KRAS Mutant Colorectal Cancer Research Articles

The relationships of OSBPL3 expression with KI-67 expression and KRAS mutations in CRC: implications for diagnosis and prognosis

BackgroundOSBPL3 is overexpressed in a variety of malignancies and is closely associated with tumor growth and metastasis. However, its expression and function in colorectal cancer (CRC) are unclear. We aimed to investigate its prognostic and therapeutic value in this disease by detecting its expression in CRC and its correlation with the clinicopathological characteristics and prognosis of patients.MethodsA total of 92 CRC samples were included in this study. According to the 2020 WHO diagnostic criteria, the criteria of the American Joint Committee on Cancer (AJCC) 8th edition staging system were used. OSBPL3 and Ki-67 expression in these samples was detected by immunohistochemistry. OSBPL3 mRNA expression was detected by qRT-PCR. KRAS/NRAS mutations were detected by an amplification refractory mutation system (ARMS). Data analysis was performed using the statistical analysis software Prism 8.ResultsOSBPL3 was found to be significantly overexpressed in CRC tumor tissues and was associated with worse progression-free survival and overall survival in patients. Additionally, OSBPL3 expression was negatively correlated with the degree of tumor differentiation. KRAS mutations were detected in approximately 32.6% of patients and were significantly associated with high OSBPL3 expression. In addition, OSBPL3 and Ki-67 expression was significantly correlated.ConclusionsOSBPL3 is highly expressed in CRC samples and predicts a worse prognosis. OSBPL3 may become a new potential therapeutic target for CRC.

Development of SOS1 Inhibitor-Based Degraders to Target KRAS-Mutant Colorectal Cancer.

Direct blockade of KRAS driver mutations in colorectal cancer (CRC) has been challenging. Targeting SOS1, a guanine nucleotide exchange factor, has arisen as an attractive approach for KRAS-mutant CRC. Here, we describe the development of novel SOS1 degraders and their activity in patient-derived CRC organoids (PDO). The design of these degraders as proteolysis-targeting chimera was based on the crystal structures of cereblon and SOS1. The synthesis used the 6- and 7-OH groups of a quinazoline core as anchor points to connect lenalidomide. Fifteen compounds were screened for SOS1 degradation. P7 was found to have up to 92% SOS1 degradation in both CRC cell lines and PDOs with excellent specificity. SOS1 degrader P7 demonstrated superior activity in inhibiting CRC PDO growth with an IC50 5 times lower than that of SOS1 inhibitor BI3406. In summary, we developed new SOS1 degraders and demonstrated SOS1 degradation as a feasible therapeutic strategy for KRAS-mutant CRC.

A PDX model combined with CD-DST assay to evaluate the antitumor properties of KRpep-2d and oxaliplatin in KRAS (G12D) mutant colorectal cancer

Patient-derived xenograft (PDX) models are more faithful in maintaining the characteristics of human tumors than cell lines and are widely used in drug development, although they have some disadvantages, including their relative low success rate, long turn-around time, and high costs. The collagen gel droplet embedded culture drug sensitivity test (CD-DST) has been used as an in-vitro drug sensitivity test for patients with cancer because of its high success rate of primary cell culture, high sensitivity, and good clinical relevance, but it is based on an in-vitro cell culture and may not simulate the tumor microenvironment accurately. This study aims to combine a PDX model with CD-DST to evaluate the efficiency of antitumor agents. KRpep-2d, a small peptide targeting KRAS (G12D), and oxaliplatin were used to verify the feasibility of this approach. Whole-exome sequencing and Sanger sequencing were first applied to test and validate the KRAS mutation status of a panel of colorectal cancer PDX tissues. One PDX model was verified to carry KRAS (G12D) mutation and was used for in-vivo and the CD-DST drug tests. We then established the PDX mouse model from the patient with the KRAS (G12D) mutation and obtained viable cancer cells derived from the same PDX model. Next, the antitumor abilities of KRpep-2d and oxaliplatin were estimated in the PDX model and the CD-DST. We found that KRpep-2d showed no significant antitumor effect on the xenograft model or on cancer cells derived from the same PDX model. In contrast, oxaliplatin showed significant inhibitory effects in both tests. In conclusion, the PDX model in combination with the CD-DST assay is a comprehensive and feasible method of evaluating the antitumor properties of compounds and could be applied for new drug discovery.

LncRNA SPINT1-AS1/miR-433-3p/E2F3 positive feedback loop promotes the KRAS-mutant colorectal cancer cell proliferation, migration and invasion.

Colorectal cancer (CRC) features high prevalence and mortality. Long non-coding RNAs (lncRNAs) exert nonnegligible roles in human cancer development. Nevertheless, the functions of most lncRNAs still remain unexplored. We currently focused on detecting the influence of SPINT1 antisense RNA 1 (SPINT1-AS1) on CRC development and investigating into the potential regulatory mechanism. RT-qPCR analysis first confirmed that SPINT1-AS1 exhibited high expression in KRAS-mutant (KRASMUT) CRC cells. Through series of functional experiments, we observed that knockdown of SPINT1-AS1 weakened KRASMUT CRC cell proliferation, migration and invasion. Afterwards, the implementation of mechanism assays help to verify that SPINT1-AS1 sequestered microRNA-433-3p (miR-433-3p) to regulate the expression of E2F transcription factor 3 (E2F3). Besides, E2F3 was validated to activate the transcription of SPINT1-AS1 in turn. Rescue experiments confirmed the functional influence of SPINT1-AS1/miR-433-3p/E2F3 on CRC cells. In summary, the molecular axis of SPINT1-AS1/miR-433-3p/E2F3 forms a positive loop which might become a potential biomarker in CRC.

Platycodin D sensitizes KRAS-mutant colorectal cancer cells to cetuximab by inhibiting the PI3K/Akt signaling pathway.

Cetuximab is a monoclonal antibody against epidermal growth factor receptor that blocks downstream signaling pathways of receptor tyrosine kinases, including Ras/Raf/MAPK and PI3K/Akt, thereby inhibiting tumor cell proliferation and inducing cancer cell apoptosis. Owing to KRAS mutations, the effectiveness of cetuximab is usually limited by intrinsic drug resistance. Continuous activation of the PI3K/Akt signaling pathway is another reason for cetuximab resistance. Platycodin-D, a bioactive compound isolated from the Chinese herb Platycodon grandiflorum, regulates Akt in different trends based on tissue types. To investigate whether platycodin-D can sensitize KRAS-mutant colorectal cancer cells to cetuximab by inhibiting the PI3K/Akt signaling pathway, HCT116 and LoVo cells were treated with cetuximab and platycodin-D. LY294002 and SC79 were used to regulate Akt to further evaluate whether platycodin-D sensitizes cells to cetuximab by inhibiting Akt. Our results confirmed that platycodin-D increased the cytotoxic effects of cetuximab, including inhibition of growth, migration, and invasion, via downregulation of PI3K and Akt phosphorylation in HCT116 and LoVo cells both in vitro and in vivo. Given these data, platycodin-D may sensitize KRAS-mutant colorectal cancer cells to cetuximab via inhibition of the PI3K/Akt signaling pathway.

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.

109P Improved accuracy of somatic variant detection from high throughput sequencing (HTS) data via site-specific noise estimation

109P Improved accuracy of somatic variant detection from high throughput sequencing (HTS) data via site-specific noise estimation

The drug efflux pump MDR1 promotes intrinsic and acquired resistance to PROTACs in cancer cells.

Proteolysis-targeting chimeras (PROTACs) are a promising new class of drugs that selectively degrade cellular proteins of interest. PROTACs that target oncogene products are avidly being explored for cancer therapies, and several are currently in clinical trials. Drug resistance is a substantial challenge in clinical oncology, and resistance to PROTACs has been reported in several cancer cell models. Here, using proteomic analysis, we found intrinsic and acquired resistance mechanisms to PROTACs in cancer cell lines mediated by greater abundance or production of the drug efflux pump MDR1. PROTAC-resistant cells were resensitized to PROTACs by genetic ablation of ABCB1 (which encodes MDR1) or by coadministration of MDR1 inhibitors. In MDR1-overexpressing colorectal cancer cells, degraders targeting either the kinases MEK1/2 or the oncogenic mutant GTPase KRASG12C synergized with the dual epidermal growth factor receptor (EGFR/ErbB)/MDR1 inhibitor lapatinib. Moreover, compared with single-agent therapies, combining MEK1/2 degraders with lapatinib improved growth inhibition of MDR1-overexpressing KRAS-mutant colorectal cancer xenografts in mice. Together, our findings suggest that concurrent blockade of MDR1 will likely be required with PROTACs to achieve durable protein degradation and therapeutic response in cancer.

Therapeutic Targeting of Cancer Stem Cells Prevents Resistance of Colorectal Cancer Cells to MEK Inhibition.

Drug resistance is a leading cause for the failure of cancer treatments. Plasticity of cancer cells to acquire stem cell-like properties enables them to escape drug toxicity through different adaptive mechanisms. Eliminating cancer stem cells (CSCs) can potentially improve treatment outcomes for patients. To determine the role of CSCs in resistance of colorectal cancer cells to targeted therapies and identify treatment strategies, we treated spheroids of BRAFmut and KRASmut colorectal cancer cells with inhibitors of the mitogen-activated protein kinase pathway and studied resistance mechanisms through gene and protein expression analyses. We found that treatments activated several oncogenic pathways and expression of CSC markers CD166 and ALDH1A3. We identified a specific combination treatment using trametinib and mithramycin A to simultaneously inhibit the CSC phenotype and activities of several pathways in cancer cells. This study demonstrates the feasibility of therapeutic targeting of CSCs as a strategy to block tumorigenic activities of cancer cells.

Modulation of Fibroblast Phenotype by Colorectal Cancer Cell-Secreted Factors Is Mostly Independent of Oncogenic KRAS.

KRAS mutations have been shown to extend their oncogenic effects beyond the cancer cell, influencing the tumor microenvironment. Herein, we studied the impact of mutant KRAS on the modulation of the pro-tumorigenic properties of cancer-associated fibroblasts (CAFs), including α-SMA expression, TGFβ1 and HGF production, extracellular matrix components and metalloproteinases expression as well as collagen contraction and migration capacities. To do so, CCD-18Co normal-like colon fibroblasts were challenged with conditioned media from control and KRAS silenced colorectal cancer (CRC) cells. Our results showed that the mutant KRAS CRC cell-secreted factors were capable of turning normal-like fibroblasts into CAF-like by modulating the α-SMA expression, TGFβ1 and HGF production and migration capacity. Oncogenic KRAS played a secondary role as its silencing did not completely impair the capacity of CRC cells to modulate most of the fibroblast properties analyzed. In summary, our work suggests that mutant KRAS does not play a major role in controlling the CRC cell-secreted factors that modulate the behavior of fibroblasts. The fact that CRC cells retain the capacity to modulate the pro-tumorigenic features of fibroblasts independently of KRAS silencing is likely to negatively impact their response to KRAS inhibitors, thus standing as a putative mechanism of resistance to KRAS inhibition with potential therapeutical relevance.

Hot-Spot-Specific Probe (HSSP) for Rapid and Accurate Detection of KRAS Mutations in Colorectal Cancer.

Detection of oncogene mutations has significance for early diagnosis, customized treatment, treatment progression, and drug resistance monitoring. Here, we introduce a rapid, sensitive, and specific mutation detection assay based on the hot-spot-specific probe (HSSP), with improved clinical utility compared to conventional technologies. We designed HSSP to recognize KRAS mutations in the DNA of colorectal cancer tissues (HSSP-G12D (GGT→GAT) and HSSP-G13D (GGC→GAC)) by integration with real-time PCR. During the PCR analysis, HSSP attaches to the target mutation sequence for interference with the amplification. Then, we determine the mutation detection efficiency by calculating the difference in the cycle threshold (Ct) values between HSSP-G12D and HSSP-G13D. The limit of detection to detect KRAS mutations (G12D and G13D) was 5–10% of the mutant allele in wild-type populations. This is superior to the conventional methods (≥30% mutant allele). In addition, this technology takes a short time (less than 1.5 h), and the cost of one sample is as low as USD 2. We verified clinical utility using 69 tissue samples from colorectal cancer patients. The clinical sensitivity and specificity of the HSSP assay were higher (84% for G12D and 92% for G13D) compared to the direct sequencing assay (80%). Therefore, HSSP, in combination with real-time PCR, provides a rapid, highly sensitive, specific, and low-cost assay for detecting cancer-related mutations. Compared to the gold standard methods such as NGS, this technique shows the possibility of the field application of rapid mutation detection and may be useful in a variety of applications, such as customized treatment and cancer monitoring.

Healthy and unhealthy plant‐based diets in relation to the incidence of colorectal cancer overall and by molecular subtypes

BackgroundPlant‐based foods have been recommended for health. However, not all plant foods are healthy, and little is known about the association between plant‐based diets and specific molecular subtypes of colorectal cancer (CRC). We examined the associations of healthy and unhealthy plant‐based diets with the incidence of CRC and its molecular subtypes.MethodsWhile 123 773 participants of the Nurses’ Health Study and the Health Professionals Follow‐up Study had been followed up (3 143 158 person‐years), 3077 of them had developed CRC. Healthy and unhealthy plant‐based diet indices (hPDI and uPDI, respectively) were calculated using repeated food frequency questionnaire data. We determined the tumoural status of microsatellite instability (MSI), CpG island methylator phenotype (CIMP), and BRAF and KRAS mutations.ResultsHigher hPDI was associated with lower CRC incidence (multivariable hazard ratio [HR] comparing extreme quartiles, 0.86, 95% confidence interval [CI]: 0.77, 0.96; P‐trend = .04), whereas higher uPDI was associated with higher CRC incidence (multivariable HR comparing extreme quartiles, 1.16, 95% CI: 1.04, 1.29; P‐trend = .005). The association of hPDI significantly differed by KRAS status (P‐heterogeneity = .003) but not by other tumour markers. The hPDI was associated with lower incidence of KRAS‐wildtype CRC (multivariable HR comparing extreme quartiles, 0.74, 95% CI: 0.57, 0.96; P‐trend = .004) but not KRAS‐mutant CRC (P‐trend = .22).ConclusionsWhile unhealthy plant‐based diet enriched with refined grains and sugar is associated with higher CRC incidence, healthy plant‐based diet rich in whole grains, fruits and vegetables is associated with lower incidence of CRC, especially KRAS‐wildtype CRC.

The cholesterol uptake regulator PCSK9 promotes and is a therapeutic target in APC/KRAS-mutant colorectal cancer

Therapeutic targeting of KRAS-mutant colorectal cancer (CRC) is an unmet need. Here, we show that Proprotein Convertase Subtilisin/Kexin type 9 (PSCK9) promotes APC/KRAS-mutant CRC and is a therapeutic target. Using CRC patient cohorts, isogenic cell lines and transgenic mice, we identify that de novo cholesterol biosynthesis is induced in APC/KRAS mutant CRC, accompanied by increased geranylgeranyl diphosphate (GGPP)─a metabolite necessary for KRAS activation. PCSK9 is the top up-regulated cholesterol-related gene. PCSK9 depletion represses APC/KRAS-mutant CRC cell growth in vitro and in vivo, whereas PCSK9 overexpression induces oncogenesis. Mechanistically, PCSK9 reduces cholesterol uptake but induces cholesterol de novo biosynthesis and GGPP accumulation. GGPP is a pivotal metabolite downstream of PCSK9 by activating KRAS/MEK/ERK signaling. PCSK9 inhibitors suppress growth of APC/KRAS-mutant CRC cells, organoids and xenografts, especially in combination with simvastatin. PCSK9 overexpression predicts poor survival of APC/KRAS-mutant CRC patients. Together, cholesterol homeostasis regulator PCSK9 promotes APC/KRAS-mutant CRC via GGPP-KRAS/MEK/ERK axis and is a therapeutic target.

Prognostic Value of KRAS Mutations in Colorectal Cancer Patients.

Simple SummaryKirsten rat sarcoma viral oncogene homolog (KRAS)-mutated colorectal cancer (CRC) seems to have a different biological behavior and therapeutic approach compared with non-KRAS mutated CRC. Except for the proven predictive significance of KRAS mutations in CRC patients, their prognostic significance is still under evaluation. Our study shows that 93.2% of RAS-mutated patients were KRAS mutants, with G12D being the most common subtype. KRAS G12D mutation is correlated with better overall survival (OS). KRAS G12C mutations may indicate worse prognosis regarding progression free (PFS) and OS, as well as exon 4 and exon 3 KRAS mutations for PFS and OS, respectively. Further studies are warranted to confirm these results.Colorectal cancer (CRC) remains a major public health issue. The detection of parameters that affect CRC prognosis is of great significance. KRAS mutations, play a crucial role in tumorigenesis with a strong predictive value. KRAS-mutated stage-IV CRC patients gain no benefit of the anti-EGFR therapy. The KRAS G12C mutation subtype is under investigation for treatment regimens. The present study aimed to detect various RAS mutations in a cohort of 578 RAS-mutated CRC patients; 49% of them had de novo metastatic disease; 60% were male; 71.4% had left-sided tumors; and 94.6% had a good performance status. KRAS mutations were detected in 93.2% of patients, with KRAS G12D being the most common subtype (30.1%). KRAS mutations presented shorter progression-free (PFS) and overall survival (OS), compared with NRAS mutations, although not significantly (PFS: 13.8 vs. 18.5 months; p = 0.552; OS: 53.1 vs. 60.9 months; p = 0.249). KRAS G12D mutations presented better OS rates (p = 0.04). KRAS G12C mutation, even though not significantly, presented worse PFS and OS rates. KRAS exon 3 and 4 mutations presented different PFS and OS rates, although these were not significant. Concluding, KRAS G12D and G12C mutations lead to better and worst prognosis, respectively. Further studies are warranted to validate such findings and their possible therapeutic implication.

Mutant KRAS-Associated Proteome Is Mainly Controlled by Exogenous Factors.

Understanding how mutant KRAS signaling is modulated by exogenous stimuli is of utmost importance to elucidate resistance mechanisms underlying pathway inhibition failure, and to uncover novel therapeutic targets for mutant KRAS patients. Hence, aiming at perceiving KRAS-autonomous versus -non autonomous mechanisms, we studied the response of two mutant KRAS colorectal cancer cell lines (HCT116 and LS174T) upon KRAS silencing and treatment with rhTGFβ1-activated fibroblasts secretome. A proteomic analysis revealed that rhTGFβ1-activated fibroblast-secreted factors triggered cell line-specific proteome alterations and that mutant KRAS governs 43% and 38% of these alterations in HCT116 and LS174T cells, respectively. These KRAS-dependent proteins were localized and displayed molecular functions that were common to both cell lines (e.g., extracellular exosome, RNA binding functions). Moreover, 67% and 78% of the KRAS-associated proteome of HCT116 and LS174T cells, respectively, was controlled in a KRAS-non-autonomous manner, being dependent on fibroblast-secreted factors. In HCT116 cells, KRAS-non-autonomously controlled proteins were mainly involved in proteoglycans in cancer, p53, and Rap1 signaling pathways; whereas in LS174T cells, they were associated with substrate adhesion-dependent cell-spreading and involved in metabolic processes. This work highlights the context-dependency of KRAS-associated signaling and reinforces the importance of integrating the tumor microenvironment in the study of KRAS-associated effects.

Mutation of KRAS in colorectal adenocarcinoma in Greenland

KRAS mutation is one of the most frequent driver mutations in colorectal cancer (CRC) and is also a prognostic biomarker. The aim of the present study was to determine the frequency of KRAS mutations over time in the Greenlandic population diagnosed with CRC. In total, 578 patients with the diagnosis of adenocarcinoma between 1988 and 2017 were identified. The status of KRAS and the mutational subtypes of KRAS mutations were determined in 102 representative samples by the Idylla™ platform in the time periods 1988-1990, 2002-2004, and 2015-2017. The results showed that the frequency of the KRAS mutations increased significantly, from 27% in 1988-1990 to 43% in 2015-2017 (p < 0.001). Furthermore, the most frequent subtypes of KRAS mutations in Greenland were G12D (c.35G > A) with 14%, G12V (c.35G > T) with 7%, and G13D (c.38G > A) with 6%. In conclusion, this study showed that the frequency of KRAS mutations in CRC has been increasing in recent decades in the specific population of Greenland. The results of this study may be used in initiatives related to targeted therapy of CRC in specific ethnicities and in investigations focusing on the environmental factors of cancer-related somatic mutations.

Abstract 2394: Discovery of a sex-specific metabolic phenotype in KRAS mutant colorectal cancer

Abstract KRAS mutations occur in 35-45% of all colorectal cancer (CRC) cases and are notoriously difficult to treat. It is known that anomalous metabolism is a hallmark for cancer, however the relationship between mutant KRAS and tumor metabolism in CRCs has not been fully explored. We recently showed sex-disparities in metabolism by primary tumor location, indicating the pressing need to investigate the role of biological sex in CRC. Our aim was therefore to determine whether mutant KRAS alters metabolism in a sex-specific manner. We performed non-targeted metabolomics and targeted metabolomics on stage I-III chemotherapy-naive KRAS-mutant and wild type CRC patient tumor tissues (n=197) using ultra-high-performance liquid chromatography (UHPLC) coupled to quadrupole Time-of-Flight Mass Spectrometry (QTOF-MS), and tandem MS (MS/MS). Our results showed that tumors from male patients were cystine(cysteine)-dependent, with altered glutathione (GSH) biosynthesis, transsulfuration activity, methionine metabolism, utilization of lipoxygenase pathways. Simultaneously, we performed comprehensive bioinformatic analysis of gene expression data from the Gene Expression Omnibus to further validate mutant KRAS effects on CRCs by sex. Our findings demonstrated differential expression to CBS, GPX4, GSS, ACSL4, ATF4, FTH1 and EGPR genes that are all involved in regulation of these metabolic pathways and indicate a role for ferroptosis. This data therefore validated our findings of decreased transsulfuration activity (regulated by CBS) and decreased GSH biosynthesis (regulated by GSS) in male patients; this finding was not seen in female patients. To further examine differences in ferroptosis by sex, it was seen that GPX4 was differentially expressed between KRAS mutant and wild type (p&amp;lt;0.05) in males, but not in females. Meanwhile, ACSL4, which appears to be a predictive marker for ferroptosis sensitivity, showed upregulated expression in males with mutant KRAS (p&amp;lt;0.00001). Further, KRAS mutant type linked to a poor 5-year overall survival only in male using Kaplan-Meier survival analysis. This is the first report to show unique sex-specific metabolic signatures by KRAS status in CRC patients indicating a role of decreased ferroptosis in males, thus a novel avenue for therapeutic approaches. Citation Format: Hong Yan, Yuping Cai, Xinyi Shen, Abhishek Jain, Philip B Paty, Marcus Bosenberg, Sajid A Khan, Caroline Johnson. Discovery of a sex-specific metabolic phenotype in KRAS mutant colorectal cancer [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 2394.

Abstract 5519: Selinexor synergizes with anti-PD-1 antibody and inhibits tumor growth and metastasis in syngeneic mouse models of KRAS mutant colorectal cancer

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer related death. In approximately half of all CRC cases, oncogenic KRAS mutations underlie poor prognosis and drug resistance. A previous multi-genomic analysis of 4,725 biological processes with 106 human non-small-cell lung cancer cells identified nuclear transport machinery as the sole process selectively required for the survival of KRAS mutant cells. Selinexor is a clinically approved oral inhibitor of nuclear export protein Exportin 1 (XPO1/CRM1). In preclinical studies, selinexor demonstrated robust synthetical lethality with native or engineered oncogenic KRAS both in vitro and in vivo. Clinical activity of selinexor monotherapy was observed in CRC patients with various KRAS oncogenic mutations (Razak 2016 JCO). We performed preclinical studies to evaluate the effectiveness of selinexor and the anti-PD-1 antibody RMP1-14 in syngeneic mouse models of KRAS mutant CRC. Methods: Two syngeneic CT-26 KRAS mutant CRC mouse models were tested: 1) metastasis model with intra-hepatically injected CT-26 cells expressing luciferase (8 mice per group); and 2) subcutaneous model with CT-26 injected into the left flank (10 mice per group). Mice were allocated to four groups: vehicle control, selinexor (15 mg/kg, PO, once weekly), RMP1-14 (300 µg/mouse, IP, once weekly), or the combination of selinexor and RMP1-14. Tumor progression and body weight of test mice were monitored throughout the experiment. Tumor samples and plasma were collected at the end of the study for further analysis. Results: Both selinexor and RMP1-14 demonstrated single agent activity against KRAS mutant CRC in the two mouse models relative to controls. In the metastasis model, tumor growth inhibition (TGI) at Day 21 was 63.5% (p=0.0001) for selinexor and 68.9% (p&amp;lt;0.0001) for RMP1-14. The combination of selinexor and RMP1-14 significantly inhibited tumor growth and metastasis with a TGI of 98.9% (p&amp;lt;0.0001). No tumors were detected in two mice from the combination group at the end of the study. A similar trend was observed in the subcutaneous xenograft models, at Day 17, TGIs for selinexor, RMP1-14 and combination treatment were 17.6% (p=0.0921), 26.9% (p=0.0059) and 70.4% (p&amp;lt;0.0001), respectively. Conclusions: Synergistic anti-cancer activity of selinexor and anti-PD-1 antibody in KRAS mutant CRC mouse models warrants further clinical investigation. Citation Format: Hua Chang, Leah Henegar, Christopher J. Walker, Trinayan Kashyap, Marie Maloof, Feng Wang, Kathleen Martyn, Shira Orr, Michael G. Kauffman, Sharon Shacham, Yosef Landesman. Selinexor synergizes with anti-PD-1 antibody and inhibits tumor growth and metastasis in syngeneic mouse models of KRAS mutant colorectal cancer [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 5519.

Abstract 338: Anti-EGF antibodies significantly improve the activity of BRAF and KRAS inhibitors in preclinical models

Abstract Background: The EGF/EGFR pathway is involved in the emergence of resistance to BRAF and MEK inhibitors and the combination of encorafenib with the anti-EGFR antibody cetuximab has been recently approved in BRAF-positive, advanced colorectal cancer (CRC). Vaccination against EGF represents an alternative to the administration of monoclonal antibodies against EGFR 1-2. In this study, we tested if antibodies generated by vaccination with IN01 (biologic fusion molecule), could improve the antitumor activity of encorafenib and sotorasib in BRAF and KRAS colorectal (CRC) and non-small cell lung cancer (NSCLC) cell lines. Methods: Cell lines with BRAF V600E (HT29, CRC) and KRAS G12C mutations (H2122 and H23, NSCLC) were used. Anti-EGF VacAbs were obtained by immunizing rabbits with recombinant human EGF. Cell lines were treated with encorafenib or sotorasib in combination with anti-EGF VacAbs and the antitumor effects compared to the combination of encorafenib/cetuximab and sotorasib/panitumumab. Cell viability was determined by MTT, changes of total and phosphorylated proteins by Western blotting and emergence of resistance by direct microscopic examination in low density cultures. Results: Anti-EGF VacAbs suppressed the EGF-induced proliferation and blocked the EGFR signaling pathways more efficiently than cetuximab and panitumumab in KRAS and BRAF cell lines. In combination, anti-EGF VacAbs significantly enhanced the antitumor activity of encorafenib (in HT29 cells) and sotorasib (in H2122 and H23 cells), further suppressing EGFR, PRAS40 and ERK 1/2 phosphorylation. Also, the antitumor effects of the anti-EGF VacAbs in combination were superior to those of cetuximab and panitumumab. Finally, the addition of anti-EGF VacAbs to the culture medium significantly delayed the emergence of resistant clones to encorafenib (in HT29) and sotorasib (in H23). Again, the effect was more pronounced than the delay observed when adding cetuximab or panitumumab. Resistant clones generated under all combinations are being analyzed by NGS and Western blotting to determine the possible mechanisms of acquired resistance. Conclusions: Anti-EGF VacAbs potentiate the antitumor effects of encorafenib and sotorasib in cancer cell lines more efficiently than cetuximab and panitumumab. Our data provide a rationale for clinical trials testing the combination of anti-EGF VacAbs with targeted inhibitors in BRAF and KRAS mutant CRC and NSCLC patients. (1)“Anti-Epidermal Growth Factor Vaccine Antibodies Enhance the Efficacy of Tyrosine Kinase Inhibitors and Delay the Emergence of Resistance in EGFR Mutant Lung Cancer Cells” García-Roman S, Molina-Vila MA, et al. J Thorac Oncol. 2018. (2) “Anti-epidermal growth factor vaccine antibodies increase the antitumor activity of kinase inhibitors in ALK and RET rearranged lung cancer cells” García-Roman S, Molina-Vila MA, et al. Transl. Oncol. 2020. Citation Format: Silvia Garcia-Roman, Miguel A. Molina-Vila, Erik d'Hondt, Rafael Rosell. Anti-EGF antibodies significantly improve the activity of BRAF and KRAS inhibitors in preclinical models [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 338.

Abstract 2665: Irreversible menin inhibitor, BMF-219, inhibits the growth of KRAS-mutated solid tumors

Abstract Introduction: KRAS (Kirsten rat sarcoma virus) is the most frequently mutated isoform amongst RAS oncogenes in human solid tumors being present in a high percentage of colorectal cancers (CRC), non-small cell lung cancers (NSCLC), and pancreatic cancers. With only one approved KRAS G12C inhibitor for NSCLC, KRAS-driven tumors continue to represent a significant unmet medical need where novel effective therapies are highly desired. Menin is a required co-factor of oncogenic transcriptional proteins with functional interactions that are critical for various malignancies including acute leukemia. We previously reported that BMF-219, a novel irreversible menin inhibitor, exhibits strong potency on acute leukemia (MOLM-13) and KRAS-mutant (MiaPaCa-2) cells. Results from MiaPaCa-2 cells prompted our exploration of the effects of BMF-219 in an expanded panel of KRAS-mutant solid tumors through in vitro and ex vivo preclinical models. Methods: BMF-219, clinical reversible menin inhibitors, or clinically approved KRAS G12C inhibitor, sotorasib, were cultured with CRC, NSCLC, pancreatic cancer cell lines for 4-days. Human ex vivo preclinical models harboring KRAS mutations were cultured with BMF-219 and reversible menin for 6-days. Cell viability was measured using CellTiter Glo and IC50 values were calculated. MiaPaCa-2 cells incubated with BMF-219 were analyzed by RNA-seq on the Illumina NextSeq 550 platform. Results: MiaPaCa-2, a KRAS G12C-mutant cell line, showed marked reduction of KRAS expression levels following 24 hours of BMF-219 treatment at 0.5 µM. An expanded panel of 14 CRC, NSCLC and pancreatic KRAS-mutant cell lines harboring G12C, G12D, G12V, and Q61L revealed single-agent BMF-219 activity after a 4-day treatment. Majority of the cell lines tested exhibited &amp;gt;90% inhibition of growth, independent of KRAS mutation type. Sotorasib reached a maximum of 90-93% growth inhibition in three of eight cell lines. By contrast, BMF-219 inhibited cell viability ≥ 90% in six of eight KRAS G12C lung cancer lines. Human CRC, NSCLC and pancreatic ex vivo preclinical models with G12C, G12D, and G12V KRAS mutations were all sensitive to BMF-219 after a 6-day treatment. Complete abrogation of growth was observed in all samples with IC50 values ranging between 0.2 μM - 0.6 μM. Conclusion: KRAS-mutant CRC, NSCLC, pancreatic cancer cell lines and ex vivo preclinical models are highly sensitive to irreversible menin inhibitor, BMF-219, where clinical reversible menin inhibitors displayed limited activity. High potency of BMF-219 was observed amongst various KRAS-mutant cell lines suggesting that BMF-219 broadly inhibits these tumors. As an irreversible menin inhibitor, BMF-219, manifests advantages over the KRAS-targeted inhibitor sotorasib in multiple cell lines tested, and displays unique potency compared with clinical reversible menin inhibitors in ex vivo preclinical models of CRC, NSCLC, and pancreatic cancer. Citation Format: Brian Law, Daniel Lu, Priyanka Somanath, James T. Palmer, Taisei Kinoshita, Thomas Butler. Irreversible menin inhibitor, BMF-219, inhibits the growth of KRAS-mutated solid tumors [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 2665.