KRAS G13D Mutation Research Articles

CRISPR/Cas9 Edited RAS & MEK Mutant Cells Acquire BRAF and MEK Inhibitor Resistance with MEK1 Q56P Restoring Sensitivity to MEK/BRAF Inhibitor Combo and KRAS G13D Gaining Sensitivity to Immunotherapy

BRAF V600E mutation drives uncontrolled cell growth in most melanomas. While BRAF V600E tumors are initially responsive to BRAF inhibitors, prolonged treatment results in inhibitor resistance and tumor regrowth. Clinical data have linked the NRAS Q61K, KRAS G13D and MEK1 Q56P mutations to the BRAF inhibitor resistance. However, development of novel therapeutics is hindered by the lack of relevant isogeneic cell models. We employed CRISPR/Cas9 genome engineering to introduce NRAS Q61K, KRAS G13D and MEK1 Q56P mutations into the A375 melanoma cell line with endogenously high expression of BRAF V600E. The resulting isogenic cell lines are resistant to BRAF inhibitors. The A375 MEK1 Q56P isogenic cells are additionally resistant to MEK inhibitors as single agent, but interestingly, these cells become sensitive to MEK/BRAF inhibitor combo. Our results suggest that resistance in the NRAS and MEK isogenic lines is driven by constitutive MEK/ERK signaling, while the resistance in the KRAS isogenic line is driven by EGFR overexpression. Interestingly, the KRAS G13D isogenic line displays elevated PD-L1 expression suggesting the KRAS G13D mutation could be a potential indication for immunotherapy. Overall, these three novel isogenic cell models with endogenous level RAS and MEK1 point mutations provide direct bio-functional evidence demonstrating that acquiring a drug-resistant gene drives tumor cell survival and may simultaneously introduce new indications for combo therapy or immunotherapy in the clinic.

KRAS G12D mutation eliminates reactive oxygen species through the Nrf2/CSE/H 2S axis and contributes to pancreatic cancer growth.

In pancreatic cancer, KRAS G12D can trigger pancreatic cancer initiation and development. Rapid tumor growth is often accompanied by excess intracellular reactive oxygen species (ROS) production, which is unfavorable to tumor. However, the regulation of intracellular ROS levels in KRAS mutant pancreatic cancer remains unclear. In this study, we establish BxPC3 stable cell strains expressing KRAS wild type (WT) and G12D mutation and find unchanged ROS levels despite higher glycolysis and proliferation viability in KRAS mutant cells than KRAS WT cells. The key hydrogen sulfide (H 2S)-generating enzyme cystathionine-γ-lyase (CSE) is upregulated in KRAS mutant BxPC3 cells, and its knockdown significantly increases intracellular ROS levels and decreases cell glycolysis and proliferation. Nuclear factor erythroid 2-related factor 2 (Nrf2) is activated by KRAS mutation to promote CSE transcription. An Nrf2 binding site (‒47/‒39 bp) in the CSE promoter is verified. CSE overexpression and the addition of NaHS after Nrf2 knockdown or inhibition by brusatol decreases ROS levels and rescues cell proliferation. Our study reveals the regulatory mechanism of intracellular ROS levels in KRAS mutant pancreatic cancer cells, which provides a potential target for pancreatic cancer therapy.

Inhibition of colon cancer K-RasG13D mutation reduces cancer cell proliferation but promotes stemness and inflammation via RAS/ERK pathway.

K-Ras is a well-studied oncogene, and its mutation is frequently found in epithelial cancers like pancreas, lung, and colorectal cancers. Cancer cells harboring K-Ras mutations are difficult to treat due to the drug resistance and metastasis properties. Cancer stem cells (CSCs) are believed the major cause of chemotherapeutic resistance and responsible for tumor recurrence and metastasis. But how K-Ras mutation affects CSCs and inflammation is not clear. Here, we compared two colon cancer cell lines, HCT-116 and HT-29, with the former being K-RasG13D mutant and the latter being wildtype. We found that HCT-116 cells treated with a K-Ras mutation inhibitor S7333 formed significantly more tumor spheroids than the untreated control, while the wild type of HT-29 cells remained unchanged. However, the size of tumor spheroids was smaller than the untreated controls, indicating their proliferation was suppressed after S7333 treatment. Consistent with this, the expressions of stem genes Lgr5 and CD133 significantly increased and the expression of self-renewal gene TGF-β1 also increased. The flow cytometry analysis indicated that the expression of stem surface marker CD133 increased in the treated HCT-116 cells. To understand the pathway through which the G13D mutation induced the effects, we studied both RAS/ERK and PI3K/Akt pathways using specific inhibitors SCH772984 and BEZ235. The results indicated that RAS/ERK rather than PI3K/Akt pathway was involved. As CSCs play the initial role in cancer development and the inflammation is a vital step during tumor initiation, we analyzed the correlation between increased stemness and inflammation. We found a close correlation of increased Lgr5 and CD133 with proinflammatory factors like IL-17, IL-22, and IL-23. Together, our findings suggest that K-RasG13D mutation promotes cancer cell growth but decreases cancer stemness and inflammation thus tumorigenesis and metastasis potential in colon cancer. Inhibition of this mutation reverses the process. Therefore, care needs be taken when employing targeted therapies to K-RasG13D mutations in clinics.

Muc16 depletion diminishes KRAS-induced tumorigenesis and metastasis by altering tumor microenvironment factors in pancreatic ductal adenocarcinoma.

MUC16, membrane-bound mucin, plays an oncogenic role in pancreatic ductal adenocarcinoma (PDAC). However, the pathological role of MUC16 in the PDAC progression, tumor microenvironment, and metastasis in cooperation with KrasG12D and Trp53R172H mutations remains unknown. Deletion of Muc16 with activating mutations KrasG12D/+ and Trp53R172H/+ in mice significantly decreased progression and prolonged overall survival in KrasG12D/+; Trp53R172H/+; Pdx-1-Cre; Muc16-/- (KPCM) and KrasG12D/+; Pdx-1-Cre; Muc16-/- (KCM), as compared to KrasG12D/+; Trp53R172H/+; Pdx-1-Cre (KPC) and KrasG12D/+; Pdx-1-Cre (KC) mice, respectively. Muc16 knockout pancreatic tumor (KPCM) displays decreased tumor microenvironment factors and significantly reduced incidence of liver and lung metastasis compared to KPC. Furthermore, in silico data analysis showed a positive correlation of MUC16 with activated stroma and metastasis-associated genes. KPCM mouse syngeneic cells had significantly lower metastatic and endothelial cell binding abilities than KPC cells. Similarly, KPCM organoids significantly decreased the growth rate compared to KPC organoids. Interestingly, RNA-seq data revealed that the cytoskeletal proteins Actg2, Myh11, and Pdlim3 were downregulated in KPCM tumors. Further knockdown of these genes showed reduced metastatic potential. Overall, our results demonstrate that Muc16 alters the tumor microenvironment factors during pancreatic cancer progression and metastasis by changing the expression of Actg2, Myh11, and Pdlim3 genes.

KRAS G12D Mutation Subtype in Pancreatic Ductal Adenocarcinoma: Does It Influence Prognosis or Stage of Disease at Presentation?

Background: KRAS G12D mutation subtype is present in over 40% of pancreatic ductal adenocarcinoma (PDAC), one of the leading global causes of cancer death. This retrospective cohort study aims to investigate whether detection of the KRAS G12D mutation subtype in PDAC patients is a determinant of prognosis across all stages of disease. Methods: We reviewed the medical records of 231 patients presenting with PDAC at a large tertiary hospital, and compared survival using the Kaplan Meier, log-rank test and Cox proportional hazards regression model. Results: KRAS G12D mutation subtype was not significantly associated with poorer survival compared across the whole population of PDAC patients (p = 0.107; HR 1.293 95% CI (0.946–1.767)). However, KRAS G12D patients who were resectable had a shorter median survival time of 356 days compared to all other genotypes (median survival 810 days) (p = 0.019; HR 1.991 95% CI (1.121–3.537)). Conclusions: KRAS G12D patients who were resectable at diagnosis had shorter survival compared to all other PDAC patients. These data suggest that KRAS G12D may be a clinically useful prognostic biomarker of PDAC.

85 (PB075) - ASP3082, a First-in-class novel KRAS G12D degrader, exhibits remarkable anti-tumor activity in KRAS G12D mutated cancer models

Background: KRAS is one of the most frequently mutated oncogenes in various cancers. Among KRAS mutations, KRAS G12D is the most frequent driver mutation and is found in approximately 34% of pancreatic ductal adenocarcinoma (PDAC), 10% to 12% of colorectal cancer, 4% of lung adenocarcinoma and also in a subset of other solid tumors. However, there is no direct KRAS G12D-targeted inhibitors used in the clinical setting. Here, we identified ASP3082, a novel KRAS G12D degrader with high potency and selectivity. Methods: We investigated (1) binding affinity of ASP3082 to KRAS G12D protein and an E3 ligase in the conditions of binary complex and ternary complex formation using surface plasmon resonance assay, (2) degradation activity of ASP3082 on KRAS G12D protein using western blotting or in-cell western method, (3) inhibitory effect of ASP3082 on the growth of human cancer cells harboring KRAS G12D mutation and KRAS wild-type, (4) specificity for ASP3082-mediated degradation using proteomics approach, (5) antitumor activity of ASP3082 in mice subcutaneously xenografted with KRAS G12D-mutated pancreatic cancer cells, (6) PK-PD profile of ASP3082 after a single intravenous administration in the xenograft model. Results: ASP3082 directly bound to KRAS G12D-mutated protein and the E3 protein to form a ternary complex. Also, ASP3082 potently degraded KRAS G12D-mutated protein and inhibited phosphorylation of ERK in KRAS G12D-mutated pancreatic cancer cells. In addition, ASP3082 showed the growth inhibitory activity of KRAS G12D-mutated pancreatic cancer cells, but not of KRAS wildtype cancer cells. Furthermore, the quantitative proteomics analysis suggested that ASP3082 selectively degraded the KRAS G12D-mutated protein over other (>9000) proteins. in vivo experiment revealed that once-weekly intravenous administration of ASP3082 induced dose-dependent and significant growth inhibition of KRAS G12D PDAC tumors, resulting in profound tumor regression without body weight loss. ASP3082 showed sustained concentrations in the xenograft tumors after a single intravenous administration and decreased KRAS G12D-mutated protein levels according to the duration of the ASP3082 concentrations. Conclusions: ASP3082 is a potential therapeutic agent for patients with tumors harboring the KRAS G12D mutation. Currently, Phase I clinical trial is underway (NCT05382559). Conflict of interest: Corporate-sponsored Research: All authors are employees of Astellas Pharma Inc.

S2163 Understanding Prolonged Survival in Advanced Colorectal Cancer Using Genomics: A Case Series

Introduction: Recent progress in molecular biology and cancer genome research had opened a new venue for clinical research and changed treatment paradigm for metastatic colorectal cancer. Case Description/Methods: Case 1: A 61-year -old Hispanic male underwent colonoscopy which revealed an mass in the proximal ascending colon with biopsy showing moderately differentiated adenocarcinoma. The patient underwent a right hemicolectomy in 2012. He subsequently completed chemotherapy with mFOLFOX-6. Further treatment was declined by patient. Follow up chest CT scan in April 2018, uncovered progression of pulmonary nodules (Figure, Panel 1). Patient was put on FOLFIRI/bevacizumab chemotherapy. NGS study showed following results; MS stable; TMB 4 mutations/Mb (low); APCR1450, BRAF D594N, FAM 123B K761, SOX9 G225fs, and TP53 R175H.The patient has stable disease on follow up. Case 2: A 73-year-old Hispanic male underwent colonoscopy showing a obstructing mass in the sigmoid colon. Biopsy showed moderately differentiated adenocarcinoma in December 2013. Patient underwent sigmoidectomy. Liver masses were found on repeat CT abdomen. The NGS reported as follows; MS-Stable, TMB 1 muts/Mb(low), APC T282fs*12/P1453fs*20, CDK8 amplification, fms like tyrosine kinase 3 (FLT3) amplification, KRAS amplification and TP53 V147D. It also detected wild type KRAS mutations in exons 2, 3 and 4. The patient was started on chemotherapy with mFOLFOX. Follow- up studies show stable disease. Case 3: A 73-year-old Hispanic male underwent colonoscopy showed a completely obstructing mass in the sigmoid colon. Biopsy showed moderately differentiated adenocarcinoma in June 2012. The tumor cells have KRAS G12D mutation in in codon 12 of KRAS. He underwent a left hemicolectomy. He completed mFOLFOX6.PET scan was done and showed a right upper lobe mass consistent with metastatic cancer. He was started on FOLFIRI/ bevacizumab. NGS reported as follows; MS-Stable, TMB 1 muts/Mb(low), APC V1452fs*21, and ATM S47fs*11. Bone Scan intense uptake in the right distal tibia (Figure, Panel 2). MRI of the right lower extremity showed a lesion in the distal tibia. He was then switched to irinotecan and bevacizumab regimen. He has stable disease. Discussion: Identification of genomic signature is key to understanding the molecular mechanism of CRC and the development of novel therapeutics.Figure 1.: Panel 1 (left) shows CT Chest for Case 1 with multiple pulmonary nodules few with cavitation, largest 9mm. Panel 2 (right) shows Bone Scan for Case 3 in February 2021 with intense uptake in the right distal tibia.

54 (PB044) - Non-clinical identification and characterization of KRAS G12D inhibitors

KRAS G12D is the most prevalent KRAS oncogenic mutant and occurs frequently in pancreatic ductal adenocarcinoma (PDAC), colorectal adenocarcinoma (CRC), and non-small cell lung cancer (NSCLC; 37%, 12% and 4%, respectively). KRAS G12D cycles between inactive (GDP) and active (GTP) states to regulate the RAS/MAPK pathway. Although the rate of intrinsic and GAP (GTPase activating protein) mediated hydrolysis is significantly slower in KRAS G12D than in KRAS wild-type (KRAS WT), we were able to build upon success of targeting the GDP state of KRAS G12C and designed novel KRAS G12D lead compounds with promising in vitro and in vivo activity. Our KRAS G12D inhibitors preferentially bind to KRAS G12D GDP as indicated by differential scanning fluorimetry (DSF), block KRAS G12D SOS1-mediated nucleotide exchange, and inhibit KRAS G12D binding to the RAS binding domain of C-RAF (RAF-RBD) with single digit nanomolar potency. Our leads effectively inhibit ERK1/2 phosphorylation (pERK) in AsPC-1 PDAC cells and inhibit cell proliferation in 3-dimensional Cell-Titer Glo assays in a panel of KRAS G12D mutant PDAC, CRC, and NSCLC cell lines. This in vivo activity translates in vitro where robust pharmacodynamic modulation in the PDAC AsPC-1 CDX model was induced. Tumor growth inhibition studies in this model revealed robust dose-dependent tumor growth inhibition and tumor regression in the absence of overt toxicity. In summary, we have identified potent and selective KRAS G12D inhibitors with robust dose-dependent tumor growth inhibition and regression activity in a PDAC CDX model. Lead optimization is ongoing with the aim of identifying a development candidate. No conflict of interest.

CRISPR/Cas9 mediated triple signal amplification platform for high selective and sensitive detection of single base mutations

Single base mutations detection is crucial for the diagnosis and treatment of cancer. However, the current methods with poor selectivity and sensitivity required large instruments, which are difficult to meet clinical demands. Herein, we develop a CRISPR/Cas9 based visual colorimetric platform to specifically detect all single base mutations. In this strategy, the Recombinase Polymerase Amplification (RPA) was firstly used to amplify the target, and introduced the PAM site in the target DNA sequence by designing the point mutation primer, thus achieving detection for all single base mutations by the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated specific recognition. With the help of CRISPR/Cas9 system, those RPA products can release single strand DNA to hybridize with the padlock probe and trigger rolling circle amplification (RCA). Based on the magnetic separation, HRP-gold nanoparticles complex (hGNPs) and biotin modified probe (Bio-probe) were further used to achieve enhanced visual variations assay by hybridizing with RCA products. Benefiting from the RPA assisted triple signal amplification, this method not only showed enhanced sensitivity with a limit of detection (LOD) as low as 0.2 fM and 0.01% of KRAS-G12D mutation percentage, but the specificity against KRAS-G12D mutation also be synergistically enhanced by combining the CRISPR/Cas9-mediated specific recognition with the specific T4 ligation reaction of RCA system. Furthermore, this system has been successfully used to visually detect genome in serum, suggesting its great potential for point-of-care diagnosis in clinical.

Cytotoxicity of combinations of the pan-KRAS SOS1 inhibitor BAY-293 against pancreatic cancer cell lines

KRAS is mutated in approximately 25% of cancer patients and first KRAS G12C-specific inhibitors showed promising responses. Pancreatic cancer has the highest frequency of KRAS mutations but the prevailing KRAS G12D mutation is difficult to target. Inhibition of the GTP exchange factor (GEF) SOS1—KRAS interaction impairs oncogenic signaling independently of the specific KRAS mutations. In general, cell lines exhibiting KRAS mutations show specific alterations in respect to glucose utilization, signal transduction and stress survival. The aim of this investigation was to check the putative synergy of the SOS1 inhibitor BAY-293 with modulators targeting specific vulnerabilities of KRAS-mutated cell lines in vitro. The cytotoxicity of BAY-293 combinations was tested against MIA PaCa-2 (G12C), AsPC1 (G12D) and BxPC3 (KRAS wildtype) cell lines using MTT tests and calculation of the combination indices (CI) according to the Chou-Talalay method. The results show that BAY-293 synergizes with modulators of glucose utilization, inhibitors of the downstream MAPK pathway and several chemotherapeutics in dependence of the specific KRAS status of the cell lines. In particular, divergent responses for BAY-293 combinations between pancreatic and NSCLC cell lines were observed for linsitinib, superior inhibitory effects of trametinib and PD98059 in NSCLC, and lack of activity with doxorubicin in case of the pancreatic cell lines. Phosphoproteome analysis revealed inhibition of distinct signaling pathways by BAY-293 for MIA PaCa-2 on the one hand and for Aspc1 and BH1362 on the other hand. In conclusion, BAY-293 exhibits synergy with drugs in dependence of the tumor type and specific KRAS mutation.

DNA Polymerase Theta Plays a Critical Role in Pancreatic Cancer Development and Metastasis.

Simple SummaryPancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers worldwide. The occurrence of oncogenic KRAS mutations is considered a signature event in PDAC, leading to genomic instability. The aim of our study was to evaluate the impact of the oncogenic KRAS G12D mutation on the activity of the error-prone alt-EJ repair mechanism, and to investigate the potential role of Polθ in the development of pancreatic cancer. We found that oncogenic KRAS increases the expression of key alt-EJ proteins in a mouse and human PDAC model. Using TLR assay, we also found increased alt-EJ activity in mouse and human cell lines upon the expression of KRAS D12D. The inactivation/impairment of alt-EJ by polymerase theta (Polθ) depletion delays the development of pancreatic cancer and prolongs the survival of experimental mice, though it does not prevent the PDAC development, which leads to full-blown PDAC with disseminated metastasis. Our studies provide a high-value target as a novel therapeutic candidate for the treatment of pancreatic and other cancers.Pancreatic ductal adenocarcinoma (PDAC), due to its genomic heterogeneity and lack of effective treatment, despite decades of intensive research, will become the second leading cause of cancer-related deaths by 2030. Step-wise acquisition of mutations, due to genomic instability, is considered to drive the development of PDAC; the KRAS mutation occurs in 95 to 100% of human PDAC, and is already detectable in early premalignant lesions designated as pancreatic intraepithelial neoplasia (PanIN). This mutation is possibly the key event leading to genomic instability and PDAC development. Our study aimed to investigate the role of the error-prone DNA double-strand breaks (DSBs) repair pathway, alt-EJ, in the presence of the KRAS G12D mutation in pancreatic cancer development. Our findings show that oncogenic KRAS contributes to increasing the expression of Polθ, Lig3, and Mre11, key components of alt-EJ in both mouse and human PDAC models. We further confirm increased catalytic activity of alt-EJ in a mouse and human model of PDAC bearing the KRAS G12D mutation. Subsequently, we focused on estimating the impact of alt-EJ inactivation by polymerase theta (Polθ) deletion on pancreatic cancer development, and survival in genetically engineered mouse models (GEMMs) and cancer patients. Here, we show that even though Polθ deficiency does not fully prevent the development of pancreatic cancer, it significantly delays the onset of PanIN formation, prolongs the overall survival of experimental mice, and correlates with the overall survival of pancreatic cancer patients in the TCGA database. Our study clearly demonstrates the role of alt-EJ in the development of PDAC, and alt-EJ may be an attractive therapeutic target for pancreatic cancer patients.

Clinicopathologic Characteristics and Outcomes for Patients With KRAS G12D-Mutant NSCLC

IntroductionCo-occurring mutations in KRAS-mutant NSCLC are associated with discrete biological properties and modulate therapeutic susceptibilities. As G12D-specific inhibitors are expected to enter the clinic, we sought to investigate the characteristics and outcomes of patients with KRAS G12D-mutant NSCLC. MethodsThis was a retrospective single-institution study. Patients with NSCLC and KRAS G12D mutations detected by the Massachusetts General Hospital SNaPshot next-generation sequencing assay were identified. Clinical and pathologic characteristics were collected by chart review. ResultsA total of 107 patients with KRAS G12D-mutant NSCLC were identified. Most patients were former smokers (80, 74.8%) and had tumors with adenocarcinoma pathologic subtype (93, 86.9%). Among 56 patients evaluated for programmed death-ligand 1 expression, tumor proportion score was less than 50% in 43 (76.8%). Concomitant mutations were identified in STK11 (17 of 107, 15.9%), KEAP1 (10 of 58, 17.2%), TP53 (36 of 107, 33.6%), and SMARCA4 (11 of 107, 10.3%). Among 57 patients treated with first-line therapy, patients with STK11 co-mutations had shorter progression-free survival (1.2 mo, 95% confidence interval [CI]: 0.6–2.9 versus 4.1 mo, 95% CI: 2.5–6.0, p = 0.0235) and overall survival (4.3 mo, 95% CI: 1.2–10.6 versus 17.9 mo, 95% CI: 8.6–31.1, p = 0.0018) compared with wild type. Patients with KEAP1 co-mutations had shorter overall survival (4.6 mo, 95% CI: 1.2–10.6 versus 17.9 mo, 95% CI: 7.1–30.1, p = 0.0125) than those without. TP53 co-mutations exerted no influence on survival. ConclusionsCo-occurring mutations were common in patients with KRAS G12D-mutant NSCLC. STK11 and KEAP1 co-mutations were associated with worse clinical outcomes, whereas co-occurring TP53 did not affect survival.

KRAS mutant-driven SUMOylation controls extracellular vesicle transmission to trigger lymphangiogenesis in pancreatic cancer.

Lymph node (LN) metastasis occurs frequently in pancreatic ductal adenocarcinoma (PDAC) and predicts poor prognosis for patients. The KRASG12D mutation confers an aggressive PDAC phenotype that is susceptible to lymphatic dissemination. However, the regulatory mechanism underlying KRASG12D mutation–driven LN metastasis in PDAC remains unclear. Herein, we found that PDAC with the KRASG12D mutation (KRASG12D PDAC) sustained extracellular vesicle–mediated (EV-mediated) transmission of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) in a SUMOylation-dependent manner and promoted lymphangiogenesis and LN metastasis in vitro and in vivo. Mechanistically, hnRNPA1 bound with SUMO2 at the lysine 113 residue via KRASG12D-induced hyperactivation of SUMOylation, which enabled its interaction with TSG101 to enhance hnRNPA1 packaging and transmission via EVs. Subsequently, SUMOylation induced EV-packaged-hnRNPA1 anchoring to the adenylate- and uridylate-rich elements of PROX1 in lymphatic endothelial cells, thus stabilizing PROX1 mRNA. Importantly, impeding SUMOylation of EV-packaged hnRNPA1 dramatically inhibited LN metastasis of KRASG12D PDAC in a genetically engineered KrasG12D/+ Trp53R172H/+ Pdx-1-Cre (KPC) mouse model. Our findings highlight the mechanism by which KRAS mutant–driven SUMOylation triggers EV-packaged hnRNPA1 transmission to promote lymphangiogenesis and LN metastasis, shedding light on the potential application of hnRNPA1 as a therapeutic target in patients with KRASG12D PDAC.

KRAS mutations as essential promoters of lymphangiogenesis via extracellular vesicles in pancreatic cancer.

Kirsten rat sarcoma virus (KRAS) gene mutations are present in more than 90% of pancreatic ductal adenocarcinomas (PDACs). KRASG12D is the most frequent alteration, promoting preneoplastic lesions and associating with a more aggressive phenotype. These tumors possess increased intratumoral lymphatic networks and frequent lymph node (LN) metastases. In this issue of the JCI, Luo, Li, et al. explored the relationship between the presence of the KRASG12D mutation and lymphangiogenesis in PDAC. The authors used in vitro and in vivo models and an elegant mechanistic approach to describe an alternative pathway for lymphangiogenesis promotion. KRASG12D induced SUMOylation of heterogenous nuclear ribonucleoprotein A1 (hnRNPA1) via SAE1 and SUMO2 activation. SUMOylated hnRNPA1 was loaded into extracellular vesicles (EVs) and internalized by human endothelial lymphatic cells (HLEC). Further, SUMOylated hnRNPA1 promoted lymphangiogenesis and LN metastasis by stabilizing prospero homeodomain protein 1 (PROX1) mRNA. These data provide mechanistic insight into cancer lymphangiogenesis with the potential for developing biomarkers and RAS pathway therapeutics.

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.

ATG7 is a haploinsufficient repressor of tumor progression and promoter of metastasis

The role of autophagy in cancer is complex. Both tumor-promoting and tumor-suppressive effects are reported, with tumor type, stage and specific genetic lesions dictating the role. This calls for analysis in models that best recapitulate each tumor type, from initiation to metastatic disease, to specifically understand the contribution of autophagy in each context. Here, we report the effects of deleting the essential autophagy gene Atg7 in a model of pancreatic ductal adenocarcinoma (PDAC), in which mutant KrasG12D and mutant Trp53172H are induced in adult tissue leading to metastatic PDAC. This revealed that Atg7 loss in the presence of KrasG12D/+ and Trp53172H/+ was tumor promoting, similar to previous observations in tumors driven by embryonic KrasG12D/+ and deletion of Trp53. However, Atg7 hemizygosity also enhanced tumor initiation and progression, even though this did not ablate autophagy. Moreover, despite this enhanced progression, fewer Atg7 hemizygous mice had metastases compared with animals wild type for this allele, indicating that ATG7 is a promoter of metastasis. We show, in addition, that Atg7+/- tumors have comparatively lower levels of succinate, and that cells derived from Atg7+/- tumors are also less invasive than those from Atg7+/+ tumors. This effect on invasion can be rescued by ectopic expression of Atg7 in Atg7+/- cells, without affecting the autophagic capacity of the cells, or by treatment with a cell-permeable analog of succinate. These findings therefore show that ATG7 has roles in invasion and metastasis that are not related to the role of the protein in the regulation of autophagy.

SEMA3C Supports Pancreatic Cancer Progression by Regulating the Autophagy Process and Tumor Immune Microenvironment.

To date, driver genes for pancreatic cancer treatment are difficult to pursue therapeutically. Targeting mutated KRAS, the most renowned driver gene in pancreatic cancer, is an active area of study. We discovered a gene named SEMA3C was highly expressed in pancreatic cancer cell lines and patients with a G12D mutation in KRAS. High expression of SEMA3C in patients was significantly associated with the decreased survival of pancreatic cancer patients based on the TCGA database. In pancreatic cancer cells, SEMA3C knockdown or inhibition exhibited growth/colony inhibition and cell cycle arrest. In addition, SEMA3C inhibition sensitized KRAS or MEK1/2 inhibition in pancreatic cancer cells. Overexpression of SEMA3C resulted in the induction of autophagy, whereas depletion of SEMA3C compromised induction of autophagy. SEMA3C modified the PD-L1 expression in tumor and immune cells and is correlated with the M2-like macrophage marker ARG1/CD163 expression, which could reshape the tumor microenvironment. Inhibition of SEMA3C decreased tumor formation in the xenograft model in vivo. Taken together, our data suggest that SEMA3C plays a substantial role in promoting cancer cell survival by regulating the autophagy process and impacting the tumor environment immune response. SEMA3C can be used as a novel target or marker with therapeutic or diagnostic potential in pancreatic cancer especially in tumors harboring the specific KRAS G12D mutation.

Abstract 3596: RM-036, a first-in-class, orally-bioavailable, Tri-Complex covalent KRASG12D(ON) inhibitor, drives profound anti-tumor activity in KRASG12D mutant tumor models

Abstract KRASG12D mutant cancers represent a significant unmet medical need with 55,000 new diagnoses annually in the US. The KRASG12D mutation occurs commonly in multiple tumor histotypes, including about 20%, 29% and 17% of KRAS mutant colorectal, pancreatic, and non-small cell lung cancers, respectively. However, there are no directly targeted inhibitors of KRASG12D in clinical development. Mutant RAS proteins exist predominantly in the GTP-bound RAS(ON) state, leading to excessive downstream signaling via interaction with effectors such as RAF kinases. Highly selective inhibitors that covalently target the KRASG12C(OFF) state and inhibit conversion to KRASG12C(ON) have been shown to be active and well tolerated in the clinic, and we have described a novel, covalent tri-complex KRASG12C(ON) inhibitor that has robust anti-tumor activity and is well tolerated in preclinical studies. Developing a covalent inhibitor of KRASG12D presents several challenges. The Asp residue in KRASG12D is significantly less reactive toward electrophiles than is Cys. And most reported covalent chemical warheads capable of reacting with Asp exhibit high nonspecific reactivity. The intrinsic GTP hydrolysis rate of KRASG12D is about 2.6-fold lower than that of KRASG12C, further biasing the cellular KRASG12D pool to the RAS(ON) state and emphasizing the importance of targeting the KRASG12D(ON) state for maximal suppression of this oncogenic driver. Based on these considerations we designed RM-036, a potent, selective, orally bioavailable, covalent KRASG12D(ON) inhibitor with attractive drug-like properties. It forms a tri-complex between the abundant intracellular chaperone cyclophilin A (CypA) and the active state of KRASG12D, positioning the warhead relative to KRASG12D and enabling selective covalent engagement of Asp. In cellular models, RM-036 exhibited efficient and selective covalent binding to KRASG12D, with no detectable reactivity toward the adjacent Asp residue in cells with the KRASG13D mutation. RM-036 showed low off-target reactivity in cell-based proteomic screens. RM-036 potently inhibited growth and induced apoptosis in KRASG12D mutant cancer cells in vitro but not in BRAFV600E-dependent cells. RM-036, as a single agent, produced deep, durable, and dose-dependent suppression of tumor RAS pathway activation with covalent target engagement in vivo following repeat oral administration. Profound tumor regressions, including complete regressions, were observed in various KRASG12D mutant xenograft models upon treatment with RM-036. All treatments regimens tested with RM-036 were well tolerated in vivo. These preclinical findings provide a strong foundation for advancing RM-036 toward clinical evaluation in patients with tumors bearing the KRASG12D mutation. Citation Format: John E. Knox, Jingjing Jiang, G. Leslie Burnett, Yang Liu, Caroline E. Weller, Zhican Wang, Laura McDowell, Shelby L. Steele, Shook Chin, Kang Jye Chou, Fang Wang, Mengqi Zhong, Elena S. Koltun, David Wildes, Mallika Singh, Adrian L. Gill, Jacqueline A. Smith. RM-036, a first-in-class, orally-bioavailable, Tri-Complex covalent KRASG12D(ON) inhibitor, drives profound anti-tumor activity in KRASG12D mutant tumor 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 3596.

Abstract 5110: Development and validation of a liquid biopsy assay with cerebrospinal fluid derived cell free DNA using droplet digital PCR for clinical applications

Abstract Background: Cerebrospinal fluid (CSF) has been recognized as an important biofluid for biomarker analysis of central nervous system (CNS) tumors due to the proximity to the disease/tumor site as compared to serum or plasma. CNS tumors have significantly enriched tumor-derived cfDNA/circulating tumor DNA (ctDNA) in CSF, however, the absolute quantities are low. Therefore, highly sensitive targeted methods such as next generation sequencing or digital droplet PCR are required to efficiently detect mutations. Recent studies have suggested the feasibility of detecting H3K27M in CSF and the potential of these mutations as biomarkers in CNS malignancies. We report the establishment of a protocol for CSF collection, cfDNA extraction and proof of concept study for the analysis of H3K27 mutations as potential clinical biomarkers for CNS tumors. Methods: CSF was collected from healthy donors (n=10) via lumbar puncture. cfDNA was isolated from approximately 2-3mL CSF from each donor using the Qiagen Circulating Nucleic Acid kit and quantified using the Qubit High Sensitivity DNA kit. Five normal donors were first tested for cfDNA recovery from CSF and then the remaining five were pooled and tested via a spike-in recovery experiment of KRAS G12D mutation ddPCR detection assay. Primer/probes specific to wildtype H3F3A/HIST1H3B and mutant to K27M were designed for ddPCR testing with cfDNA from the CSF of CNS tumor patients (n=10). Results: cfDNA was successfully isolated from CSF of five healthy samples. The mean cfDNA concentration was 7.6ng DNA per mL CSF with a range of 3.89-11.27 ng/mL CSF. The low yield of cfDNA further confirm the need for an extremely sensitive detection method for biomarkers in CSF. Synthetic DNA for the KRAS G12D mutation was spiked into a pool of healthy volunteer CSF at quantities as low as 1fg and then measured successfully downstream in ddPCR, illustrating the ability of this workflow to recover and measure very low levels the mutant DNA. The 1fg spike in amount yielded an average of 0.77copies/uL. Multiple ddPCR assays were designed for the HIST1H3B assay but after several iterations, no successful assay could be established for this mutation: a result consistent with other reported findings. However, an assay to measure H3F3A p.K27M was identified and is currently being optimized. Conclusion: Collectively, we demonstrated the use of CSF as a matrix for biomarker testing in a clinical setting. Using commercial kits, ctDNA can be extracted from CSF and used downstream in biomarker detection assays. On-going work includes expanding the tested cohort to CNS tumor samples using a developed ddPCR assay to detect the H3F3A p.K27M mutation. We anticipate the establishment of a reliable CSF cfDNA detection method to enable the study of CNS tumor for genomic evolution, minimal residual disease monitoring and the characterization of tumors at recurrence. Citation Format: Dennis J. O'Rourke, Danyi Wang, Zheng Feng. Development and validation of a liquid biopsy assay with cerebrospinal fluid derived cell free DNA using droplet digital PCR for clinical applications [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 5110.

Abstract 2468: Genetic metastatic colorectal cancer model with in vivo imaging capabilities

Abstract Introduction: Colorectal cancer (CRC) is the second leading cancer related cause of death worldwide, responsible for nearly 2 million deaths annually, most commonly due to complications from metastasis. A mouse model that reproduces human CRC is critically important to support pre-clinical research in the field. Recently a novel inducible genetically engineered mouse model in an immune competent B6 background was developed that metastasizes to the lung, liver, and peritoneum recapitulating what is seen in CRC patients. This model uses the Lgr5 (L) promoter to drive tamoxifen inducible Cre recombinase with the following conditional alleles: Apc (A), mutant Kras G12D (K), Tgfbr2 (T), and p53 (P) (LAKTP). After establishing the model in our center, we found mice developing tumors in the absence of tamoxifen induction. Because the original model lacked the ability to monitor the tumor development and progression in vivo in real time we created a LAKTP based mouse model that would allow for easy monitoring of tumor growth over time, allowing us to determine the frequency and time course of tumor development in the absence of tamoxifen induction. Methods: C57BL/6J mice, with each individual mutation (L, A, K, T, and P), were purchased from Jackson Laboratory and used for breeding in-house. Following breeding, congenic C57BL/6J LAKTP mice were characterized prior to breeding in conditional Luciferase (Rosa26 LSL Luciferase) which was also purchased from Jackson Laboratory. Luciferin was provided by intraperitoneal injection prior to capturing the bioluminescence signal using an imager from Spectral Imaging Systems. Tissue samples were fixed in formalin and embedded in paraffin prior sectioning for histology and immunohistochemistry. Results: Approximately 30% of our LAKTP mice died or required sacrifice due to meeting humane endpoints around 12 weeks of life in the absence of tamoxifen induction. Upon dissection, these mice exhibited large tumors located in the small intestine that led to non-survival bowel obstructions. Ten percent of these mice had liver metastases as well. Breeding in the conditional Luciferase allele allowed for detection of developing, non-induced tumors as early as 6 weeks of life. In addition to being useful for monitoring tumor growth in vivo, immunostaining of tumor tissue with anti-luciferase antibodies allows for detection of cells derived from Lgr5 positive intestinal stem cells. Conclusion: The LAKTP mouse model is an important tool for studying CRC, but exhibits tumor development due to leaky Cre activity in the absence of tamoxifen in approximately 30% of mice by 12 weeks. Our strategy of breeding in conditional luciferase allows for in vivo imaging such that mice can be partitioned based on bioluminescence for trials that require equivalent tumor burden at the beginning of interventions. In addition, bioluminescence imaging also allows for tumor size to be monitored over time. Citation Format: Tyler Leiva, James Griffith, Shaoxuan Guo, Megan Lerner, Lacey McNally, William Berry, Katherine Morris. Genetic metastatic colorectal cancer model with in vivo imaging capabilities [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 2468.