Merkel cell carcinoma (MCC) is a poorly differentiated neuroendocrine carcinoma with limited treatment options, primarily immunotherapy, to which only ~50% of patients respond. Lineage plasticity drives its poorly differentiated phenotype, in turn promoting tumor aggressiveness and treatment resistance. Targeting the mechanisms underlying lineage plasticity could help induce differentiation, reduce proliferation, and potentially sensitize the tumor to existing therapies, yet such strategies are underdeveloped in MCC. Here, we integrate scRNA-seq and bulk ATAC-seq data to generate Boolean networks, simulate their dynamics, and predict a key regulator of differentiation, which we validated in vitro. Using CytoTRACE2 across two independent datasets, we revealed the existence of tumor subpopulations with distinct developmental potency states. We then constructed and refined transcription factor regulatory networks using BooleaBayes and expanded them with ATAC-seq inferred regulatory interactions. Across multiple network constructions, simulations of single-gene perturbations consistently identified the Notch effector RBPJ as the key regulator predicted to shift MCC cells toward a more differentiated state. Experimental knockdown of RBPJ in an MCC cell line altered expression of differentiation-associated genes, reduced the expression of MCC markers, and drastically reduced cell growth. These findings identify RBPJ as a regulator of MCC lineage plasticity and candidate for targeted treatment, while highlighting the utility of probabilistic network modeling for prioritizing therapeutic targets in translational cancer research.

e15073 Background: Colorectal cancer (CRC) remains a significant clinical challenge due to its intrinsic heterogeneity, the paucity of personalized treatment options, and the commonality of chemoresistance. Methods: To better understand drug response dynamics at the single-cell level, we have developed a high-throughput, image-based phenotypic profiling pipeline using CRC patient-derived organoid (PDO) monolayer cultures. Diverse CRC PDOs representing multiple Consensus Molecular Subtypes were treated with a combination of 5-Fluorouracil and Oxaliplatin (FOX), imaged utilizing a biomarker panel identified using transcriptomic data, and analyzed using our newly developed phenotypic profiling pipeline. To further elucidate individual tumor response in vivo, we have also developed protocols to implant our PDOs into mice to create Patient Derived Xenograft (PDX) models. Results: Using our new analysis pipeline, we have identified a subpopulation of CRC cells that, after acute exposure to FOX treatment, exhibit elevated AKT signaling and increased expression of cancer stem cell markers. We observe this subpopulation across multiple PDOs, including across distinct CRC molecular subtypes, suggesting that chemotherapy itself may contribute to the enrichment of drug-resistant phenotypes driven by PI3K/AKT survival signaling. Targeting this survival phenotype, we also demonstrate that transient pre-treatment with the clinically utilized PI3K/mTOR dual inhibitor Dactolisib effectively sensitizes CRC PDOs to FOX, synergizing with this standard-of-care chemotherapy regimen and reducing the fraction of chemoresistant cells. Synergy score profiling revealed that this combination was broadly effective across diverse PDOs, with the strongest response noted in the organoid exhibiting the highest baseline AKT signaling. Preliminary data in our PDX models suggest this combination is synergistic in vivo as well. Conclusions: Our findings highlight the power of personalized organoid-based phenotypic profiling for dissecting molecular mechanisms of therapeutic resistance and support the rationale for transient PI3K/mTOR inhibition as a strategy to improve CRC treatments and outcomes.

Abstract African Americans (AAs) have among the highest incidence and mortality rates of colorectal cancer (CRC) in the US. They present with more right-sided, microsatellite stable (MSS) tumors and are diagnosed at earlier ages than non-Hispanic Whites (NHW). While DNA methylation changes and their significance have been previously described in CRC, much less is known about the unique DNA methylation changes that occur in AA CRCs compared to NHW CRCs. In the current study, we analyzed DNA methylation changes in AA patients and compared those changes to data from NHWs. AA patients diagnosed with a MSS colorectal tumor at the Yale New Health System were included in the study. A pathologist reviewed archival slides, and selected areas from CRCs (n=160) and adjacent normal tissue (n=42) were cored (median age=59; 82 females, 78 males). DNA was bisulfite converted and analysis performed using EPIC arrays. The 450K methylation array from NHW CRCs was also analyzed for 217 CRCs and 20 normal tissues obtained from TCGA (median age=68; 103 females, 114 males). Differentially methylated regions (DMRs) with |Δβ|>0.1, FDR corrected p-value <0.05 between tumor and normal tissue were identified using SeSAMe. We identified 4881 DMRs (57% hyper- and 44% hypomethylated) between AA CRC and normal tissue. Of these, 46% were in CpG Islands, 15% in CpG Shores, 4% in CpG shelves, and 36% in open sea. Most DMRs (71%) were in promoter regions, with 18% in gene bodies, 5% in enhancer regions, and 6% in intergenic regions. Next, we performed additional DMR analysis using overlapping probes from both arrays to compare NHW with AA data. We identified 2239 DMRs between AA CRC and normal tissue compared to 4138 in NHW. Of the 2239 AA DMRs, 1419 (66%) were common with NHW DMRs, 790 (34%) were unique to AAs. There were many overlapping pathways and genes; however, the DMRs impacting these pathways differed between AA and NHW CRCs. Hypermethylation of LCK, a tyrosine kinase involved in T-cell receptor signaling, was found in 60% of AA CRCs compared to 5.9% of NHW CRCs. LCK hypermethylation leads to CD4/8 T-cell inactivation and linked to poor outcomes in lung cancer. We observed AA-specific hypermethylation of SSTR1 in 57.2% of AA CRCs compared to 27.6% of NHW CRCs. Somatostasin (SST) suppresses tumor growth by regulating certain factors involved in cell proliferation. Hypermethylation of SSTR1 has been associated with various malignancies including gastric cancer. Lastly, promoter hypermethylation of 3 protocadherin genes was found in 65.1% of AAs compared to 33.1% NHWs. Protocadherins regulate WNT signaling and hypermethylation is associated with poor outcomes in multiple cancers. Taken together, these results show AA-specific CRC methylation changes compared with NHW CRCs. Further work is warranted to investigate the potential functional and clinical implications of these methylation alterations, some of which may represent therapeutic targets and biomarkers. Citation Format: Seeta Rajpara, David N. Buckley, Rosa Xicola, Reger Mikaeel, Mary K. Yagle, Baris Kerimoglu, Curtis Thorne, Dante A. Bellomo, Megha Padi, Xavier Llor, Nathan Ellis, Bodour Salhia. DNA Methylation analysis of African American colorectal cancers reveal race-specific alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7004.

Abstract Background: The incidence of early-onset colorectal cancer (EO-CRC) has been alarmingly increasing, particularly among non-Hispanic Whites (NHWs), resulting in similar rates to ones in African Americans (AAs). Overall, the median age of diagnosis has decreased from 72 to 66 years. The molecular features that could potentially distinguish this phenotype are still not well-established. This study aimed to identify genetic, epigenetic, and transcriptomic factors associated with EO-CRC. Methods: 510 patients with microsatellite-stable tumors analyzed for somatic mutations, targeting 20 cancer driver genes. We included a cohort of 175 AAs from the Yale New Haven Health System, a previously studied cohort of 51 AAs from the Chicago Colorectal Cancer Consortium, and the publicly available molecular data from TCGA COAD-READ PanCancer cohort (50 AAs and 229 NHWs). We analyzed the transcriptomic and methylation profiles from the Yale cohort using Tempo-SeqTM and Illumina EPIC array, respectively. Results: Of the 510 patients, 18% were diagnosed with EO-CRC. Using a multiple variant analysis, adjusting for sex, race, tumor location, and stage, EO-CRC patients were less likely to be male (OR = 0.59, 95% CI: 0.37-0.94, p-value = 0.03), their tumors lacked mutations in APC (OR = 0.37, 95% CI: 0.22-0.63, p-value = 0.0002), and they were more likely to have mutations in FBXW7 (OR = 3.1, 95% CI: 1.60-6.00, p-value = 0.0007) and BCL9L (OR = 3.1, 95% CI: 1.00-8.72, p-value = 0.04). We compared tumors with and without mutations in FBXW7 or BCL9L in two independently analyses. Combining transcriptomics and methylation data, we identified 10 candidate genes that showed significant gene downregulation with hypermethylation or upregulation with hypomethylation. Among FBXW7 mutant tumors, we found the Cartilage oligomeric matrix protein (COMP) gene was significantly downregulated and hypermethylated. In CRC, COMP has been associated with a crosstalk between the TGF-β pathway and immune cell infiltration. Moreover, inactivation of FBXW7 resulted in the accumulation of phosphorylated TGIF1 molecules and repression of TGFβ-dependent transcription in cancer cell lines. Deficiency of FBXW7 is a key element in the phosphorylation-dependent ubiquitination and subsequent proteasome degradation of oncoproteins, such as c-MYC, NOTCH, and Cyclin E. Among BCL9L mutant tumors, keratin 20 (KRT20), the intermediate filament protein and marker of intestinal differentiation in CRC, was downregulated and hypermethylated. BCL9L plays an important role in tumorigenesis induced by aberrant Wnt signaling or aneuploidy tolerance. Conclusion: Our findings further demonstrate the importance of specific etiologic mechanisms in EO-CRC at the level of somatic alterations, providing new ways to understand the biology of EO-CRC and prospects for the development of targeted therapies. Citation Format: Reger Mikaeel, Dante Bellomo, Seeta Rajpara, David Buckley, Mary Yagle, Baris Kerimoglu, Curtis Thorne, Megha Padi, Bodour Salhia, Nathan A. Ellis, Xavier Llor, Rosa Xicola. FBXW7 and BCL9L altered pathways as hallmarks of early-onset colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4409.

African Americans (AAs) have among the highest incidence and mortality rates of colorectal cancer (CRC) in the US. They present with more right-sided, microsatellite stable tumors and are diagnosed at earlier ages than non-Hispanic Whites (NHW). While DNA methylation changes and their significance have been previously described in CRC, much less is known about the unique DNA methylation changes that occur in AA CRCs compared to NHW CRCs. In the current study, we analyzed DNA methylation changes in AA patients and compared those changes to data from NHWs. AA patients diagnosed with a microsatellite-stable colorectal tumor at the Yale New Health System were included in the study. A pathologist reviewed archival slides, and selected areas from tumor (n=160) and adjacent normal tissue (n=42) were cored. The median age of patients was 59 from 82 females and 78 males. Extracted DNA was bisulfite converted and tested using EPIC arrays. DNA methylation (450K) from NHW CRCs was also analyzed for 217 CRCs and 20 normal tissues obtained from TCGA (median age=70; 114 males, 103 females). Differentially methylated regions (DMRs) with |Db|>0.1, FDR corrected p-value <0.05 between tumor and normal tissue were identified using SeSAMe. We identified 4881 DMRs (57% hyper- and 44% hypomethylated) between AA CRC and normal tissue. Of these, 46% were in CpG Islands, 15% in CpG Shores, 4% in CpG shelves, and 36% in open sea. Most DMRs (71%) were in promoter regions, compared with 18% in gene bodies, 5% in enhancer regions, and 6% in intergenic regions. Next, we performed an additional DMR analysis using overlapping probes from the 450K and EPIC arrays to compare TCGA NHW with Yale AA data. We identified 2290 DMRs between AA CRC and normal tissue compared to 4657 in NHW. Of the 2290 AA DMRs, 1405 (61%) were common with NHW DMRs, 885 (39%) were unique to AAs. There were many overlapping pathways and genes involving metabolic functions, DNA damage response, immune response, and WNT signaling. However, the DMRs impacting these pathways differed between AA and NHW CRCs. Several AA-specific DMRs were associated with immune response and included LCK and GPSM3 genes. Hypermethylation of LCK, a tyrosine kinase involved in T-cell receptor signaling, was found in 60% of AA CRCs compared to 8% of NHW CRCs. LCK hypermethylation leads to CD4/8 T-cell inactivation and is linked to poor outcomes in lung cancer. We also observed AA-specific hypermethylation of GPSM3 in 60% of AA CRCs compared to 18% of NHW CRCs. GPSM3 is associated with CD4/8 T-cell infiltration. Lastly, promoter hypermethylation of 6 protocadherin genes was found in 59% of AAs compared to 20% NHWs. Protocadherins play a role in regulating WNT signaling and hypermethylation has been associated with poor outcomes in multiple cancers. Taken together, these results show AA-specific CRC methylation changes compared with NHW CRCs. Further work is warranted to investigate the potential functional and clinical implications of these methylation alterations, some of which may represent therapeutic targets and biomarkers. Citation Format: David N. Buckley, Rosa Xicola, Mikaeel Reger, Mary K. Yagle, Baris Kerimoglu, Curtis Thorne, Dante Bellomo, Megha Padi, Xavier Llor, Nathan Ellis, Bodour Salhia. DNA Methylation analysis of African American colorectal cancers reveal race-specific alterations [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C060.

Glycoconjugation strategies in anticancer drug discovery exploit the high expression of glucose transporters in malignant cells to achieve preferential uptake and hence attractive pharmacological characteristics of increased therapeutic windows and decreased unwanted toxicity. Here we present the design of glycoconjugated prochelators of aroylhydrazone AH1, an antiproliferative scavenger that targets the increased iron demand of rapidly proliferating malignant cells. The constructs feature a monosaccharide (d-glucose, d-glucosamine, or glycolytic inhibitor 2-deoxy-d-glucose) connected at the C2 or C6 position via a short linker, which masks the chelator through a disulfide bond susceptible to intracellular reduction. Cellular assays showed that the glycoconjugates rely on the GLUT1 transporter for uptake, lead to intracellular iron deprivation, and present antiproliferative activity. Ectopic overexpression of GLUT1 in malignant and normal cells increased the uptake and toxicity of the glycoconjugated prochelators, demonstrating that these compounds are well suited for targeting cells overexpressing glucose transporters and therefore for selective iron sequestration in malignant cells.

Supplementary Data from Cyst(e)inase–Rapamycin Combination Induces Ferroptosis in Both In Vitro and In Vivo Models of Hereditary Leiomyomatosis and Renal Cell Cancer

Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible disease characterized by an increase in differentiation of fibroblasts to myofibroblasts and excessive accumulation of extracellular matrix in lung tissue. Pharmacological activation of NRF2 has proved to be a valuable antifibrotic approach, however the detailed mechanisms of how NRF2 mediates anti-fibrotic function remain unclear. In this study, we found that the anti-fibrotic function of sulforaphane (SFN), an NRF2 activator, was largely dependent on LOC344887, a long noncoding RNA. Two functional AREs were identified in both the promoter and intron 1 of LOC344887, which defines LOC344887 as a novel anti-fibrotic NRF2 target gene. RNA-seq analysis revealed that LOC344887 controls genes and signaling pathways associated with fibrogenesis. Deletion or downregulation of LOC344887 enhanced expression of CDH2/N-cadherin, as well as a number of other fibrotic genes and blunted the anti-fibrotic effects of SFN. Furthermore, LOC344887-mediated downregulation of fibrotic genes may involve the PI3K-AKT signaling pathway, as pharmacologic inhibition of PI3K activity blocked the effects of LOC344887 knockdown. Our findings demonstrate that NRF2-mediated LOC344887 upregulation contributes to the antifibrotic potential of SFN by repressing the expression of CDH2 and other fibrotic genes, providing novel insight into how NRF2 controls the regulatory networks of IPF. This study provides a better understanding of the molecular mechanisms of NRF2 activators against pulmonary fibrosis and presents a novel therapeutic axis for prevention and intervention of fibrosis-related diseases.

OBJECTIVE: S-Allyl-L-cysteine (SAC) is a biological active organosulfur component of garlic and has various pharmacological effects. SAC has displayed anti-cancer activity but the mechanism is unresolved. This study has focused on investigating the possible apoptotic and autophagic effects of SAC on two human leukemia cell lines: acute promyelocytic leukemia (HL-60) and chronic myeloid leukemia (K562).MATERIAL AND METHODS: Cell cytotoxicity was evaluated via MTT test. Bax, Bcl-2, caspase 3, mTOR, AKT, and PI3K gene expression amounts were identified via Real time quantitative reverse transcription polymerase chain reaction (qRT-PCR). HL-60 and K562 cells were incubated with SAC at three diverse doses (5 mM, 10 mM, and 20 mM) (3,75 mM, 7,5 mM, and 15 mM), respectively.RESULTS: SAC caused a cytotoxic effect on HL-60 and K562 cells with IC50 values of approximately 11.525 mM and 10.025 mM, respectively. In HL-60 cells, an increase in Bax expression levels was detected at doses of 5 mM and 10 mM SAC (p=0.027, p=0.000). Treatment with 10 mM SAC increased the expression level of caspase 3 in HL-60 cells as compared with the control and 5 mM SAC treated cells (p=0.000, p=0.020). In K562 cells, SAC induced a significant decrease in mTOR, AKT, and PI3K expression levels in at all doses (p=0.000, p=0.000, p=0.000).CONCLUSIONS: In conclusion, our data indicates that SAC induces autophagy in K562 cells by downregulating the PI3K/AKT/mTOR signaling pathway. Furthermore, increased Bax and caspase 3 gene expression levels suggest that SAC may be an effective active ingredient with which to induce apoptosis in HL-60 cells.

Overcrowding conditions and temperatures shifts regularly manifest in large-scale infections of farmed fish, resulting in economic losses for the global aquaculture industries. Increased understanding of the functional mechanisms of fish antimicrobial host defenses is an important step forward in prevention of pathogen-induced morbidity and mortality in aquaculture setting. Like other vertebrates, macrophage-lineage cells are integral to fish immune responses and for this reason, much of the recent fish immunology research has focused on fish macrophage biology. These studies have revealed notable similarities as well as striking differences in the molecular strategies by which fish and higher vertebrates control their respective macrophage polarization and functionality. In this review, we address the current understanding of the biological mechanisms of teleost macrophage functional heterogeneity and immunity, focusing on the key cytokine regulators that control fish macrophage development and their antimicrobial armamentarium.

Overcrowding conditions and temperatures shifts regularly manifest in large-scale infections of farmed fish, resulting in economic losses for the global aquaculture industries. Increased understanding of the functional mechanisms of fish antimicrobial host defenses is an important step forward in prevention of pathogen-induced morbidity and mortality in aquaculture setting. Like other vertebrates, macrophage-lineage cells are integral to fish immune responses and for this reason, much of the recent fish immunology research has focused on fish macrophage biology. These studies have revealed notable similarities as well as striking differences in the molecular strategies by which fish and higher vertebrates control their respective macrophage polarization and functionality. In this review, we address the current understanding of the biological mechanisms of teleost macrophage functional heterogeneity and immunity, focusing on the key cytokine regulators that control fish macrophage development and their antimicrobial armamentarium.

In the investigation for alternative chemotherapeutic strategies against leukemia, Pd(II) complexes were synthesized and investigated for cytotoxic and apoptotic properties on two human leukemia cell lines (HL-60 and K562). Pd(II) complexes (Pd-5a and Pd-6a) with 5a and 6a as ligands were synthesized and characterized by H-1-NMR and F-TIR. The cytotoxicity of the compounds was quantified using MTT method. Bax, Bcl-2, and caspase 3 gene expression levels were estimated using RT-qPCR. Here we show that Pd(II) complexes have important cytotoxic activity on human leukemia cell lines. RT-qPCR indicated that Bax and caspase 3 gene expression levels were increased after 24h treatment with Pd-5a and Pd-6a complexes in both HL-60 and K562 cells at some selected dose. Furthermore, Bcl-2 gene expression level decreased after 24h treatment with Pd-5a and Pd-6a complexes in K562 cells at all selected dose. In HL-60 cells, only one selected Pd-5a dose (25 mu M) decreased the gene expression level of Bcl-2. The results obtained in the present investigation indicate that these two newly synthesized Pd(II) complexes have apoptotic effects at appropriate doses through caspase 3 and Bax genes and might represent a novel potentially active agents for the management of human leukemia cell lines.

BACKGROUND/AIM DNA topoisomerases are ubiquitous enzymes that regulate conformational changes in DNA topology during essential cellular processes, and, for this reason, have been characterized as the cellular targets of a number of anticancer drugs. Bortezomib is a powerful proteasome inhibitor used in the treatment of hematological malignancies. In this study, we investigated the inhibitory effects of bortezomib on human topoisomerase I and II enzymes both alone and in combination modes with camptothecin and etoposide. MATERIALS AND METHODS The interactions of these drugs with topoisomerase enzymes were evaluated by relaxation assay in cell-free systems. IC50 values of the drugs on topoisomerase enzymes were calculated using the S probit analysis program. RESULTS Bortezomib showed a very weak inhibition effect on topoisomerase I (IC50 = 87.11 mM). On the other hand, it had a strong inhibitory effect on topoisomerase II (IC50 = 1.41 mM). Our results indicated that bortezomib is effective not only on proteasome but also on topoisomerase II. In addition, bortezomib possesses an increased synergistic effect when used in combination with camptothecin and etoposide than when used alone. CONCLUSION The results of this study point out that these data may build a framework for combination studies with bortezomib, camptothecin, and etoposide in the treatment of cancer.