Zhang, Wu

198 Background: The Hedgehog (Hh) signaling pathway regulates tissue patterning, growth and differentiation, including within gastrointestinal tract. Mammalian homologs include Sonic (SHH), Indian (IHH) and Desert (DHH) Hedgehog, which signal through PTCH and SMO and the GLI transcription factors. In the digestive tract, both SHH and IHH are expressed, but the functional role of Hh signaling in mCRC remains incompletely defined. Methods: We analyzed 433 mCRC patients treated with either bevacizumab (bev = 226) or cetuximab (cet = 207) plus first line chemotherapy in CALGB/SWOG 80405 (NCT00265850). RNA expression (Illumina HiSeq 2500) of 9 Hh pathway genes (SHH, IHH, DHH, PTCH1, PTCH2, SMO, GLI1, GLI2, GLI3) was evaluated. Median overall survival (mOS) and progression free survival (mPFS) in months (mo) were compared across expression tertiles (low [L], medium [M], high [H]). Likelihood ratio tests, hazard ratios and 95% confidence intervals were computed from multivariable Cox proportional hazards models, adjusting for age, sex, ethnicity, ECOG PS, tumor location, number of metastatic sites, KRAS , microsatellites instability status and treatment. Prognostic effects were modeled as marginal terms; predictive effects were modeled as multiplicative interactions. Results: Overall, IHH and GLI2 demonstrated prognostic value for OS after adjusting for multiple testing (Benjamini-Hochberg False Discovery Rate (FDR) < 0.05). High IHH expression (H group, n = 143) correlated with improved mOS (H: 36.0 mo vs M: 32.8 mo vs L: 23.5 mo; p= 0.031), whereas low GLI2 expression (L group, n = 145) was associated with longer mOS (H: 25.6 mo vs M: 33.9 mo vs L: 33.7 mo; p= 0.0016). Moreover, IHH showed predictive value for cet benefit in both OS (mOS H: 38.1 mo vs M: 34.0 mo vs L: 18.4 mo; p= 0.0032) and PFS (mPFS H: 12.6 mo vs M: 12.9 mo vs L: 8.8 mo; p= 0.0025) with significant treatment interaction for OS and PFS (p= 0.0046 and p= 0.0049, respectively; FDR< 0.05). No significant associations were found in bev treated pts. No other Hh genes showed significant prognostic or predictive effects after FDR correction. Conclusions: These results highlight a dual role of Hh pathway in mCRC. Consistent with prior preclinical evidence, reduced IHH expression appears to mark poor prognosis, likely reflecting the loss of IHH-mediated restraint on Wnt signaling, which drives proliferation and dedifferentiation. Conversely, low GLI2, a downstream transcription factor, was linked with favorable outcomes, underscoring context-specific effects of Hh signaling. The strong predictive association of IHH with cet efficacy supports its potential as a biomarker in RAS wild type mCRC. These results suggest that Hh pathway modulation, particularly IHH signaling, may influence disease biology and treatment response. Validation in independent cohorts and mechanistic studies is warranted to clarify implications.

179 Background: Neuroligins are a family of postsynaptic cell adhesion molecules that play a crucial role in synapse formation and function. Recent studies suggest Neuroligin family genes (NLGN1,2,3,4X) may promote cancer cell growth and metastasis in various cancers, including colorectal cancer. Here, we investigated the association between neuroligin family gene expression and first-line treatment outcomes in mCRC. Methods: We evaluated the expression of 4 genes in the neuroligin family (NLGN1, NLGN2, NLGN3 and NLGN4X) on treatment outcomes of 433 pts with mCRC treated with either bevacizumab (Bev, n = 226) or cetuximab (Cet, n = 207) in combination with first-line chemotherapy within the CALGB/SWOG 80405 trial. mRNA was isolated from FFPE tumor samples and sequenced on the HiSeq 2500 (Illumina). Overall survival (OS) and progression-free survival (PFS) were compared between groups of pts categorized by low expression (Q1) (≤25 percentile), medium expression (Q2-3) (25th-75th percentile) and high expression (Q4)(>75th percentile) of NLGN family genes. The associations between gene expression and clinical outcomes were assessed via the multivariable Cox model and plotted with Kaplan-Meier curve. Treatment-gene interactions were evaluated using the likelihood ratio test. False discovery rate (FDR) was controlled through the Benjamini-Hochberg procedure. Results: High NLGN2 expression was associated with shorter PFS (median 10.0 mos, 95% CI (8.3, 11.3)) and OS (median 25.2mos, 95%CI (20.9, 30.0)) compared to low NLGN2 expression (PFS: median 11.2mos, 95% CI (9.8,14.3))(HR 1.41, 95% CI (1.06, 1.89)), P = 0.029). (OS: (median 31.8mos, 95%CI (26.8, 37.5)( HR 1.49, 95% CI (1.10, 2.01) P =0.0095) in multivariate analysis. Patients treated in the cetuximab arm(n=207), high HLGN2 expression significantly associated with shorter PFS (median 8.0 vs 13.4, HR 2.02, 95% CI (1.35, 3.03), P = 0.0021) and OS (median19.6 vs 36.3, HR 2.30, 95% CI (1.50, 3.53), P =8.3e-05). High NLGN3 expression was associated with shorter OS (23.9 vs 36.0 mos, P =0.0083) in cetuximab treatment arms, no significance found in PFS. We found significant treatment interaction for OS with NLGN2 and NLGN3 favoring cetuximab. In pts treated with cetuximab, High NLGN2 expression was significantly associated with shorter OS (median 29.1vs 36.3) (p=0.024) compared to pts treated with bevacizumab. For NLGN3, pts treated with cetuximab, high NLGN3 expression was significantly associated with shorter OS (median 25.0 vs 36.0) (p=0.018) compared to pts treated with bevacizumab. No significant association was found in NLGN1 and NLGN4X. Conclusions: Our results suggest NLGN2/3 gene expression may predict mCRC treatment outcomes. Potential therapeutic drug target NLGN in combination with anti-EGFR treatment in mCRC is warranted.

Background. MicroRNAs are small, single-stranded non-coding RNA molecules that play major role in gene regulation. miRNAs have been reported as both tumor suppressive and oncogenic and are promising candidates for cancer prognosis and treatment. miR-494 is known to promote cell migration and invasion in CRC. Our study aimed to further explore the role of miR-494 modulation (inhibition and overexpression) in CRC. Methods. We evaluated the impact of tumor gene expression of MIR494 on outcomes (overall survival: OS; progression-free survival: PFS) in data for 433 metastatic CRC (mCRC) patients (pts) (bevacizumab [bev] (N = 226) vs cetuximab [cet] (N = 207)) enrolled in randomized phase III CALGB/SWOG 80405 trial obtained from Alliance for Clinical Trials in Oncology. RNA extracted from FFPE tumor samples was sequenced using the HiSeq 2500 (Illumina). Gene expression was divided into high, medium and low. Associations were assessed by log-rank test and multivariable Cox proportional hazards regression model. Treatment-gene interactions were analyzed using the likelihood ratio test. CRC cell lines HCT116 and HT29 were transfected with miR-494 inhibitors and mimics for 48 hours and total RNA was collected, followed by RT-qPCR (miR-494 and target genes BMAL1, PAK1, CDH1, CDH2, SNAI1, TWIST) and clonogenic assays. Results. High miR-494 was significantly associated with worse PFS in overall (median survival: high vs low: 9.3 vs 11.3 months, P&lt;0.025) and cet-treated (median survival high vs low: 9.2 vs 9.6 months, P&lt;0.025) mCRC cohorts, while no significance was observed in bev-treated mCRC. Multivariable Cox model showed significant association between high miR-494 and worse PFS and OS in the cet-treated cohort; however, treatment-gene interaction was not statistically significant. Expression analysis revealed miR-494 was significantly reduced and increased following inhibitor and mimic transfection, respectively. Differential gene expression of the target genes analyzed was observed between the miR-494 inhibitor and mimic-treated HCT116 and HT29 samples. In HCT116 cells, inhibition of miR-494 significantly (P &lt; 0.01) reduced expression of PAK1, while increased the expression of CDH1. The number of colonies formed relatively increased after transfection with miR-494 inhibitor compared to miR-494 mimic. Conclusion. Our findings showed that high miR-494 expression correlated with worse PFS overall and in cet-treated mCRC pts. It would be interesting to check for potential associations with EGFR signaling (direct or indirect). Further studies are warranted for efficient targeting and to gain better understanding of the associated signaling involved in miR-494 modulation in CRC. Support: U10CA180821, U10CA180882; U10CA180888 (SWOG): Genentech. https://acknowledgments.alliancefound.org; P30CA014089, UG1CA180830 [HJL]. Clinicaltrials.gov Identifier: NCT00265850 Citation Format: Pooja Mittal, Joshua Millstein, Shivani Soni, Francesca Battaglin, Karam Ashouri, Sandra Algaze, Priya Jayachandran, Yan Yang, Lesly T. Gonzalez, Unnati H. Shah, Wu Zhang, Fang-Shu Ou, Alan P. Venook, Indrakant K. Singh, Heinz J. Lenz. Effect of miR-494 modulation in colorectal cancer (CRC): Clinical outcome, target expression and cell characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2787.

254 Background: SLFN11 was recently identified as a dominant determinant of response to DNA-damaging anticancer agents. Several studies showed SLFN11 expression led to better sensitivity to topoisomerase inhibitors and PARP inhibitors. However, clinical evidence in mCRC has been lacking. Methods: Two independent cohorts (CALGB/SWOG 80405 trial and a real-world patient [Caris CODEai] cohort) were included in this study. Patients were stratified into SLFN11 -high and -low groups based on the median SLFN11 gene expression levels. Survival outcomes were compared between SLFN11 -high and SLFN11 -low in irinotecan-treated and oxaliplatin-treated patients. Median time was estimated using Kaplan-Meier method and Cox models were used for covariate adjustment. Endpoints of interest were progression-free survival (PFS) and overall survival (OS). Results: In the CALGB/SWOG 80405 trial cohort, 433 patients were included (110 treated with FOLFIRI-based chemotherapy and 323 treated with FOLFOX-based chemotherapy). In patients treated with FOLFIRI-based chemotherapy, SLFN11 -low patients exhibited numerically better PFS (median PFS, 13.4 vs 11.2 months, log-rank p = 0.03, adjusted hazard ratio [HR] = 0.86, 95% confidence interval [CI] 0.55-1.39, p = 0.50) and OS (median OS, 39.6 vs 30.3 months, log-rank p = 0.02, adjusted HR = 0.73, 95% CI 0.46-1.16, p = 0.19) compared to SLFN11 -high patients. In patients treated with FOLFOX-based chemotherapy, SLFN11 -low patients exhibited numerically better PFS (median PFS 11.2 vs 9.8 months, log-rank p = 0.03, adjusted HR = 0.83, 95% CI 0.65-1.05, p = 0.12) and OS (31.1 vs 26.1 months, log-rank p = 0.07, adjusted HR = 0.84, 95% CI 0.65-1.08, p = 0.16) compared to SLFN11 -high patients. No significant interaction was observed between treatment and SLFN11 expression in terms of PFS ( p = 0.69) and OS ( p = 0.44). In the Caris CODEai cohort, a better OS in SLFN11 -low patients than in SLFN11 -high patients was observed in irinotecan-treated patients ( N = 2906) (median OS, 25.5 vs 21.7 months, HR = 0.90, 95% CI 0.82-1.00, p = 0.048). However, no significant difference in OS between SLFN11 -low and SLFN11-high was observed in oxaliplatin-treated patients ( N = 4428) (median OS, 39.5 vs 35.8 months, HR = 0.98, 95% CI 0.90-1.06, p = 0.56). Conclusions: Low SLFN11 expression showed a potential for better prognosis in mCRC patients receiving irinotecan- and oxaliplatin-containing chemotherapies. These findings are not consistently aligned with preclinical data and clinical evidence in other cancer types previously reported. Further investigation is warranted to better understand the biological role and clinical implication of SLFN11 in mCRC.

3588 Background: The brain-gut axis, a bidirectional network between the central and enteric nervous systems, plays a critical role in modulating the gastrointestinal tract function. Increasing evidence suggests that neuronal signaling molecules can promote gastrointestinal cancers; however, the mechanisms remain unclear. Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter and dysregulated GABA signaling has been found in CRC, affecting both tumor and tumor microenvironment. Here, we investigated the association between tumor GABA pathway gene expression and first-line treatment outcomes in mCRC. Methods: We evaluated the impact of the expression of 25 genes in the GABA pathway ( GABBR1, GABBR2, GABRA1, GABRA2, GABRA4, GABRA5, GABRA6, GABRB1, GABRB3, GABRD, GABRE, GABRG1, GABRG2, GABRG3, GABRQ, GABRR1, GABRR2, ABAT, ALDH5A1, GAD1, GAD2, SLC6A1, SLC6A13, SLC6A11, SLC6A12) on treatment outcomes (overall survival: OS; progression free survival: PFS) of 433 pts enrolled in the randomized, phase III CALGB/SWOG 80405 trial. RNA extracted from FFPE tumor samples was sequenced using the HiSeq 2500 (Illumina). The associations between gene expression (continuous, per standard deviation increase) and outcomes were assessed via the multivariable Cox proportional framework, with hazard ratios (HR) and 95% confidence intervals (CI) computed. The likelihood ratio test was used for interaction tests. False discovery rate (FDR) was controlled at 5% with the Benjamini-Hochberg method. Results: Overall, low GAD1 (encoding for glutamate decarboxylase 1, the enzyme responsible for catalyzing the production of GABA from L-glutamic acid) was associated with longer PFS (HR: 1.26, 95% CI [1.13, 1.41], P = 3.2E-05) and OS (HR 1.27 [1.13, 1.43], P = 5.9E-05) across all treatment arms. High ABAT (encoding for the aminotransferase responsible for GABA catabolism) was also associated with longer OS (HR 0.81 [0.72, 0.91], P = 6E-04). To evaluate the predictive value, interactions with targeted therapies bevacizumab (n = 226) vs cetuximab (n = 207) were tested. We found significant interactions for PFS with ABAT ( P= 0.0017) and OS with SLC6A13 ( P= 0.0014 ).In pts treated with cetuximab, high ABAT expression was significantly associated with longer PFS (HR 0.73 [0.61, 0.87], P = 2.8E-04), and high SLC6A13 was marginally associated with worse OS (HR 1.18 [1.00, 1.39], P = 0.052). We did not observe similar associations in pts treated with bevacizumab. Conclusions: Our results highlight a potential role for GABA signaling in affecting mCRC treatment outcomes, suggesting that GABA pathway genes may be promising novel predictive and prognostic biomarkers and supporting the rationale to investigate GABA as a target for drug development. ClinicalTrials.gov Identifier: NCT00265850

3586 Background: HSP90 mediated chaperoning is a well-conserved biological mechanism for stabilization and activation of kinases. More than 60% of human kinases including VEGFR, CRAF, CSF1R, and FGFR are target of HSP90 (client kinases), whereas EGFR is non-client. CDC37 is a specific co-chaperone determining selectivity of client kinases recognized by HSP90. We hypothesized that gene expression levels of CDC37 have predictive values for anti-angiogenic therapies in mCRC. Methods: The subjects of this study were mCRC patients treated with regorafenib (REGO, Japanese retrospective cohort) and those treated with bevacizumab (BEV) or cetuximab (CET) in combination with first-line chemotherapy (CALGB/SWOG 80405 trial cohort). CDC37 expression levels were measured using RNA isolated from FFPE samples by nCounter gene expression profiling (Nanostring) and HiSeq 2500 (Illumina) in the Japanese and CALGB/SWOG 80405 cohorts, respectively. Overall survival (OS) and progression-free survival (PFS) were compared between patients with high CDC37 expression ( CDC37-H) and those with low expression ( CDC37-L), grouped by median cutoff value in each cohort. Results: In total, 484 patients were included (50 treated with REGO, 227 treated with BEV, and 207 treated with CET). In REGO-treated patients, CDC37-H showed significantly better OS (median 11.3 vs 6.0 months, adjusted hazard ratio [HR] 0.24, 95% confidence interval [CI] 0.11-0.54, p <0.01) and PFS (median 3.5 vs 1.9 months, adjusted HR 0.51, 95% CI 0.28-0.94, p = 0.03) compared to CDC37-L. Similarly, in BEV-treated patients, CDC37-H showed significantly better PFS (median 13.5 vs 9.6 months, adjusted HR 0.59, 95% CI 0.43-0.79, p <0.01) and numerically better OS (median 34.1 vs 29.4 months, adjusted HR 0.81, 95% CI 0.60-1.11, p = 0.20) compared to CDC37-L. However, in CET-treated patients, CDC37-H and CDC37-L patients showed similar OS (median 33.7 vs 26.1 months, adjusted HR 1.00, 95% CI 0.73-1.38, p = 0.98) and PFS (median 11.3 vs 11.0 months, adjusted HR 1.08, 95% CI 0.81-1.45, p = 0.60). Significant interaction was observed between CDC37 expression and treatment in terms of PFS in the CALGB/SWOG 80405 cohort ( p = 0.01). Conclusions: Our results suggest patients with CDC37-dependent ( CDC37-H) tumors may derive more benefit from REGO and BEV both of which target HSP90 client kinases or signaling pathways, but not from CET which target HSP90 non-client kinase. Further validation studies are warranted to develop a novel personalized approach for targeted therapies based on CDC37 expression in mCRC patients. Support: U10CA180821, U10CA180888; Pfizer, Genentech; https://acknowledgments.alliancefound.org. ClinicalTrials.gov Identifier: NCT00265850.