53-reporter activity in a dose-dependent manner within a range of compound concentrations from 0 to 600 µM. We investigated the capability of a set of the CB002analogues to induce apoptosis as indicated by Propidium Iodide (PI) staining Sub-G1 population.
As shown in Figure 1C, treatment of tumor cells with CB002-analogues at an IC50 concentration (100 µM) resulted in a significant increase in Sub-G1 content in SW480 cells. Moreover, the most potent CB002-analogue #4, was found to increase cleaved-PARP and cytochrome C release from the mitochondria to the cytosol providing further evidence for apoptosis induction in SW480 tumor cells ( Figure 1D-E, S15). We investigated whether the p53-family member p73 may be a mediator of apoptosis and responsible for inducing p53 transcriptional targets by CB002-analogues.

CB002 and structural analogues induce Noxa in an ATF3/4-dependent manner, independent of p73
As we previously showed for CB002 (19), p53-targets Noxa and DR5 were induced independently of p73 and PARP cleavage occurred despite effective p73 knockdown in CB002-analogue #4 treated SW480 tumor cells ( Figure 1F). Our previously published CB002 data indicated that Noxa plays a key role in mediating CB002-induced apoptosis (19). Thus, we sought to determine if CB002 analogues induce Noxa expression in 4 human colorectal cancer cell lines. In DLD-1 (p53 S241F ), SW480(p53 R273H,P309S ), HCT116(p53 WT ), and HCT116 p53 -/tumor cells expressing the exogenous R175H p53 mutant, Noxa protein expression was found to be induced, though some variation across cell lines was observed ( Figure 1G). As these CB002-analogues are xanthine derivatives, we investigated whether other known xanthine derivatives, i.e., caffeine, pentoxifylline and theophylline can induce Noxa expression. However, we found that only the p53-pathway restoring CB002-analogue xanthine compounds and not caffeine, pentoxifylline and theophylline, induce Noxa protein expression ( Figure 1H). Since Noxa can be transcriptionally activated independently of p53 we sought to explore other transcription factors involved in Noxa induction. We performed a knockdown of integrated stress response transcription factors ATF3/4 on SW480 cells. Knockdown of ATF3/4 upon treatment with 100 µM CB002 or 25 µM CB002analogue #4 abrogated Noxa protein induction ( Figure 1I). Hence our data suggests that ATF3/4 plays a role in regulating Noxa expression.

CB002 analogue #4 treatment of human tumor cells enriches for cell cycle genes in addition to genes involved in the p53-pathway including apoptosis, indicating p53-pathway functional restoration
In order to understand how the CB002-analogue molecules restore the p53-pathway, we performed a transcriptomic and proteomic analysis in SW480 cells treated with analogue #4. To verify the quality of our data, the principal component (PC) plot was obtained. PC plots show that the factor with most variability within the samples was the difference between control and treatment ( Figure S2A-C). Significant differentially expressed genes were defined by a false discovery rate (FDR) < 0.05, and a total of 3,362 genes met these criteria ( Figure S2D). We then sought to identify the differentially expressed genes involved in the p53 pathway. To do this, a comprehensive known p53 target gene set used for comparison were the genes that have been previously shown to be directly regulated by p53 through chromatin immunoprecipitation assays (CHIP) assays and genes that were protein-coding genes in at least 3 of the 17 genome wide data sets (Table S3 from Fisher's analysis [20]). Out of the 343 genes in the known p53 target gene set, 334 genes were tested in the microarray but only 197 genes met the low expression cutoff.
From the 197 genes that met the low expression criteria, 102 genes were found to be differentially expressed (Figure 2A). Gene ontology (GO) analysis of the 102 differentially expressed genes indicated that these genes are highly enriched in the regulation of programmed cell death ( Table   1). A gene expression heatmap of these genes is shown in Figure 2B, and the majority of the genes are found to be upregulated by analogue #4 treatment of tumor cells. We then performed a transcription factor analysis of all 3,362 differentially expressed genes. Transcription factor analysis defined by direct binding of predictive binding motifs revealed E2F transcription factors as having the highest normalized enrichment score (NES) (Figure 2C). Because the transcription factor ATF4 was shown to be important for Noxa induction in Figure 1I, we compared a known ATF4 gene set (Table S3 from ref. [21]), along with an E2F gene set (Table S1 from together with the known p53 gene set and the differentially expressed genes in our analogue #4 treatment ( Figure 2D). The resulting Venn diagram of this comparison shows that both ATF4 and E2F targets genes are not unique to these transcription factors and also share common targets with p53 (~5%). Analyzing the ratio of differentially expressed genes to the transcription factor gene set did not show an obvious gene enrichment regulation of one transcription factor ( Table 2).
Despite p53 not being the top predictive transcription factor in our analysis, ingenuity pathway analysis (IPA) determined p53 to be activated as an upstream regulator with a z-score value of 3.3 and p-value of 2.9x10 -34 . A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for the p53-pathway signaling was obtained with an adjusted p value (adjp) equal to 1.18 x 10 -1 that despite not reflecting a significant enrichment of the p53 pathway, it indicates the presence of a total of 31 differentially expressed genes out of the 52 genes tested and present in the KEGG analysis. Thus, this accounts for 60% of differentially expressed genes in the KEGG p53-pathway analysis. Differentially expressed genes involved in the KEGG analysis fold change is described by color and additional genes not shown in the p53-pathway figure and yet involved in the KEGG analysis are shown as a heatmap ( Figure S3 and Figure S4). In line with the GO terms results, p53 target genes involved in apoptosis such as Noxa, Puma, and DR5 were upregulated by the analogue #4 treatment. Taken together, this data indicates that although a large set of genes differentially expressed are not predicted to be directly regulated through direct p53 binding, a subset of these are enriched in the p53-pathway, indicative of p53-pathway restoration.  Figure S5, Figure   S7, Figure S8, and Figure S9). Additional genes not shown in the pathway KEGG figures and yet involved in the KEGG analysis are shown as a heatmap ( Figure S6, Figure S8, Figure S10, and Figure S11). Gene ontology (GO) terms in biological processes also reflected enrichment of genes that participate in cell cycle regulation ( Table 3). Taken  suggests that the identified family of small molecules represent a unique mechanism of action that involves S-phase delay perturbation and p53-pathway restoration. In order to show that the stimulation of the p53 pathway at the transcriptional level was restoring the p53 pathway at the protein level, a comparative label-free quantitative proteomic analysis of SW480 colon cancer cells in response to DMSO and analogue #4 (T4) treated for 24 hours was performed. Figure Figure 3B). In particular, CDK4, CKS1B, ERCC6L, MAPK3, and MAX are significantly decreased in analogue #4 treatment than in CB002 ( Figure   3C).
As the CB002-analogue molecules were discovered as p53 pathway restoring compounds, we compared the proteomic data, with the known p53 target gene set used in our transcriptomic analysis (Table S3 from  should be considered as preliminary and warrants further optimization. Moreover, differences were observed at the level of protein expression between parental compound CB002 and its analogue #4 both downregulated and to a lesser extent, upregulated proteins ( Figure S14). This indicates that these small molecules can have different effects in tumor cells albeit they have >50% homology in their proteomic composition.

CB002 and analogues perturb S-Phase but not G2-checkpoint unlike other xanthines
Caffeine is a G2-checkpoint deregulator through inhibition of ATM/ATR. Thus, combination of chemotherapy agents with caffeine results in enhanced cancer cell cytotoxicity. Nonetheless, it was not pursued due to caffeine's lack of achievable required concentrations in plasma. We investigated whether CB002 and its analogues can deregulate the G2-checkpoint, like caffeine, pentoxifylline and theophylline. We synchronized SW480 colon cancer cells using double thymidine block, released and treated with CB002 analogue compound alone or in combination with etoposide and probed for key G2/M-phase cell cycle markers. As expected, we observed that etoposide treatment enhances protein expression of pcdc2 (Tyr15) and pcdc25c (Ser16) indicating cell cycle arrest due to DNA damage. The combination of etoposide with caffeine resulted in G2deregulation as indicated by decreased expression of pcdc2 (Tyr15) and pcdc25c (Ser16) . Similarly, the combination of etoposide with CB002 or CB002-analogue #4 showed a decrease in expression of pcdc2 (Tyr15) and pcdc25c (Ser16) . Nonetheless, CB002 or CB002-analogue #4 do not increase Mphase marker pH3 (Ser10) as would be expected for a G2-deregulator like caffeine ( Figure 5A). This data suggests that CB002 and CB002-analogue #4 either do not deregulate the G2-checkpoint or that these compounds delay cells going into M-phase. Moreover, CB002 and its analogues increase p-Cdc25c and p-Cdc2 in combination with etoposide indicating cell cycle arrest. A similar experiment was performed as a time course after cell synchronization release to further elucidate the cell cycle effects of CB002-analogue #4. As seen in Figure 5D, cell cycle markers pcdc2 (Tyr15) and pcdc25c ( BrdU-positive cells in Figure 5 and Figure S16.

CB002-analogue #4 is has anti-tumor effects in vitro and in vivo
We focused on lead CB002-analogue #4 and investigated its therapeutic index in vitro and in vivo.
We treated an isogenic HCT116 cell line panel with varying p53 mutation-status were treated with 100 µM CB002 and 25 µM CB002-analogue #4 and established IC50 values by the Cell-Titer glow cytotoxicity assay. Across this panel, CB002-analogue #4 has a 20-to 30-fold range in IC50 values, independently of the HCT116 p53-status ( Figure 6A). Thus, the results indicate that the restoration of the p53-pathway by CB002 or analogue #4 is p53-independent. SW480 cells treated with CB002-analogue #4 showed a significant increase of Sub-G1 content as compared to vehicle control, whereas treatment with CB002-analogue #4 of normal human WI38 lung fibroblast cells did not significantly increase the sub-G1 cell population indicating that CB002-analogue #4 is safe to normal cells in vitro ( Figure 6B).
We further investigated the anti-cancer cytotoxicity potential of CB002-analogue #4. We treated a colorectal cancer patient-derived organoid with CB002-analogue #4 and performed cellular cytotoxicity analysis in vitro and immunofluorescence staining of ethidium homodimer, calcein, caspase-3 and Ki-67 to distinguish between dead, live, apoptotic and proliferating cells, respectively. CB002-analogue #4 enhances cytotoxicity as compared to the CB002 parent compound in the tested colorectal cancer patient-derived organoid as indicated by the cell viability response curve ( Figure 6C). Moreover, the immunofluorescence assay staining for ethidium homodimer and calcein shows an increase of ethidium homodimer staining of CB002 and CB002analogue #4 to a larger extent as compared to vehicle control indicating an enhanced killing of cells. Calcein staining shows that organoids treated with CB002-analogue #4 are smaller in size indicating that CB002-analogue #4 decreases the growth of the patient-derived organoid ( Figure   6D). Cleaved caspase-3 staining indicates that both CB002 and CB002-analogue #4 treatment at IC50 doses increases apoptotic cells ( Figure 6D). CB002-analogue #4 treatment also results in an inverse relationship with Ki-67 staining with respect to drug concentration, indicating that CB002analogue #4 decreases the population of proliferating cells ( Figure 6E).
We investigated CB002-analogue #4 in vivo for anti-tumor efficacy as well as toxicity in NSG mice. Mice were xenografted with human SW480 colorectal cancer cells treated with CB002analogue #4 at 50 mg/kg by oral gavage 3 times per week. Our data suggests that CB002-analogue #4 is well-tolerated as indicated by the mouse body weights during the duration of the experiment ( Figure 6F). At 5-weeks of treatment, CB002-analogue #4 treated tumors have a statistically significant lower tumor volume as compared to vehicle control ( Figure 6G). To determine the importance of Noxa in vivo, mice were xenografted with SW480 cell containing a stable knockdown of Noxa. Mice xenografted with SW480 shNoxa cells did not have a significant difference in tumor volume after CB002-analogue #4 treatment compared to vehicle control treated tumors indicating that Noxa is important for reduced tumor volume in vivo ( Figure 6H).

Discussion
We describe a novel class of anti-tumor agents with a unique mechanism of action involving restoration of p53 pathway signaling, independently of p53, in tumors with mutated-p53 and characteristics of an S-phase checkpoint. The defining members of this class that best exemplify the novel mechanistic properties are CB002-analogues #4 and #10. The properties of these CB002analogue xanthine compounds are different from other xanthines such as caffeine, pentoxifylline, and theophylline that do not restore p53 pathway signaling in tumors with mutant p53 and which deregulate a G2-checkpoint rather than induce an S-phase checkpoint.
Our approach to discovering p53 pathway restoring compounds involved cell-based screening for functional restoration of p53-regulated reporter activity, coupled with cell death induction. Thus, small molecule lead compounds and structural-analogues were not expected to act directly on mutant p53 or restore binding of mutant p53 to genes normally regulated by p53. In the case of the compounds described here, activation of p53 target genes such as Noxa or DR5 occurred independently of p53 and this was observed in tumor cells with different p53 mutations. Thus, there is no expectation that CB002 or analogues #4 or #10 will cause mutant p53 to bind to DNA or chromatin in the regulatory regions of Noxa or DR5 in a manner that wild-type p53 does.
Moreover, the induction of p53 targets occurred independently of p53 family member p73, but in a manner that requires integrated stress response transcription factor proteins ATF3/4. These results provide a molecular mechanism for activation of p53 target genes in a manner that substitutes transcription factors such as ATF3/4 for defective p53. This mechanism results in tumor suppression through induction of pro-apoptotic factors despite p53 mutation, and therefore acts as a bypass mechanism to prevent tumor growth in drug-treated cells.
CB002-analogue #4 is 20-30 times more potent and like the CB002 parental-compound restores the p53-pathway and induces apoptosis independently of p73. The twelve p53 pathway restoring structural analogues of CB002 tested were similar in that they resemble the structure of a xanthine.
Our transcriptional analysis identified 102 genes involved in the p53-pathway and IPA determined p53 to be activated as an upstream regulator with a z-score value of 3.3 and p-value of 2.9x10 -34 .
This data further validates the novel anti-cancer class of small molecules as p53-restoring drugs.  Our proteomic data shows activation of the integrated stress response as indicated by the increase of genes involved in the unfolded protein response, tRNA aminoacylation and increase of ATF3/4 protein expression by western blot (Figure 3A, Figure 1I). Whether the S-phase perturbation is a result of cellular stress remains to be addressed. shown that CB002 induces p21 expression [19], as well as analogue #4 in this study thus it is possible that the observed S-phase perturbation is through p53-independent p21 stimulation that binds to DREAM complexes. Therefore, it will be interesting to see if ATF3/4 regulate p21 expression and the effect of p21 knockdown on cell cycle genes and effect on the S-phase perturbation observed by CB002 analogues.
We show that CB002-analogue #4 induces apoptosis in colorectal cancer patient-derived organoid cells and that it is safe both in vitro and in vivo as indicated by lack of a statistically significant increase in the Sub-G1 population in normal human fibroblasts and also a healthy NSG mice body weight throughout treatment, respectively. The observed decrease in tumor volume was statistically significant at 5-weeks. This effect was suboptimal than desired and further optimization will be required to reach optimal effects. Importantly, the decrease in tumor volume by CB002-analogue #4 is dependent on Noxa. As Noxa is not commonly mutated in human cancer, its induction by the CB002-analogues offers a feasible therapeutic advantage leading to tumor cell death and its expression may be used as a pharmacodynamic biomarker to predict therapeutic response. Taken together, our data suggests that CB002-analogues #4 and #10 represent a novel class of anti-tumor agents that provide a unique therapeutic strategy that can be clinically translated.

CB002 analogue small molecule drug screening
CB002 structural analogues were obtained from Chembridge Library and screening was performed in the human SW480 colorectal cancer cell line that stably expresses a p53-regulated luciferase reporter previously generated in our laboratory [28]. Cells were seeded at a density of 1x10 4 cells per well in 96-well plates (Greiner Bio-One) and treated with the indicated compound from 0 to 600 µM. p53 transcriptional activity was imaged using an IVIS imaging system at 6 hours. A total of three biological replicates per condition were performed.

CellTiter-Glo® luminescent cell viability assay
SW480 Cells were seeded in 96-well plates at a density of 5x10 3 cells per well. A total of three biological replicates per condition were performed. 20 µL of CellTiter-Glo® reagent was added directly to the wells, according to the manufacturer's protocol, and bioluminescence signal was determined using an IVIS imaging system at a period of 48-72 hours after treatment.

Cell synchronization
When indicated, cells were synchronized by double thymidine block. Cells were treated with 2 µM Thymidine for 16 hours, drug was removed and replaced by complete growth media for 8 hours.
Cells were treated for the second time with 2 µM Thymidine for 16 hours, at this point cells were treated and harvested as indicated.

Propidium Iodide and BrdU Flow Cytometry Assay
Cells were seeded at a density of 5 BrdU analysis gating-Cell aggregates were gated out in the PI Peak vs DNA PI histogram. BrdU lower limit intensity was set on upper limit of the negative control. No BrdU antibody in Figure   3E and no goat-anti mouse Alexa Fluor 488 antibody in Figure S9

Knockdown of expression of p73 using siRNA
A total of 1x10 5 cells/well were plated per well in a 12-well plate in medium with 10% FBS without antibiotic. Forward transfection of p73 siRNA (s14319, Ambion®) was performed using the Lipofectamine® RNAiMAX Transfection Reagent (Life Technologies) and incubated for 48 hours before drug treatments.

Microarray analysis
SW480 cells were seeded at a density of 1x10 6 in 10 cm dishes and once adhered, treated with DMSO vehicle control or CB002-analogue #4 for a total of two biological replicates per condition.
Floating cells were collected and adherent cells were trypsinized at 12 hours of treatment. Cells were pelleted and RNA was isolated using a Quick-RNA™ MiniPrep (#R1055, Zymo Research) according to manufacturer instructions. RNA quality was tested using an Agilent Bioanalyzer RNA kit. Once RNA quality was sufficient, RNA was amplified and labeled using the low RNA input linear amplification kit (Agilent). Labeled cDNA was hybridized onto Affymetrix Human Gene 2.0-ST array. Significant changes in gene expression were determined as follows: the low expression cutoff of probe signal intensity was set at 50 (unless at least one sample did not meet this criteria for that particular probe). Normalization was performed using the RMA method and Limma eBayes for the statistical method using R studio programming software. Genes with an FDR of <0.05 were determined as significant in DMSO vehicle control versus analogue #4. Experiments were performed at least twice and more than three technical replicates were obtained, a representation of one is shown.

Drug efficacy using in vivo tumor xenografts
In vivo drug efficacy studies were performed on 10 NSG female randomized mice per cohort. Mice

Statistical analysis
To assess the statistical significance, Two-way ANOVA or Unpaired t-test for two comparisons was performed, with p < 0.05 defined as statistically significant. Data are presented as means ± SEM (three biological replicates). Comparisons were made against the DMSO vehicle control.               Ki-67 (green) Figure S1. CB002 and its analogue #2 -#11 chemical structures. Identified family of CB002 and its analogue are xanthine derivates. Figure S2. Transcriptomic analysis quality control principal component (PC) plots and false discovery rate (FDR) bar graph. PC1 accounts for the highest variability factor being the differences between control and analogue #4 treatment. Statistically significant changes in gene expression were determined as FDR < 0.05. Figure S3. Kyoto Encyclopedia of Genes and Genomes (KEGG) for the p53-pathway signaling. Analogue #4 differentially regulated genes that overlapped with the known p53 target gene set were used to perform a KEGG analysis for the p53 pathway.      (T4) samples. Data represents the close clustering of protein abundance of each replicates under the same group, however showed variability among the treatments. B-D, Volcano plot of fold change versus q-value of the total of 3743 proteins quantified from SW480 cell lines in response to DMSO, CB and T4 treatments. Red and green circles represent the significant (q < 0.05) up and down regulated proteins. Gray circles (q = 0.05) are non-significant and below the threshold of fold expression. E, heat map and clustering analysis of the total proteins (3743) identified from DMSO, CB and T4 samples.   Figure S13