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Background: Disruption of circadian processes has been linked to cancer initiation, progression, metastasis, resistance, and mortality. Clock proteins are an emerging target for therapy in breast cancer. Circadian rhythms are controlled by a network of transcription/translation feedback loops primarily driven by BMAL and CLOCK and the transcriptional repressors period (PER1-3) and cryptochrome (CRY1-2). We investigated the molecular and clinical associations of clock genes in breast cancer. Methods: A total of 9563 breast tumors underwent molecular profiling (Caris Life Sciences). Analyses included next-generation sequencing of DNA (592 genes-NextSeq, WES-NovaSeq) and RNA (NovaSeq). Clock gene Score (CS) was determined using expression of clock pathway gene Z scores (positives of BMAL, CLOCK and negatives of PER1/2 and CRY1/2) and then stratified into quartiles. xCell was used to quantify immune cell infiltration in the tumor microenvironment (TME). ER/PR was tested by IHC and HER2 was tested by either IHC or CISH. Significance was determined as P values adjusted for multiple comparison (Q) of < 0.05. Real-world survival information was obtained from insurance claims data and was calculated from either tissue collection to last contact or time on treatment (TOT); comparison was done by Kaplan-Meier test. Results: TNBC had the highest median CS score, while HR+/HER2- had the lowest CS (0.96 vs 0.26 q Citation Format: Priya Jayachandran, Yasmine Baca, Joanne Xiu, Yuanzhong Pan, Phil Walker, Francesca Battaglin, Hiroyuki Arai, Moh’d Khushman, Janice Lu, Darcy Spicer, Shannon Mumenthaler, Richard Goldberg, Benjamin Weinberg, Emil Lou, Michael Hall, Arielle L. Heeke, W. Michael Korn, Steve A. Kay, Heinz-Josef Lenz, Evanthia T. Roussos Torres. Clock Genes in Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-06.

Exploring the relationship between various neurotransmitters and breast cancer cell growth has revealed their likely centrality to improving breast cancer treatment. Neurotransmitters play a key role in breast cancer biology through their effects on the cell cycle, epithelial mesenchymal transition, angiogenesis, inflammation, the tumor microenvironment and other pathways. Neurotransmitters and their receptors are vital to the initiation, progression and drug resistance of cancer and progress in our biological understanding may point the way to lower-cost and lower-risk antitumor therapeutic strategies. This review discusses multiple neurotransmitters in the context of breast cancer. It also discusses risk factors, repurposing of pharmaceuticals impacting neurotransmitter pathways, and the opportunity for better integrated models that encompass exercise, the intestinal microbiome, and other non-pharmacologic considerations. Neurotransmitters’ role in breast cancer should no longer be ignored; it may appear to complicate the molecular picture but the ubiquity of neurotransmitters and their wide-ranging impacts provide an organizing framework upon which further understanding and progress against breast cancer can be based.

The brain-gut axis, a bidirectional network between the central and enteric nervous system, plays a critical role in modulating the gastrointestinal tract function and homeostasis. Recently, increasing evidence suggests that neuronal signaling molecules can promote gastrointestinal cancers, however, the mechanisms remain unclear. Aberrant expression of neurotransmitter signaling genes in colorectal cancer supports the role of neurotransmitters to stimulate tumor growth and metastatic spread by promoting cell proliferation, migration, invasion, and angiogenesis. In addition, neurotransmitters can interact with immune and endothelial cells in the tumor microenvironment to promote inflammation and tumor progression. As such, pharmacological targeting of neurotransmitter signaling represent a promising novel anticancer approach. Here, we present an overview of the current evidence supporting the role of neurotransmitters in colorectal cancer biology and treatment.

Reactive oxygen species activate EGFR/RAS/MAPK signaling either through the inactivation of phosphatases or by direct oxidation of kinases. We hypothesized that functional single-nucleotide polymorphisms (SNPs) in antioxidant genes link to the efficacy of cetuximab in patients with metastatic colorectal cancer (mCRC).We analyzed genomic and clinical data from FIRE-3, a phase III trial comparing cetuximab and bevacizumab along with FOLFIRI in untreated mCRC patients. Genomic DNA extracted from blood samples was genotyped. Thirteen functional SNPs in antioxidant genes were tested for associations with clinical outcomes.In total, 236 patients were included (FOLFIRI/cetuximab arm, n = 129; FOLFIRI/bevacizumab arm, n = 107). In univariate analysis, two SNPs (TXN2 rs4821494 and GPX4 rs4807542) were significantly associated with overall survival (OS) in the FOLFIRI/cetuximab arm. Multivariate analysis confirmed the significant association of TXN2 rs4821494 (T/T vs. any G allele, hazard ratio = 2.47, 95% confidence interval = 1.06-5.72, P = .03). In the FOLFIRI/bevacizumab arm, no SNPs were significantly associated with clinical outcomes. Treatment-by-SNP interaction test confirmed the predictive value of TXN2 rs4821494 (OS: P = .03).TXN2 rs4821494 involved in the antioxidant system may predict the efficacy of cetuximab-based first-line chemotherapy in mCRC, warranting further validation studies.

Associations between LIN28B rs314277/rs314276 and clinical outcomes in RAS wild type patients

Copy number alteration frequency in appendiceal adenocarcinoma, NET and GCC

Associations between selected SNPs and gene expression status in colon tissue from GTEx analysis

MCP-counter results for the three appendiceal tumor groups

Details about the imputation

RNA-binding proteins (RBPs) regulate many posttranscriptional cellular activities. Accumulating evidence suggests associations between RBPs with colonic tumorigenesis and chemosensitivity. We investigated the prognostic and predictive values of SNPs of genes encoding RBPs in metastatic colorectal cancer (mCRC), using clinical and genomic data from three randomized clinical trials of standard first-line chemotherapy for mCRC (TRIBE, FIRE-3, and MAVERICC). Genomic DNA extracted from blood samples was genotyped using an OncoArray. We tested 30 candidate SNPs of 10 major RBP-related genes with additive models. Prognostic values were estimated by meta-analysis approach. Treatment-by-SNP interactions were tested to estimate predictive values for targeted drugs and cytotoxic backbone chemotherapies. This study included 884 patients. The meta-analysis revealed prognostic values of LIN28B rs314277 [HR, 1.26; 95% confidence interval (CI), 1.06–1.49, P = 0.005, FDR-adjusted P = 0.072 for overall survival (OS)] and LIN28B rs314276 (HR, 1.25; 95% CI, 1.08–1.44, P = 0.002, FDR-adjusted P = 0.062 for OS). Although some SNPs showed potentially predictive values, these associations were not confirmed after FDR adjustment. In conclusion, the results of this study are warranting additional studies to provide the evidence that RBP-related SNPs may be associated with the prognosis of patients with mCRC treated with standard first-line chemotherapies. In addition, further studies are warranted to study the predictive value.

Goblet cell carcinoid (GCC) is a distinct subtype of appendiceal neoplasm that exhibits unique clinical and pathologic features. We aimed to reveal the molecular profiles of GCC compared with other appendiceal tumors, such as adenocarcinomas and neuroendocrine tumors. A total of 495 appendiceal tumor samples (53 GCCs, 428 adenocarcinomas, and 14 neuroendocrine tumors) were tested with next-generation sequencing (NGS) on a 592-gene panel and IHC. Microsatellite instability (MSI)/mismatch repair (MMR) status was tested with a combination of NGS, IHC, and fragment analyses. Tumor mutational burden (TMB) was evaluated by NGS, and PD-L1 expression was tested by IHC (SP142). The most prevalent mutated genes within GCCs were TP53 (24.0%), ARID1A (15.4%), SMAD4 (9.4%), and KRAS (7.5%). Pathway-specific alterations were dominantly observed in cell cycle, MAPK, epigenetic, and TGFβ signaling pathways. GCCs as compared with adenocarcinomas exhibited significantly lower mutation rates in KRAS, GNAS, and APC, and significantly higher mutation rates in CDH1, CHEK2, CDC73, ERCC2, and FGFR2. GCCs as compared with neuroendocrine tumors showed significantly lower mutation rates in KRAS, APC, BRCA2, and FANCA. In GCCs, MSI high/MMR deficient, TMB high (≥17 mutations/Mb), and PD-L1 expression were seen in 0.0%, 0.0%, and 2.0% of tumors, respectively. No significant differences were observed in any immunotherapy-related markers examined when compared with adenocarcinomas and neuroendocrine tumors. In conclusion, GCCs had considerably distinct mutational profiles compared with appendiceal adenocarcinomas and neuroendocrine tumors. Understanding these molecular characteristics may be critical for the development of novel and more effective treatment strategies for GCC.

The methods in assessing microsatellite instability (MSI) and mismatch repair (MMR) status

Supplementary Table from Multi-omic Characterization of Pancreatic Ductal Adenocarcinoma Relates CXCR4 mRNA Expression Levels to Potential Clinical Targets

Supplementary Figure from Multi-omic Characterization of Pancreatic Ductal Adenocarcinoma Relates CXCR4 mRNA Expression Levels to Potential Clinical Targets

Supplementary Data from Multi-omic Characterization of Pancreatic Ductal Adenocarcinoma Relates CXCR4 mRNA Expression Levels to Potential Clinical Targets

Purpose:Chemokines are essential for immune cell trafficking and are considered to have a major impact on the composition of the tumor microenvironment. CX-chemokine receptor 4 (CXCR4) is associated with poor differentiation, metastasis, and prognosis in pancreatic ductal adenocarcinoma (PDAC). This study provides a comprehensive molecular portrait of PDAC according to CXCR4 mRNA expression levels.Experimental Design:The Cancer Genome Atlas database was used to explore molecular and immunologic features associated with CXCR4 mRNA expression in PDAC. A large real-word dataset (n = 3,647) served for validation and further exploratory analyses. Single-cell RNA analyses on a publicly available dataset and in-house multiplex immunofluorescence (mIF) experiments were performed to elaborate cellular localization of CXCR4.Results:High CXCR4 mRNA expression (CXCR4high) was associated with increased infiltration of regulatory T cells, CD8+ T cells, and macrophages, and upregulation of several immune-related genes, including immune checkpoint transcripts (e.g., TIGIT, CD274, PDCD1). Analysis of the validation cohort confirmed the CXCR4-dependent immunologic TME composition in PDAC irrespective of microsatellite instability–high/mismatch repair–deficient or tumor mutational burden. Single-cell RNA analysis and mIF revealed that CXCR4 was mainly expressed by macrophages and T-cell subsets. Clinical relevance of our finding is supported by an improved survival of CXCR4high PDAC.Conclusions:High intratumoral CXCR4 mRNA expression is linked to a T cell– and macrophage-rich PDAC phenotype with high expression of inhibitory immune checkpoints. Thus, our findings might serve as a rationale to investigate CXCR4 as a predictive biomarker in patients with PDAC undergoing immune checkpoint inhibition.

POLE and DDR mutations in MSS/pMMR patients

Supplementary Table from Comprehensive Analysis of R-Spondin Fusions and RNF43 Mutations Implicate Novel Therapeutic Options in Colorectal Cancer

Next generation sequencing Immunohistochemistry for HER-2 and MET Dual color silver in situ hybridization (SISH) for HER-2 and MET

Details of NGS, WTS, CMS, and MSI/MMR status

Purpose:Gene fusions involving R-spondin (RSPOfp) and RNF43 mutations have been shown to drive Wnt-dependent tumor initiation in colorectal cancer. Herein, we aimed to characterize the molecular features of RSPOfp/RNF43 mutated (mut) compared with wild-type (WT) colorectal cancers to gain insights into potential rationales for therapeutic strategies.Experimental Design:A discovery cohort was classified for RSPOfp/RNF43 status using DNA/RNA sequencing and IHC. An independent cohort was used to validate our findings.Results:The discovery cohort consisted of 7,245 colorectal cancer samples. RSPOfp and RNF43 mutations were detected in 1.3% (n = 94) and 6.1% (n = 443) of cases. We found 5 RSPO fusion events that had not previously been reported (e.g., IFNGR1–RSPO3). RNF43-mut tumors were associated with right-sided primary tumors. No RSPOfp tumors had RNF43 mutations. In comparison with WT colorectal cancers, RSPOfp tumors were characterized by a higher frequency of BRAF, BMPR1A, and SMAD4 mutations. APC mutations were observed in only a minority of RSPOfp-positive compared with WT cases (4.4% vs. 81.4%). Regarding RNF43 mutations, a higher rate of KMT2D and BRAF mutations were detectable compared with WT samples. Although RNF43 mutations were associated with a microsatellite instability (MSI-H)/mismatch repair deficiency (dMMR) phenotype (64.3%), and a tumor mutation burden ≥10 mt/Mb (65.8%), RSPOfp was not associated with MSI-H/dMMR. The validation cohort replicated our genetic findings.Conclusions:This is the largest series of RSPOfp/RNF43-mut colorectal cancers reported to date. Comprehensive molecular analyses asserted the unique molecular landscape associated with RSPO/RNF43 and suggested potential alternative strategies to overcome the low clinical impact of Wnt-targeted agents and immunotherapy.

Supplementary Figure S1. Histopathological subtypes of AA. A: adenocarcinoma, not otherwise specified (NOS: common colonic type). B: low-grade mucinous adenocarcinoma (MU) with pseudomyxoma peritonei (PMP). C: mucinous adenocarcinoma (MU). D: signet ring cell carcinoma (SR). All images are with hematoxylin and eosin stain.

Supplementary Table S2. Patients characteristics.

Purpose:Defective DNA damage response (DDR) is a hallmark of cancer leading to genomic instability and is associated with chemosensitivity. Although the mismatch repair system has been extensively studied, the clinical implications of other mechanisms associated with DDR alterations in patients with colorectal cancer remain unclear. This study aimed to understand DDR pathways alterations and their association with common clinical features in patients with colorectal cancer.Experimental Design:Next-generation sequencing and whole-transcriptome sequencing were conducted using formalin-fixed paraffin-embedded samples submitted to a commercial Clinical Laboratory Improvement Amendments–certified laboratory. Samples with pathogenic or presumed pathogenic mutations in 29 specific DDR-related genes were considered as DDR-mutant (DDR-MT) and the remaining samples as DDR-wild type (DDR-WT).Results:Of 9,321 patients with colorectal cancer, 1,290 (13.8%) were DDR-MT. The frequency of DDR-MT was significantly higher in microsatellite instability-high (MSI-H) cases than in microsatellite stable cases (76.4% vs. 9.5%). The DDR-MT genotype was higher in the right-sided, RAS-wild, BRAF-mutant, and CMS1 subgroups. However, these associations were primarily confounded by the distribution of MSI status. Compared with the DDR-WT tumors, the DDR-MT tumors had a higher mutational burden and gene expression levels in the immune-related pathway, which were independent of MSI status.Conclusions:We characterized a distinct subgroup of patients with colorectal cancer with tumors harboring mutations in the DDR-related genes. These patients more commonly had MSI-H tumors and exhibited an activated immune signature regardless of their tumor's MSI status. These findings warrant further investigations to develop personalized treatment strategies in this significant subgroup of patients with colorectal cancer.

Supplementary Figure 1: Patients with acquired MET amplification have shorter PFS as compared to those with MET-negative tumors. PFS duration expressed as month time units is shown for individual patients with acquired MET amplification (blue histograms) and MET-negative (red histograms) tumors (panel A). Kaplan Meier curves show shorter median PFS of MET-amplified (blue) as compared with MET-negative (red) patients; moreover, around 40% and 15% patients MET-negative (but none of MET-amplified subjects) were still progression-free at 10 and even 20 months, respectively (panel B).

Table S1-S12

Purpose: Even if RAS-BRAF wild-type and HER2/MET–negative metastatic colorectal cancer (mCRC) patients frequently respond to anti-EGFR mAbs, acquired resistance almost invariably occurs. Mechanisms of resistance to EGFR blockade include the emergence of KRAS, NRAS, and EGFR extracellular domain mutations as well as HER2/MET alterations. However, these findings derive from retrospective studies that analyzed one single resistance mechanism at a time; moreover, it is still unclear how molecular heterogeneity affects clonal evolution in patients. In this work, we aimed at extensively characterizing and correlating the molecular characteristics of tissue- and blood-based data in a prospective cohort of patients with mCRC who received anti-EGFR antibodies.Experimental design: Twenty-two RAS-BRAF wild-type, HER2/MET–negative mCRC patients progressing on anti-EGFR therapy after initial response underwent rebiopsy. Next-generation sequencing and silver in situ hybridization (SISH)/IHC analyses were performed both on archival tumors and postprogression samples. Circulating tumor (ctDNA) molecular profiles were obtained in matched tissue–plasma samples.Results: RAS mutations and HER2/MET amplification were the most frequently detected resistance mechanisms in both tissue and blood sample analysis. On the other hand, BRAF and EGFR ectodomain mutations were much rarer. Patients with acquired MET amplification showed worse PFS on anti-EGFRs. We detected both intralesion heterogeneity, as suggested by co-occurrence of different resistance mechanisms in the same sample, and interlesion heterogeneity. The combined analysis of tissue and blood (ctDNA) results highlights the complexity of clonal evolution triggered by EGFR blockade.Conclusions: Our results indicate that it may be extremely challenging to target the complex landscape of molecular heterogeneity associated with emergence of resistance to targeted therapies in patients with mCRC. Clin Cancer Res; 23(10); 2414–22. ©2016 AACR.

Purpose:The natural history and prognosis of appendiceal adenocarcinomas differ from those of adenocarcinomas arising in other large bowel sites. We aimed to compare the molecular profiles exhibited by appendiceal adenocarcinomas and colorectal cancers, or between the histopathologic subtypes of appendiceal adenocarcinoma.Experimental Design:A total of 183 samples from appendiceal adenocarcinoma [46 adenocarcinoma, not otherwise specified (NOS), 66 pseudomyxoma peritonei (PMP), 44 mucinous adenocarcinoma (MU), and 27 signet ring cell carcinoma (SR)], 994 from right-sided colorectal cancer (R-CRC), and 1,080 from left-sided CRC (L-CRC) were analyzed by next-generation sequencing (NGS) and IHC markers. Microsatellite instability (MSI) and tumor mutational burden (TMB) were tested by NGS, and programmed death ligand 1 (PD-L1) by IHC.Results:We observed high mutation rates in appendiceal adenocarcinoma samples for KRAS (55%), TP53 (40%), GNAS (31%), SMAD4 (16%), and APC (10%). Appendiceal adenocarcinoma exhibited higher mutation rates in KRAS and GNAS, and lower mutation rates in TP53, APC, and PIK3CA (6%) than colorectal cancers. PMP exhibited much higher mutation rates in KRAS (74%) and GNAS (63%), and much lower mutation rates in TP53 (23%), APC (2%), and PIK3CA (2%) than NOS. Alterations associated with immune checkpoint inhibitor response (MSI-high, TMB-high, PD-L1 expression) showed similar frequency in appendiceal adenocarcinoma compared with L-CRC, but not R-CRC, and those of NOS were higher than other subtypes of appendiceal adenocarcinoma and L-CRC.Conclusions:Molecular profiling of appendiceal adenocarcinoma revealed different molecular characteristics than noted in R-CRC and L-CRC, and molecular heterogeneity among the histopathologic subtypes of appendiceal adenocarcinoma. Our findings may be critical to developing an individualized approach to appendiceal adenocarcinoma treatment.

Supplementary Figure from Comprehensive Analysis of R-Spondin Fusions and RNF43 Mutations Implicate Novel Therapeutic Options in Colorectal Cancer

Frequent mutations of genes in the histone-lysine N-methyltransferase 2 (KMT2) family members were identified in gastric cancers (GCs). Understanding how gene mutations of KMT2 family affect cancer progression and tumor immune microenvironment may provide new treatment strategies. A total of 1245 GCs were analyzed using next-generation sequencing, whole transcriptome sequencing, immunohistochemistry (Caris Life Sciences, Phoenix, AZ). The overall mutation rate of genes in the KMT2 family was 10.6%. Compared to KMT2-wild-type GCs, genes involved in epigenetic modification, receptor tyrosine kinases/MAPK/PI3K, and DNA damage repair (DDR) pathways had higher mutation rates in KMT2-mutant GCs (p

Cancer stem cells (CSCs) are primarily maintained by a network of pluripotency transcription factors (PTFs). Given a close relationship of CSC regulation with epidermal growth factor receptor and vascular endothelial growth factor signalling, we investigated whether single-nucleotide polymorphisms (SNPs) in PTF genes are related to the efficacy of cetuximab and/or bevacizumab in patients with metastatic colorectal cancer (mCRC).Genomic and clinical data from three independent clinical trial cohorts were tested: cetuximab cohort (FOLFIRI/cetuximab arm in FIRE-3, n = 129), bevacizumab cohort 1 (FOLFIRI/bevacizumab arm in FIRE-3, n = 107) and bevacizumab cohort 2 (FOLFIRI/bevacizumab arm in TRIBE, n = 215). Genomic DNA extracted from blood samples was genotyped, and ten SNPs were tested for association with clinical outcomes.In the cetuximab cohort, four SNPs were significantly associated with progression-free survival in univariate analysis: NANOG rs11055767 (any A allele vs C/C, hazard ratio [HR] = 0.62, 95% confidence interval [CI] = 0.42-0.94, p = 0.02), NANOG rs10744044 (any A allele vs G/G, HR = 0.59, 95% CI = 0.39-0.90, p = 0.01), NANOGP8 rs2168958 (any C allele vs A/A, HR = 2.12, 95% CI = 1.36-3.29, p 0.001) and NANOGP8 rs2279066 (any C allele vs T/T, HR = 1.80, 95% CI = 1.06-1.68, p = 0.03). Multivariate analysis confirmed the significant associations for NANOGP8 rs2168958 and NANOGP8 rs2279066. In either bevacizumab cohort, no significant associations were observed in univariate analysis.Germ line polymorphisms in the PTF genes could be predictive markers for cetuximab in mCRC.

Disruption of the cellular pathway modulating endogenous 24-hour rhythms, referred to as ‘the circadian clock’, has been recently proven to be associated with cancer risk, development and progression. This pathway operates through a complex network of transcription-translation feedback loops generated by a set of interplaying proteins. The expression of core circadian clock genes is frequently dysregulated in human tumors; however, the specific effects and underlying mechanisms seem to vary depending on the cancer types and are not fully understood. Additionally, specific oncogenes may differentially induce the dysregulation of the circadian clock in tumors. Pharmacological modulation of clock components has been shown to result in specific lethality in certain types of cancer cells, and thus holds great promise as a novel anti-cancer therapeutic approach. Here we present an overview of the rationale and current evidence for targeting the clock in cancer treatment.

Background: Mitochondrial MAO A and MAO B isoenzymes catalyze oxidative deamination of neuroactive and vasoactive monoamines in CNS and peripheral tissues. MAO A inhibitors have been used as antidepressants; MAO B inhibitors have been used for Parkinson’s disease. Recently, we and others have shown MAO inhibitors can be repurposed for cancer therapy. This study focused on the MAO inhibitors as a novel therapeutic strategy for CRC utilizing patient derived xenograft (PDX) platform which recapitulates the patient’s molecular characteristics. Methods and Results: CRC PDX models were selected from our repository based on MAO A and B expression and activity. To establish two PDX models with high or low MAO A/B activity we implanted patient’s tumor samples (F0 generation) with high MAO A/B activity (MAO high) or low MAO A/B activity (MAO low) in 8 weeks NSG mice (F1 generation). F1 MAO low PDX model showed substantially slower tumor growth rates as compared to MAO high PDX model (200 days vs 65 days), suggesting the role of MAOs in CRC growth. For each PDX model trial, tumors were pooled from 6 F1 PDX mice and implanted in 30 NSG mice (F2 generation). There were five mice in each of experimental groups: one control arm (vehicle: 67% PEG 400, 33% saline) and treatment arms (i.p. for 21 days): 1. MAO A inhibitors: clorygyline: 50mg/kg; 2. Near infrared dye conjugated MAO A inhibitor (NMI): 5 mg/kg; 3. MAO B inhibitor: deprenyl 10mg/kg;4. MAO A & B inhibitor: phenelzine (30mg/kg). MAO high PDX cohort showed significant reduction in tumor volume in mice treated with MAO A inhibitors clorygyline (p=0.009); NMI (p=0.053) and MAO B inhibitor deprenyl (p=0.022), phenelzine (p= 0.097) when compared to control. Angiogenesis marker CD31 staining of tumor tissues showed significant reduction in mice treated with clorygyline and NMI. Additionally, Ki 67 staining demonstrated considerable decrease in cell proliferation and TUNEL assay exhibited increase in apoptosis in tumor treated with clorygyline and NMI. RNA sequencing revealed 50 differentially expressed genes (DEGs) between clorygyline treated tumors and control, 171 between NMI treated and control group (padj =1.5). Ingenuity Pathway Analysis showed alterations in pathways including oxidative phosphorylation, sirtuin pathway, estrogen receptor signaling, and mitochondrial dysfunction. NMI showed same efficacy with no toxicity compared to clorygyline even at 10 times lower dose in MAO high PDX model. No significant difference in tumor size was found among treatment groups in MAO low PDX cohort. Conclusion: Our CRC PDX studies showed both MAO A and B are important for tumor growth and are potential target for therapy. Both MAO A and B inhibitors and NMI (NIR-conjugated MAO an inhibitor, clorgyline) reduced tumor growth. IHC and RNA seq data shows MAO A inhibitor and NMI has similar mechanisms, though NMI is more effective than clorygyline and also can be used for diagnosis. Citation Format: Shivani Soni, Hui Ju Tseng, Yan Yang, Goar Smbatayan, Unnati Hemant Shah, Jae Ho Lo, Joshua Millstein, Francesca Battaglin, Pooja Mittal, Lesly Torres Gonzalez, Wu Zhang, Jean Chen Shih, Heinz Josef Lenz. MAO A, MAO B inhibitors and NMI for colon cancer therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4671.

Background. Immune checkpoint inhibitors have substantial clinical success in MSI-H/dMMR metastatic CRC. However, only a subset of patients exhibits durable responses, emphasizing the importance of identifying mechanism of innate and acquired resistance. The relation between the circadian clock and the immune system is well established. We used a syngeneic mouse model of MSI-H CRC to investigate whether a targeted enhancement of clock repressor proteins cryptochrome 1/2 (CRY 1/2) may affect tumor growth and response to anti-PD1 (aPD1). Methods. 1 × 106 MC38 cells were subcutaneously implanted into the right flank of 8 weeks old C57BL/6 mice. Mice were randomized into 1 control and 3 treatment arms (10 mice/group): A. CRY stabilizer SHP656(10 mg/kg PO, 5 days/week); B. aPD1 (100 μg/mouse IP, twice/week administered for 2 weeks); C. combination SHP656 + aPD1. Tumors from 4 mice/group were processed for tumor infiltration studies. Remaining 6 mice/group were kept for 3 weeks on maintenance aPD1 (twice/week) for survival studies. Treatment response was measured by tumor growth and survival. Treatment effects on immune cell infiltration in the tumor microenvironment (TME), functional myeloid derived suppressor cells (MDSCs) and T cells markers were investigated via flow cytometry. Results. Single agent SHP656 significantly improved survival when compared with vehicle treated mice. Similarly, combination treatment with SHP656 + aPD1 improved survival, however no additional benefit was observed over treatment with aPD1 alone. All treatment groups had a response rate of 67% (4/6 mice/treatment group showed 2-fold decrease in tumor volume 24 days after tumor challenge vs vehicle). In responders, SHP656 as a single agent or in combination with aPD1 were more effective in suppressing tumor growth as compared to aPD1 alone. Flow cytometry of 14 tumor samples demonstrated that monocytic-MDSC infiltration was significantly decreased in the combination therapy and aPD1 alone treatment arms (p = .016) as compared to vehicle suggestive of a less suppressive TME. T cell analysis suggested a decrease in intratumoral exhausted CD8+ T cells (TIM3−PD-1+, p = .02) in SHP656 + aPD1 vs vehicle, while infiltration of CD4+ T cells and T regulatory cells were not significantly affected by any treatment. Nanostring profiling on treated tumors also confirmed immune activation in responders. Conclusions. Our results show for the first time that targeting the clock pathway through modulation of CRY affects TME and tumor progression in in vivo models of MSI-H CRC. A decrease in infiltration of mMDSCs and exhausted CD8+ T cells seems to play a critical role in combination treatment efficacy but needs to be studied further. These results, suggests that pharmacological targeting of the circadian machinery holds great potential and could significantly impact the efficacy of immunotherapy in metastatic CRC. Citation Format: Shivani Soni, Francesca Battaglin, Sofi Castanon, Jae Ho Lo, Goar Smbatyan, Priscilla Chan, Meng Qu, Priya Jayachandran, Hiroyuki Arai, Natsuko Kawanishi, Wu Zhang, Steve A. Kay, Heinz Josef Lenz, Evanthia T. Torres. Targeting the clock pathway to modulate immune response in MSI-high colorectal cancer (CRC): evidence from a preclinical in vivo model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4124.