KRAS Mutant Colorectal Cancer Cells Research Articles

150 (PB030) - MEKi-based combination strategies for targeting KRAS-driven cancer

Background: KRAS is one of the most frequently mutated oncogenes in human cancer. Recent successes with KRAS G12C-specific inhibitors are changing the treatment paradigm in this subset of cancer patients. An alternative strategy is targeting downstream KRAS effectors, such as MEK. However, MEK inhibitors have historically proven unsuccessful as monotherapy in KRAS mutant cancers, highlighting the need for combinatory approaches. One possibility is the combination of MEK and SOS1 inhibition, which may overcome adaptive resistance to MEKi by blocking SOS1-mediated reactivation of MAPK signaling. Another promising approach is to combine MEKi with inhibitors of Bromodomain and extra-terminal (BET) proteins. While BET inhibitors exhibit broad anti-tumor activity in preclinical cancer models, BETi monotherapy demonstrated only moderate clinical activity. Resistance to BETi has been, at least partially, attributed to MAPK pathway upregulation, suggesting that a combination with MEKi may help to overcome resistance. Material and Methods: Here, we present BI 3011441, a potent and selective inhibitor of human MEK1/2. Using in vitro assays, we compare BI 3011441 to other clinical stage MEK inhibitors. We also combine BI 3011441 with BI 1701963 and BI 894999 - two potent, selective, and orally bioavailable inhibitors of SOS1 and BET, respectively. Both combinations are tested in vitro and/or in vivo across a panel of KRAS mutant pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) models. Results: BI 3011441 efficiently inhibits ERK phosphorylation in cell-based assays and demonstrates the strongest in vitro growth inhibition in a panel of BRAF or KRAS mutant cell lines among the MEK inhibitors tested. In vivo, BI 3011441 leads to a dose-dependent modulation of MAPK pathway, which translates into anti-tumor activity in xenograft models of KRAS-driven cancer. The combination of BI 3011441 with SOS1i BI 1701963 synergistically decreases MAPK signaling resulting in enhanced efficacy in xenograft models, including NSCLC, CRC and PDAC. In NSCLC models this combination results in 10% of partial responses (PR) and 70% of stable disease (SD) as defined by modified Response Evaluation Criteria In Solid Tumors (mRECIST). In PDAC and CRC models the combination leads to 30% and 43% of SD, respectively. When combined with clinically relevant concentrations of BETi BI 894999, BI 3011441 strongly decreases in vitro proliferation and induces apoptosis in KRAS mutant cancer cells. Synergy is observed in 17 out of 44 cell lines across different indications and harboring different KRAS mutations. Conclusions: In summary, we show that combining BI 3011441 with SOS1 or BET inhibitors may lead to improved responses in KRAS mutant tumors, likely by circumventing MAPK pathway-related adaptive resistance. Conflict of interest: Other Substantive Relationships: The authors are either employees or have financial relationships with Boehringer Ingelheim.

Abstract 1056: Determining efficacy of combining the MEK inhibitor trametinib with vincristine identified by unbiased high throughput screening in RAS-mutated colorectal cancer cells

Abstract Background: Colorectal cancer (CRC) is a heterogeneous disease with various driver genetic mutations. Metastatic colorectal cancer (mCRC) is the second leading cause of cancer related deaths in the US. Greater than 50% of patients with mCRC harbor mutations in the oncogenic drivers KRAS or NRAS. While direct targeting of specific mutations in RAS is in the early stages of development in patients with mCRC, targeting most mutations in RAS is technically challenging. Hence, prior efforts have been focused on targeting MEK, a downstream mediator of RAS. Unfortunately, several MEK inhibitors that have entered clinical trials have failed to exhibit clinical efficacy as a monotherapy or in combination with select agents in patients with mCRC. Thus, identifying combination therapies with unbiased screening methods has the potential to improve the efficacy of MEK targeting in patients with mCRC. Aims: We aimed to perform unbiased high throughput screening (HTS) to identify drugs that, when combined with the MEK inhibitor trametinib, would enhance its efficacy. Methods: We performed unbiased HTS with KRAS mutated CRC cells grown in 3D using the MEK inhibitor trametinib as the base drug, and two different “clinically ready” compound libraries; 1) the NCI oncology set V, and, 2) a custom clinical compound set composed of drugs either approved by the FDA or in clinical trials. Using the Bliss model of synergy, vincristine was identified to be synergistic with trametinib. Effects of combining trametinib with vincristine were analyzed in vitro by 1) cell growth assays, and 2) measuring changes in cell signaling and apoptosis mediators by RPPA and western blotting. The efficacy of this combination was also examined in vivo using KRAS mutated patient derived xenografts (PDXs). Results: HTS studies demonstrated that combining trametinib with vincristine was synergistic in 3D CRC cell culture. This combination was further validated by MTT and colony formation assays. Analyses of markers for apoptosis by RPPA and western blotting demonstrated that the combination increases cell death in multiple CRC cell lines when compared to single agents. Furthermore, combining trametinib with vincristine led to significant inhibition in tumor growth when compared to the monotherapies using RAS-mutated PDXs in vivo. The combination was well tolerated by mice in vivo with minor decreases in body weight. Conclusions: Our unbiased HTS along with in vitro and in vivo validation studies demonstrated that combining trametinib with vincristine can enhance the efficacy of MEK targeted therapy in RAS mutated CRC cells. Furthermore, our in vivo studies were performed using drug doses that reflect clinically achievable doses in humans and thus serve as basis for future clinical studies to determine the efficacy of this drug combination in patients with RAS mutated mCRC. Citation Format: Susmita Ghosh, Fan Fan, Jason Roszik, Reid Powell, Yong Park, Clifford Stephan, Lee M. Ellis, Rajat Bhattacharya. Determining efficacy of combining the MEK inhibitor trametinib with vincristine identified by unbiased high throughput screening in RAS-mutated colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1056.

Abstract 3187: Understanding the role of cancer-associated fibroblasts (CAFs) on pathogenesis of KRAS mutated cancers by defining differences in the basal state proteome and secretome

Abstract Background: KRAS is commonly mutated in solid tumors. Cancer-associated fibroblasts (CAFs) are a key tumor stroma component, which can affect cancer progression and drug response. We hypothesized that defining the differential secreted proteins between CAFs and KRAS mutant-cancer cells could reveal potential mechanisms by which CAFs influence cancer progression and drug resistance. Methods: We studied 12 cell lines: 3 KRAS mutant-colorectal cancer cell lines (H747, LIM2099 and SW620), 3 KRAS mutant-lung cancer cell lines (H1792, H2030 and H23), 3 KRAS mutant-pancreatic cancer cell lines (CAPAN1, DANG, MIAPACA2) and 3 CAF cell line models (colorectal and lung CAF and pancreatic stellate cells). We quantified proteins in cell lines and serum-free media after 24 hour incubation in cells using mass spectrometry in a multiplexed manner (12plex) in triplicates to characterize the proteome and the profile of secreted proteins (secretome), respectively. Prediction software (signalP, secretomeP, TMHMM) and databases (surfaceome and cell surface protein atlas) were used to annotate secreted or transmembrane proteins. Results: Our proteome analysis quantified 9307 proteins. T-test between the cell types (CAF vs cancer) identified 1770 differentially expressed proteins, where 712 proteins were CAF-enriched. This included known CAF markers (e.g α smooth muscle actin) but also proteins not known to be CAF markers such as heat shock protein beta-5/6 (HSPB5/6). The secretome analysis identified 2330 secreted or transmembrane proteins where 202 were differentially expressed between cancer cells and CAFs. 190 proteins were CAF-enriched with the highest ranking being syndecan-2 (SDC2), collagen α-28(I) chain (COL28A1), decorin (DCN) and wnt family member 5B (WNT5B). Interestingly, basal proteome analysis of KRAS mutant-cancer cells showed expression of receptors corresponding to the CAF-enriched ligands, such as receptor tyrosine kinase-like orphan receptor 1/2 (ROR1/2) which respond to WNT5B. This suggests that CAF-enriched ligands may activate certain downstream pathways, including non-canonical WNT signaling, in KRAS mutant-cancer cells. Conclusion: We undertook deep quantitative proteomics where we identified potential novel CAF markers HSPB5/6. We have also found proteins in the CAF secretome, such as WNT5B, which may drive drug resistance in KRAS mutant-cancer cells. Citation Format: Rachel Lau, Lu Yu, Theodoros Roumeliotis, Lisa Pickard, Adam Stewart, Jyoti Choudhary, Udai Banerji. Understanding the role of cancer-associated fibroblasts (CAFs) on pathogenesis of KRAS mutated cancers by defining differences in the basal state proteome and secretome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3187.

SERS characterization of colorectal cancer cell surface markers upon anti-EGFR treatment.

Colorectal cancer (CRC) is the third most diagnosed and the second lethal cancer worldwide. Approximately 30-50% of CRC are driven by mutations in the KRAS oncogene, which is a strong negative predictor for response to anti-epidermal growth factor receptor (anti-EGFR) therapy. Examining the phenotype of KRAS mutant and wild-type (WT) CRC cells in response to anti-EGFR treatment may provide significant insights into drug response and resistance. Herein, surface-enhanced Raman spectroscopy (SERS) assay was applied to phenotype four cell surface proteins (EpCAM, EGFR, HER2, HER3) in KRAS mutant (SW480) and WT (SW48) cells over a 24-day time course of anti-EGFR treatment with cetuximab. Cell phenotypes were obtained using Raman reporter-coated and antibody-conjugated gold nanoparticles (SERS nanotags), where a characteristic Raman spectrum was generated upon single laser excitation, reflecting the presence of the targeted surface marker proteins. Compared to the KRAS mutant cells, KRAS WT cells were more sensitive to anti-EGFR treatment and displayed a significant decrease in HER2 and HER3 expression. The SERS results were validated with flow cytometry, confirming the SERS assay is promising as an alternative method for multiplexed characterization of cell surface biomarkers using a single laser excitation system.

KRAS Mutants Upregulate Integrin β4 to Promote Invasion and Metastasis in Colorectal Cancer.

Knocking-out ITGB4, which is overexpressed in KRAS-mutant colorectal cancer and promotes tumor aggressiveness, diminishes local invasiveness and rates of pulmonary metastasis.

Liver Endothelium Promotes HER3-Mediated Cell Survival in Colorectal Cancer with Wild-Type and Mutant KRAS.

This body of work highlighted a potential strategy of using HER3 antibodies in combination with standard chemotherapy agents for treating patients with either KRAS wild-type or KRAS mutant metastatic colorectal cancer.

Dual blockade of macropinocytosis and asparagine bioavailability shows synergistic anti-tumor effects on KRAS-mutant colorectal cancer

Mutations of KRAS gene are found in various types of cancer, including colorectal cancer (CRC). Despite intense efforts, no pharmacological approaches are expected to be effective against KRAS-mutant cancers. Macropinocytosis is an evolutionarily conserved actin-dependent endocytic process that internalizes extracellular fluids into large vesicles called macropinosomes. Recent studies have revealed macropinocytosis's important role in metabolic adaptation to nutrient stress in cancer cells harboring KRAS mutations. Here we showed that KRAS-mutant CRC cells enhanced macropinocytosis for tumor growth under nutrient-depleted conditions. We also demonstrated that activation of Rac1 and phosphoinositide 3-kinase were involved in macropinocytosis of KRAS-mutant CRC cells. Furthermore, we found that macropinocytosis was closely correlated with asparagine metabolism. In KRAS-mutant CRC cells engineered with knockdown of asparagine synthetase, macropinocytosis was accelerated under glutamine-depleted condition, and albumin addition could restore the glutamine depletion-induced growth suppression by recovering the intracellular asparagine level. Finally, we discovered that the combination of macropinocytosis inhibition and asparagine depletion dramatically suppressed the tumor growth of KRAS-mutant CRC cells in vivo. These results indicate that dual blockade of macropinocytosis and asparagine bioavailability could be a novel therapeutic strategy for KRAS-mutant cancers.

Cetuximab promotes RSL3-induced ferroptosis by suppressing the Nrf2/HO-1 signalling pathway in KRAS mutant colorectal cancer

Cetuximab is approved for the treatment of metastatic colorectal cancer (mCRC) with RAS wild-type. Nevertheless, the prognosis remains poor and the effectiveness of cetuximab is limited in KRAS mutant mCRC. Recently, emerging evidence has shown that ferroptosis, a newly discovered form of nonapoptotic cell death, is closely related to KRAS mutant cells. Here, we further investigated whether cetuximab-mediated regulation of p38/Nrf2/HO-1 promotes RSL3-induced ferroptosis and plays a pivotal role in overcoming drug resistance in KRAS mutant colorectal cancer (CRC). In our research, we used two KRAS mutant CRC cell lines, HCT116 and DLD-1, as models of intrinsic resistance to cetuximab. The viability of cells treated with the combination of RSL3 and cetuximab was assessed by the CCK-8 and colony formation assays. The effective of cetuximab to promote RSL3-induced ferroptosis was investigated by evaluating lipid reactive oxygen species accumulation and the expression of the malondialdehyde and the intracellular iron assay. Cetuximab therapy contributed to regulating the p38/Nrf2/HO-1 axis, as determined by western blotting and transfection with small interfering RNAs. Cetuximab promoted RSL3-induced ferroptosis by inhibiting the Nrf2/HO-1 in KRAS mutant CRC cells, and this was further demonstrated in a xenograft nude mouse model. Our work reveals that cetuximab enhances the cytotoxic effect of RSL3 on KRAS mutant CRC cells and that cetuximab enhances RSL3-induced ferroptosis by inhibiting the Nrf2/HO-1 axis through the activation of p38 MAPK.

Oncolytic Virus Affects the RAS Pathway in Cancer: RNA Sequence Analysis.

Approximately 45% of individuals diagnosed with Colorectal Cancer (CRC) also possess KRAS mutations. One developing therapeutic method for this disease is reovirus treatment. It is theorized that reovirus treatment on patients with KRAS mutated CRC cells would be successful due to the virus' innate oncolytic properties [1]. Reovirus, a stable form of nonenveloped double-stranded RNA, causes minor infections in humans under normal circumstances. However, when the virus encounters KRAS mutated cells, it has the potential to lyse them [2]. While this method of treatment to CRC has shown signs of success, we are still some ways from universal administration of reovirus as a treatment. This review seeks to utilize various studies, as well as our original research data, to investigate reovirus as an efficient method of treatment, with a focus on select growth, apoptotic and RAS-related genes, and their effectiveness of mitigating KRAS mutated CRC post reovirus treatment. Furthermore, the review highlights transcriptome analysis as an effective tool to examine these genes and their activity. It has been shown that reovirus treatment induces apoptosis and mitigates growth related gene activity. This review confirms the novelty of our findings on the efficacy of reovirus in CRC treatment. The study that this review article discusses concluded that 10 apoptotic and lymphocyte-related genes were found to be upregulated and 6 angiogenesis and Ras-related genes were found to be downregulated post reovirus treatment. These findings enforce the notion that reovirus could be used as a novel treatment for KRAS mutated CRC.

RAS specific protease induces irreversible growth arrest via p27 in several KRAS mutant colorectal cancer cell lines

Ras-specific proteases to degrade RAS within cancer cells are under active development as an innovative strategy to treat tumorigenesis. The naturally occurring biological toxin effector called RAS/RAP1-specific endopeptidase (RRSP) is known to cleave all RAS within a cell, including HRAS, KRAS, NRAS and mutant KRAS G13D. Yet, our understanding of the mechanisms by which RRSP drives growth inhibition are unknown. Here, we demonstrate, using isogenic mouse fibroblasts expressing a single isoform of RAS or mutant KRAS, that RRSP equally inactivates all isoforms of RAS as well as the major oncogenic KRAS mutants. To investigate how RAS processing might lead to varying outcomes in cell fate within cancer cells, we tested RRSP against four colorectal cancer cell lines with a range of cell fates. While cell lines highly susceptible to RRSP (HCT116 and SW1463) undergo apoptosis, RRSP treatment of GP5d and SW620 cells induces G1 cell cycle arrest. In some cell lines, growth effects were dictated by rescued expression of the tumor suppressor protein p27 (Kip1). The ability of RRSP to irreversibly inhibit cancer cell growth highlights the antitumor potential of RRSP, and further warrants investigation as a potential anti-tumor therapeutic.

Quadruple-editing of the MAPK and PI3K pathways effectively blocks the progression of KRAS-mutated colorectal cancer cells.

Mutated KRAS promotes the activation of the MAPK pathway and the progression of colorectal cancer (CRC) cells. Aberrant activation of the PI3K pathway strongly attenuates the efficacy of MAPK suppression in KRAS‐mutated CRC. The development of a novel strategy targeting a dual pathway is therefore highly essential for the therapy of KRAS‐mutated CRC. In this study, a quadruple‐depleting system for the KRAS, MEK1, PIK3CA, and MTOR genes based on CRISPR/SaCas9 was developed. Adenovirus serotype 5 (ADV5) was integrated with two engineered proteins, an adaptor and a protector, to form ADV‐protein complex (APC) for systemic delivery of the CRISPR system. Quadruple‐editing could significantly inhibit the MAPK and PI3K pathways in CRC cells with oncogenic mutations of KRAS and PIK3CA or with KRAS mutation and compensated PI3K activation. Compared with MEK and PI3K/MTOR inhibitors, quadruple‐editing induced more significant survival inhibition on primary CRC cells with oncogenic mutations of KRAS and PIK3CA. The adaptor specifically targeting EpCAM and the hexon‐shielding protector could dramatically enhance ADV5 infection efficiency to CRC cells and significantly reduce off‐targeting tropisms to many organs except the colon. Moreover, quadruple‐editing intravenously delivered by APC significantly blocked the dual pathway and tumor growth of KRAS‐mutated CRC cells, without influencing normal tissues in cell‐ and patient‐derived xenograft models. Therefore, APC‐delivered quadruple‐editing of the MAPK and PI3K pathways shows a promising therapeutic potential for KRAS‐mutated CRC.

4-Acetyl-Antroquinonol B Improves the Sensitization of Cetuximab on Both Kras Mutant and Wild Type Colorectal Cancer by Modulating the Expression of Ras/Raf/miR-193a-3p Signaling Axis.

The KRAS mutation is one of the leading driver mutations in colorectal cancer (CRC), and it is usually associated with poor prognosis and drug resistance. Therapies targeting the epidermal growth factor receptor (EFGR) are widely used for end-stage CRC. However, patients with KRAS mutant genes cannot benefit from this therapy because of Ras signaling activation by KRAS mutant genes. Our previous study revealed the anti-proliferative effect of 4-acetyl-antroquinonol B (4-AAQB) on CRC cells, but whether the drug is effective in KRAS-mutant CRC remains unknown. We screened CRC cell lines harboring the KRAS mutation, namely G12A, G12C, G12V and G13D, with one wild type cell line as the control; SW1463 and Caco-2 cell lines were used for further experiments. Sulforhodamine B assays, together with the clonogenicity and invasion assay, revealed that KRAS-mutant SW1463 cells were resistant to cetuximab; however, 4-AAQB treatment effectively resensitized CRC cells to cetuximab through the reduction of colony formation, invasion, and tumorsphere generation and of oncogenic KRAS signaling cascade of CRC cells. Thus, inducing cells with 4-AAQB before cetuximab therapy could resensitize KRAS-mutant, but not wild-type, cells to cetuximab. Therefore, we hypothesized that 4-AAQB can inhibit KRAS. In silico analysis of the publicly available GEO (GSE66548) dataset of KRAS-mutated versus KRAS wild-type CRC patients confirmed that miR-193a-3p was significantly downregulated in the former compared with the latter patient population. Overexpression of miR-193a-3p considerably reduced the oncogenicity of both CRC cells. Furthermore, KRAS is a key target of miR-193a-3p. In vivo treatment with the combination of 4-AAQB and cetuximab significantly reduced the tumor burden of a xenograft mice model through the reduction of the expression of oncogenic markers (EGFR) and p-MEK, p-ERK, and c-RAF/p-c-RAF signaling, with the simultaneous induction of miR-193a-3p expression in the plasma. In summary, our findings provide strong evidence regarding the therapeutic effect of 4-AAQB on KRAS-mutant CRC cells. Furthermore, 4-AAQB effectively inhibits Ras singling in CRC cells, through which KRAS-mutant CRC can be resensitized to cetuximab.

Abstract 961: FGFR3 and eIF4E are overexpressed and interact with PRMT5 and KRAS in CRC

Abstract Introduction Nearly 45% of colorectal cancer (CRC) patients harbor a mutation in their KRAS gene for which, despite many years of research, there are still no targeted therapies available. Protein Arginine Methyltransferase 5 (PRMT5) is a transcription regulator for multiple cellular processes that is currently being tested as a potential therapeutic target in several cancer types. Previous studies have shown that PRMT5 depletion leads to a reduction in FGFR3 and eIF4E expression. FGFR3 and eIF4E are oncogenes which play an important role in cellular proliferation and tumorigenesis. Furthermore, our group's previous research has demonstrated that PRMT5 inhibition shows significantly greater therapeutic effects in KRAS mutant CRC cells when compared to KRAS wild-type (WT) CRC cells. Methodology The Gene Expression Profiling Interactive Analysis (GEPIA) database was used to analyze the RNA-Seq data of CRC patients from The Cancer Genome Atlas (TCGA) database. We first used the GEPIA database to determine whether eIF4E and FGFR3 are overexpressed in colon and rectum patient tumor samples when compared to normal colon and rectum patient samples. We then used the GEPIA database to determine whether eIF4E and FGFR3 gene expression are correlated with PRMT5 and KRAS gene expression in colon and rectum patient tumor samples. We next used the STRING database to analyze whether eIF4E and FGFR3 interact with PRMT5 and KRAS. Finally, we used Biorender to develop a proposed molecular mechanism outlining how eIF4E and FGFR3 interact with PRMT5 and KRAS in CRC. Results • eIF4E is 2.4 and 2.2-Fold overexpressed in colon and rectum patient tumor samples, respectively, compared to normal colon and rectum patient samples (q < 0.01). • FGFR3 is 2.3 and 3.7-Fold overexpressed in colon and rectum patient tumor samples, respectively, compared to normal colon and rectum patient samples (q < 0.01). • eIF4E gene expression is positively correlated with PRMT5 (p < 0.01, R = 0.85) and KRAS (p < 0.01, R = 0.86) gene expression in colon and rectum patient tumor samples. • FGFR3 gene expression is positively correlated with PRMT5 (p < 0.01, R = 0.61) and KRAS (p < 0.01, R = 0.63) gene expression in colon and rectum patient tumor samples. • eIF4E and FGFR3 are shown to interact with PRMT5 and KRAS in the STRING database (Interaction score > 0.150). • A molecular mechanism is proposed suggesting that PRMT5 inhibition may show greater therapeutic effects in KRAS mutant CRC, when compared to KRAS WT CRC, through PRMT5's interactions with eIF4E and FGFR3. Conclusion eIF4E and FGFR3 have been shown to be overexpressed and to interact with PRMT5 and KRAS in CRC. This significant observation can be therapeutically utilized towards developing new effective treatments for KRAS mutant CRC patients. Further analysis is currently in progress to verify the proposed molecular mechanism outlining how eIF4E and FGFR3 interact with PRMT5 and KRAS in CRC. Citation Format: David Shifteh, Tzuriel Sapir, Moshe Pahmer, Sanjay Goel, Radhashree Maitra. FGFR3 and eIF4E are overexpressed and interact with PRMT5 and KRAS in CRC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 961.

Abstract 2148: Transcriptome analysis reveals antitumorigenic alterations of seven KRAS-related pathways in reovirus-treated colorectal cancer patients

Abstract Introduction: Reovirus propagates with high efficiency in KRAS mutated colorectal cancer (CRC). About 40-45% of CRC patients possess KRAS mutation. Oncolytic reovirus treatment in combination with chemotherapy was tested in patients possessing KRAS mutated metastatic CRC. This study evaluates the biological responses to reovirus treatment by determining the gene expression patterns in RAS-related signaling pathways. Methodology: Reovirus was administered as a 60-min intravenous infusion for 5 consecutive days every 28 days, at tissue culture infective dose (TCID50) of 3x1010. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood pre and post reovirus administration at 48hr, Day-8, and Day-15. Clariom D Human Assay was used to determine the expression of vital genes compared to pre-reovirus treatment by RNA sequencing. Using exported sample signals, ΔΔCt method was used to analyze the fold changes of 2,083 genes within seven KRAS-related pathways. Of these genes, 155 had significance, 16 of which were further analyzed. Significance was calculated by students-Two-tail-T test. A hierarchical clustering dendrogram was constructed using Pearson's correlation coefficient. Results: Fold changes [p<0.05] are normalized to Pre-treatment and compared to untreated controls. Several genes were common to multiple pathways. Conclusion: Our transcriptome assay displayed that reovirus treatment causes meaningful alterations in gene expression, showing that reovirus is a successful therapy for KRAS mutated CRC. Reovirus in combination with chemotherapy leads to the regression of tumor growth and KRAS mutated CRC cell proliferation. Our results corroborate that reovirus treatment leads to immune responses, which include increased lymphocytic maturation and activity, increased levels of apoptosis, and a decrease in angiogenesis. While reovirus is propagating, NF-κB, SOS1, and RRAS promote this immune response. Results: Fold Changes of Genes Analyzed with Correlated PathwaysSelected GenesPathways48hr FCD8 FCD15 FCSOS1RAS, MAPK, EGFR, PI3K, Angiogenesis2.49--RRASRAS, MAPK2.24--PIK3CBRAS, PI3K-2.273.16NOS3EGFR, PI3K--0.61MIR 16-2miR In lymphocytes, Angiogenesis--1.7CHORDC1miR In lymphocytes, Angiogenesis1.89-4.54RTN4miR In lymphocytes, Angiogenesis4.66--FAM96AmiR In lymphocytes, Angiogenesis4.54--SYNEmiR In lymphocytes, Angiogenesis-0.780.71ANGPT1Angiogenesis, PI3K-0.62-VEGFBAngiogenesis0.440.280.28NFKBApoptosis/Immune Response, RAS, MAPK, PI3K-191.42NFKB1(NFKB Subunit)Apoptosis/Immune Response, RAS, MAPK, PI3K--1.42NFKB1A (NFKB Subunit)Apoptosis/Immune Response, RAS, MAPK, PI3K-19-JUNApoptosis/Oncogene, MAPK, EGFR--0.69CASP8Apoptosis-2.111.77CASP9Apoptosis, PI3K-1.45-IGF2Apoptosis, PI3K-0.73- Citation Format: Elisha Fogel, Avishai Samouha, Sanjay Goel, Radhashree Maitra. Transcriptome analysis reveals antitumorigenic alterations of seven KRAS-related pathways in reovirus-treated colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2148.

Oncogenic KRAS mutations enhance amino acid uptake by colorectal cancer cells via the hippo signaling effector YAP1.

Oncogenic KRAS mutations develop unique metabolic dependencies on nutrients to support tumor metabolism and cell proliferation. In particular, KRAS mutant cancer cells exploit amino acids (AAs) such as glutamine and leucine, to accelerate energy metabolism, redox balance through glutathione synthesis and macromolecule biosynthesis. However, the identities of the amino acid transporters (AATs) that are prominently upregulated in KRAS mutant cancer cells, and the mechanism regulating their expression have not yet been systematically investigated. Here, we report that the majority of the KRAS mutant colorectal cancer (CRC) cells upregulate selected AATs (SLC7A5/LAT1, SLC38A2/SNAT2, and SLC1A5/ASCT2), which correlates with enhanced uptake of AAs such as glutamine and leucine. Consistently, knockdown of oncogenic KRAS downregulated the expression of AATs, thereby decreasing the levels of amino acids taken up by CRC cells. Moreover, overexpression of mutant KRAS upregulated the expression of AATs (SLC7A5/LAT1, SLC38A2/SNAT2, and SLC1A5/ASCT2) in KRAS wild‐type CRC cells and mouse embryonic fibroblasts. In addition, we show that the YAP1 (Yes‐associated protein 1) transcriptional coactivator accounts for increased expression of AATs and mTOR activation in KRAS mutant CRC cells. Specific knockdown of AATs by shRNAs or pharmacological blockage of AATs effectively inhibited AA uptake, mTOR activation, and cell proliferation. Collectively, we conclude that oncogenic KRAS mutations enhance the expression of AATs via the hippo effector YAP1, leading to mTOR activation and CRC cell proliferation.

PRMT1 enhances oncogenic arginine methylation of NONO in colorectal cancer

Arginine methylation is an important posttranslational modification catalyzed by protein arginine methyltransferases (PRMTs). However, the role of PRMTs in colorectal cancer (CRC) progression is not well understood. Here we report that non-POU domain-containing octamer-binding protein (NONO) is overexpressed in CRC tissue and is a potential marker for poor prognosis in CRC patients. NONO silencing resulted in decreased proliferation, migration, and invasion of CRC cells, whereas overexpression had the opposite effect. In a xenograft model, tumors derived from NONO-deficient CRC cells were smaller than those derived from wild-type (WT) cells, and PRMT1 inhibition blocked CRC xenograft progression. A mass spectrometry analysis indicated that NONO is a substrate of PRMT1. R251 of NONO was asymmetrically dimethylated by PRMT1 in vitro and in vivo. Compared to NONO WT cells, NONO R251K mutant-expressing CRC cells showed reduced proliferation, migration, and invasion, and PRMT1 knockdown or pharmacological inhibition abrogated the malignant phenotype associated with NONO asymmetric dimethylation in both KRAS WT and mutant CRC cells. Compared to adjacent normal tissue, PRMT1 was highly expressed in the CRC zone in clinical specimens, which was correlated with poor overall survival in patients with locally advanced CRC. These results demonstrate that PRMT1-mediated methylation of NONO at R251 promotes CRC growth and metastasis, and suggest that PRMT1 inhibition may be an effective therapeutic strategy for CRC treatment regardless of KRAS mutation status.

ERK1/2 Signaling Induces Upregulation of ANGPT2 and CXCR4 to Mediate Liver Metastasis in Colon Cancer.

Carcinoma development in colorectal cancer is driven by genetic alterations in numerous signaling pathways. Alterations in the RAS-ERK1/2 pathway are associated with the shortest overall survival for patients after diagnosis of colorectal cancer metastatic disease, yet how RAS-ERK signaling regulates colorectal cancer metastasis remains unknown. In this study, we used an unbiased screening approach based on selection of highly liver metastatic colorectal cancer cells in vivo to determine genes associated with metastasis. From this, an ERK1/2-controlled metastatic gene set (EMGS) was defined. EMGS was associated with increased recurrence and reduced survival in patients with colorectal cancer tumors. Higher levels of EMGS expression were detected in the colorectal cancer subsets consensus molecular subtype (CMS)1 and CMS4. ANGPT2 and CXCR4, two genes within the EMGS, were subjected to gain-of-function and loss-of-function studies in several colorectal cancer cell lines and then tested in clinical samples. The RAS-ERK1/2 axis controlled expression of the cytokine ANGPT2 and the cytokine receptor CXCR4 in colorectal cancer cells, which facilitated development of liver but not lung metastases, suggesting that ANGPT2 and CXCR4 are important for metastatic outgrowth in the liver. CXCR4 controlled the expression of cytokines IL10 and CXCL1, providing evidence for a causal role of IL10 in supporting liver colonization. In summary, these studies demonstrate that amplification of ERK1/2 signaling in KRAS-mutated colorectal cancer cells affects the cytokine milieu of the tumors, possibly affecting tumor-stroma interactions and favoring liver metastasis formation. SIGNIFICANCE: These findings identify amplified ERK1/2 signaling in KRAS-mutated colorectal cancer cells as a driver of tumor-stroma interactions that favor formation of metastases in the liver.

Targeting antioxidant enzymes enhances the therapeutic efficacy of the BCL-XL inhibitor ABT-263 in KRAS-mutant colorectal cancers

Cancer chemotherapeutic drugs exert cytotoxic effects by modulating intracellular reactive oxygen species (ROS) levels. However, whether ROS modulates the efficacy of targeted therapeutics remains poorly understood. Previously, we reported that upregulation of the anti-apoptotic protein, BCL-XL, by KRAS activating mutations was a potential target for KRAS-mutant colorectal cancer (CRC) treatment. Here, we demonstrated that the BCL-XL targeting agent, ABT-263, increased intracellular ROS levels and targeting antioxidant pathways augmented the therapeutic efficacy of this BH3 mimetic. ABT-263 induced expression of genes associated with ROS response and increased intracellular ROS levels by enhancing mitochondrial superoxide generation. The superoxide dismutase inhibitor, 2-methoxyestradiol (2-ME), exhibited synergism with ABT-263 in KRAS-mutant CRC cell lines. This synergistic effect was attributed to the inhibition of mTOR-dependent translation of the anti-apoptotic MCL-1 protein via caspase 3-mediated cleavage of AKT and S6K. In addition, combination treatment of ABT-263 and 2-ME demonstrated a synergistic effect in in vivo patient-derived xenografts harboring KRAS mutations. Our data suggest a novel role for ROS in BH3 mimetic-based targeted therapy and provide a novel strategy for treatment of CRC patients with KRAS mutations.

Palladium (II) Complex Enhances ROS-Dependent Apoptotic Effects via Autophagy Inhibition and Disruption of Multiple Signaling Pathways in Colorectal Cancer Cells.

Inhibition of autophagy is reported to be a therapeutically effective strategy in overcoming resistance that is a deadly outcome in cancer. One of the most common reasons for chemo-resistance to treatment is the patients with tumors exhibiting a KRAS mutation, which occurs in approximately 40% of colorectal cancer patients. Hence, we assessed whether a Palladium (Pd)(II) complex is a promising anticancer complex, compared to 5-fluorouracil in KRAS wt HT-29 and KRAS mutant HCT-15 cells. HCT-15 and HT-29 cells were used for colorectal cancer and Chloroquine (CQ) was used as an inhibitor of autophagy. In this context, cells were treated with Pd(II) complex and 5-FU in combination with CQ for 48h and cell viability was measured by SRB assay. Cell death mode was examined with M30 and M65 ELISA assays, using annexin V/propidium iodide. Autophagy was determined by Acridine Orange (AO) staining. Furthermore, the expressions of various autophagy and apoptosis-related proteins were evaluated with Western blotting. Luminex assay and the level of Reactive Oxygen Species (ROS) were examined. Cell viability was found to decrease in a dose-dependent manner and CQ enhanced cytotoxic effect in Pd(II) and 5-FU treated cells in colorectal cancer cells. Our data showed that inhibition of autophagic flux significantly increased intrinsic apoptosis through the activation of ROS. We showed that combinatorial treatment with CQ induced apoptosis via the caspase-dependent mitochondrial pathway. Luminex analysis revealed that the combination resulted in a down-regulation of NF-κB/AKT/CREB signaling pathways in both cell lines, however, decreased Erk1/2 protein expression was only observed after treatment with CQ combination in HCT-15 cells. We suggest that the inhibition of autophagy along with Pd(II) and 5-FU treatment has a synergistic effect on KRAS-mutant colorectal cancer cells. Autophagy inhibition by CQ promotes apoptosis via blockade of the NF-κB/AKT/CREB and activation of ROS.

In-Vitro and In-Silico Evaluations of Heterocyclic-Containing Diarylpentanoids as Bcl-2 Inhibitors Against LoVo Colorectal Cancer Cells.

In the present study, we investigated the in-vitro anti-cancer potential of six diarylpentanoids against a panel of BRAF- and KRAS-mutated colorectal cancer cell lines including T84, SW620, LoVo, HT29, NCI-H508, RKO, and LS411N cells. Structure–activity relationship study suggested that the insertions of tetrahydro-4H-thiopyran-4-one and brominated phenyl moieties are essential for better cytotoxicity. Among the evaluated analogs, 2e has been identified as the lead compound due to its low IC50 values of approximately 1 µM across all cancer cell lines and high chemotherapeutic index of 7.1. Anti-proliferative studies on LoVo cells showed that 2e could inhibit cell proliferation and colony formations by inducing G2/M cell cycle arrest. Subsequent cell apoptosis assay confirmed that 2e is a Bcl-2 inhibitor that could induce intrinsic cell apoptosis by creating a cellular redox imbalance through its direct inhibition on the Bcl-2 protein. Further molecular docking studies revealed that the bromophenyl moieties of 2e could interact with the Bcl-2 surface pocket through hydrophobic interaction, while the tetrahydro-4H-thiopyran-4-one fragment could form additional Pi-sulfur and Pi-alkyl interactions in the same binding site. In all, the present results suggest that 2e could be a potent lead that deserves further modification and investigation in the development of a new Bcl-2 inhibitor.