Chemoresistance In Patients Research Articles

USP1 deubiquitinates PARP1 to regulate its trapping and PARylation activity.

PARP inhibitors (PARPi) represent a game-changing treatment for patients with ovarian cancer with tumors deficient for the homologous recombination (HR) pathway treated with platinum (Pt)-based therapy. PARPi exert their cytotoxic effect by both trapping PARP1 on the damaged DNA and by restraining its enzymatic activity (PARylation). How PARP1 is recruited and trapped at the DNA damage sites and how resistance to PARPi could be overcome are still matters of investigation. Here, we described PARP1 as a substrate of the deubiquitinase USP1. At molecular level, USP1 binds PARP1 to remove its K63-linked polyubiquitination and controls PARP1 chromatin trapping and PARylation activity, regulating sensitivity to PARPi. In both Pt/PARPi-sensitive and -resistant cells, USP1/PARP1 combined blockade enhances replicative stress, DNA damage, and cell death. Our work dissected the biological interaction between USP1 and PARP1 and recommended this axis as a promising and powerful therapeutic choice for not only sensitive but also chemoresistant patients with ovarian cancer irrespective of their HR status.

Targeting ketone body metabolism in mitigating gemcitabine resistance.

Chemotherapy is often combined with surgery for muscle invasive and non-muscle invasive bladder cancer. However, 70% of the patients recur within 5 years. Metabolic reprogramming is an emerging hallmark in cancer chemoresistance. Here, we report a gemcitabine resistance mechanism which promotes cancer reprogramming via the metabolic enzyme, OXCT1. This mitochondrial enzyme, responsible for the rate-limiting step in β-hydroxybutyrate (βHB) catabolism, was elevated in muscle invasive disease and in chemo-resistant bladder cancer patients. Resistant orthotopic tumors presented an OXCT1-dependent rise in mitochondrial oxygen consumption rate, ATP, and nucleotide biosynthesis. In resistant bladder cancer, knocking out OXCT1 restored gemcitabine sensitivity, and administering the non-metabolizable βHB, enantiomer (S-βHB) only partially restored gemcitabine sensitivity. Suggesting an extra-metabolic role for OXCT1, multi-omics analysis of gemcitabine sensitive and resistant cells revealed an OXCT1-dependent signature with the transcriptional repressor, OVOL1, as a master regulator of epithelial differentiation. The elevation of OVOL1 target genes was associated with its cytoplasmic translocation and poor prognosis in a chemotherapy-treated BCa patient cohort. The knockout of OXCT1 restored OVOL1 transcriptional repressive activity by its nuclear translocation. Orthotopic mouse models of bladder cancer supported OXCT1 as a mediator of gemcitabine sensitivity through ketone metabolism and regulating cancer stem cell differentiation.

Targeting a chemo-induced adaptive signaling circuit confers therapeutic vulnerabilities in pancreatic cancer

Advanced pancreatic ductal adenocarcinomas (PDACs) respond poorly to all therapies, including the first-line treatment, chemotherapy, the latest immunotherapies, and KRAS-targeting therapies. Despite an enormous effort to improve therapeutic efficacy in late-stage PDAC patients, effective treatment modalities remain an unmet medical challenge. To change the status quo, we explored the key signaling networks underlying the universally poor response of PDAC to therapy. Here, we report a previously unknown chemo-induced symbiotic signaling circuit that adaptively confers chemoresistance in patients and mice with advanced PDAC. By integrating single-cell transcriptomic data from PDAC mouse models and clinical pathological information from PDAC patients, we identified Yap1 in cancer cells and Cox2 in stromal fibroblasts as two key nodes in this signaling circuit. Co-targeting Yap1 in cancer cells and Cox2 in stroma sensitized PDAC to Gemcitabine treatment and dramatically prolonged survival of mice bearing late-stage PDAC, whereas simultaneously inhibiting Yap1 and Cox2 only in cancer cells was ineffective. Mechanistically, chemotherapy triggers non-canonical Yap1 activation by nemo-like kinase in 14-3-3ζ-overexpressing PDAC cells and increases secretion of CXCL2/5, which bind to CXCR2 on fibroblasts to induce Cox2 and PGE2 expression, which reciprocally facilitate PDAC cell survival. Finally, analyses of PDAC patient data revealed that patients who received Statins, which inhibit Yap1 signaling, and Cox2 inhibitors (including Aspirin) while receiving Gemcitabine displayed markedly prolonged survival compared to others. The robust anti-tumor efficacy of Statins and Aspirin, which co-target the chemo-induced adaptive circuit in the tumor cells and stroma, signifies a unique therapeutic strategy for PDAC.

The Impact and Mechanism of Action of Peptostreptococcus anaerobius on Chemotherapy Resistance in Human Colorectal Cancer.

Peptostreptococcus anaerobius plays an important role in the development of colorectal cancer, and previous studies by our group have demonstrated that Peptostreptococcus anaerobius promotes resistance to 5-Fu chemotherapy in animal models of colorectal cancer. In this study, the effects of Peptostreptococcus anaerobius on chemotherapy resistance in colorectal cancer and its possible mechanism of action were investigated from the clinical point of view. Patients were selected according to exclusion and inclusion criteria and divided into sensitive and chemotherapy groups (n = 20/group). Fecal samples were collected from the patients. The bacterial 16S rRNA genes in the samples were sequenced and the abundance and varieties in the fecal bacteria were compared between the 2 groups. Immunohistochemistry and Western blotting were used to assess interleukin 23 levels in tumor tissues. Significantly elevated abundance of Peptostreptococcus was observed in fecal samples from chemoresistant colorectal cancer patients compared to those from chemosensitive individuals. Immunohistochemistry and Western blotting results showed that chemoresistant patients had higher levels of interleukin 23 relative to chemosensitive patients and the levels were positively associated with Peptostreptococcus. Peptostreptococcus may mediate the development of chemoresistant colorectal cancer by promoting the upregulation of interleukin 23. Efforts to target Peptostreptococcus thus have the potential to alter the prognosis of colorectal cancer patients.

GATA factor TRPS1, a new DNA repair protein, cooperates with reversible PARylation to promote chemoresistance in patients with breast cancer

Resistance to DNA-damaging agents is a major unsolved challenge for breast cancer patients undergoing chemotherapy. Here, we show that elevated expression of transcriptional repressor GATA binding 1 (TRPS1) is associated with lower drug sensitivity, reduced response rate, and poor prognosis in chemotherapy-treated breast cancer patients. Mechanistically, elevated TRPS1 expression promotes hyperactivity of DNA damage repair (DDR) in breast cancer cells. We provide evidence that TRPS1 dynamically localizes to DNA breaks in a Ku70- and Ku80-dependent manner, and that TRPS1 is a new member of the DDR protein family. We also discover that the dynamics of TRPS1 assembly at DNA breaks is regulated by its reversible PARylation in the DDR, and that mutations of the PARylation sites on TRPS1 lead to increased sensitivity to chemotherapeutic drugs. Taken together, our findings provide new mechanistic insights into the DDR and chemoresistance in breast cancer patients and identify TRPS1 as a critical DDR protein. TRPS1 may also be considered as a target to improve chemo-sensitization strategies and, consequently, clinical outcomes for breast cancer patients.

Single-cell analysis of chemotherapy-resistant microenvironment identifies a chemo-response biomarker for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal, aggressive cancer due to limited response chemotherapy. The tumor microenvironment (TME) has emerged as a key player in the development of chemoresistance and in malignant progression. In this study, we hypothesized that chemotherapy response is predictable by chemotherapy-related cell types and their differentially expressed genes (DEGs). DEGs of chemoresistance cell types were identified via single cell analysis and Wilcoxon test. A chemotherapy response signature was established using a random forest model and validated with public datasets. Bulk cell fraction was analyzed using BayesPrism algorithms. Log-rank test was used to analyze survival of PDAC patients. We found that natural killer (NK) cells, myeloid cells, and erythroid cells were highly infiltrated in chemo-resistant TME. A total of 36 chemoresistance-related DEGs of chemo-resistant cells were identified in chemo-resistant PDAC. Functional enrichment analysis showed that chemoresistance upregulated various inflammation-related pathways, including TGF-β signaling. Based on these features, we constructed a random forest model to predict the response and survival for PDAC patients, which accurately distinguished high-risk and chemoresistant patients with significantly poorer prognosis in both the training and independent validation datasets. Cox regression analysis indicated that predicted labels were an independent prognostic factor in PDAC. Moreover, deconvolution of TME confirmed higher infiltration levels of M2 macrophage and NK cells in predicted chemoresistance. When combined with chemotherapy response related tumor mutations, the model showed excellent ability in predicting chemotherapy response and survival. The TME was closely associated with the chemotherapy response and prognosis of PDAC. Our TME-based random forest model predicted chemotherapy response with complementary knowledge to the response-related genetic mutations.

Single-cell RNA sequencing and cell-cell communication analysis reveal tumor microenvironment associated with chemotherapy responsiveness in ovarian cancer.

To investigate the impact of the tumor microenvironment (TME) on the responsiveness to chemotherapy in ovarian cancer (OV). We integrated single cell RNA-seq datasets of OV containing chemo-response information, and characterize their clusters based on different TME sections. We focus on analyzing cell-cell communication to elaborate on the mechanisms by which different components of the TME directly influence the chemo-response of tumor cells. scRNA-seq datasets were annotated according to specific markers for different cell types. Differential analysis of malignant epithelial cells revealed that chemoresistance was associated with the TME. Notably, distinct TME components exhibited varying effects on chemoresistance. Enriched SPP1+ tumor-associated macrophages in chemo-resistant patients could promote chemoresistance through SPP1 binding to CD44 on tumor cells. Additionally, the overexpression of THBS2 in stromal cells could promote chemoresistance through binding with CD47 on tumor cells. In contrast, GZMA in the lymphocytes could downregulate the expression of PARD3 through direct interaction with PARD3, thereby attenuating chemoresistance in tumor cells. Our study indicates that the non-tumor cell components of the TME (e.g. SPP1+ TAMs, stromal cells and lymphocytes) can directly impact the chemo-response of OV and targeting the TME was potentially crucial in chemotherapy of OV.

CACNA1H restrains chemotherapy resistance in ovarian clear cell carcinoma cells by repressing autophagy.

Ovarian clear cell carcinoma (OCCC) is a subtype of ovarian cancer and is highly malignant with high chemoresistance. CACNA1H is pivotal in tumor development. However, the role of CACNA1H in the acquisition process of chemotherapeutic resistance in OCCC cells is rarely reported. Therefore, this study aimed to explore the role of CACNA1H in chemotherapy resistance of OCCC cells and its related mechanism. Based on bioinformatics analysis, we found that CACNA1H was downregulated in chemoresistant OCCC patients compared to chemosensitive OCCC patients. Comparing DDP-resistant and sensitive OCCC cell lines, the resistant strain showed lower CACNA1H mRNA expression. CACNA1H expression was associated with calcium signaling pathways in chemoresistant OCCC patients. CACNA1H mRNA expression was significantly downregulated in OCCC cells compared to normal ovarian epithelial cells. When CACNA1H was overexpressed, intracellular Ca2+ concentration and protein levels of p-CaMKII and p-Akt were significantly upregulated, while protein levels of LC3-II/LC3-I and Beclin1 were downregulated, indicating a repression of autophagy. The rescue experiment revealed that CACNA1H overexpression in drug-resistant OCCC cells reduced autophagy-induced DDP resistance via CaMKII/Akt signaling. Overall, CACNA1H increased intracellular Ca2+ concentration and activated CaMKII/Akt signaling pathway in OCCC, thereby repressing autophagy to maintain the sensitivity of OCCC cells to DDP.

Targeting estrogen metabolism in high-grade serous ovarian cancer shows promise to overcome platinum resistance

The high mortality rate due to chemoresistance in patients with high-grade ovarian cancer (HGSOC) emphasizes the urgent need to determine optimal treatment strategies for advanced and recurrent cases. Our study investigates the interplay between estrogens and chemoresistance in HGSOC and shows clear differences between platinum-sensitive and -resistant tumors. Through comprehensive transcriptome analyzes, we uncover differences in the expression of genes of estrogen biosynthesis, metabolism, transport and action underlying platinum resistance in different tissues of HGSOC subtypes and in six HGSOC cell lines. Furthermore, we identify genes involved in estrogen biosynthesis and metabolism as prognostic biomarkers for HGSOC. Additionally, our study elucidates different patterns of estrogen formation/metabolism and their effects on cell proliferation between six HGSOC cell lines with different platinum sensitivity. These results emphasize the dynamic interplay between estrogens and HGSOC chemoresistance. In particular, targeting the activity of steroid sulfatase (STS) proves to be a promising therapeutic approach with potential efficacy in limiting estrogen-driven cell proliferation. Our study reveals potential prognostic markers as well as identifies novel therapeutic targets that show promise for overcoming resistance and improving treatment outcomes in HGSOC.

MicroRNA-605-3p Inhibited the Growth and Chemoresistance of Osteosarcoma Cells via Negatively Modulating RAF1.

Osteosarcoma (OS) is the leading cancer-associated mortality in childhood and adolescence. Increasing evidence has demonstrated the key function of microRNAs (miRNAs) in OS development and chemoresistance. Among them, miRNA-605-3p acted as an important tumor suppressor and was frequently down-regulated in multiple cancers. However, the function of miR-650-3p in OS has not been reported. The aim of this work is to explore the novel role of miR-605-3p in osteosarcoma and its possible involvement in OS chemotherapy resistance. The expression levels of miR-605-3p in OS tissues and cells were assessed by reverse transcription quantitative PCR (RT-qPCR). The relevance of miR-605-3p with the prognosis of OS patients was determined by the Kaplan-Meier analysis. Additionally, the influence of miR-605-3p on OS cell growth was analyzed using the cell counting kit-8, colony formation assay, and flow cytometry. The mRNA and protein expression of RAF1 were detected by RT-qPCR and western blot. The binding of miR-605-3p with the 3'-UTR of RAF1 was confirmed by dual-luciferase reporter assay. Our results showed that miR-605-3p was markedly decreased in OS tissues and cells. A lower level of miR-605-3p was strongly correlated with lymph node metastasis and poor 5-year overall survival rate of OS patients. In vitro assay found that miR-605-3p suppressed OS cell proliferation and promoted cell apoptosis. Mechanistically, the proto-oncogene RAF1 was seen as a target of miR-605-3p and strongly suppressed by miR-605-3p in OS cells. Restoration of RAF1 markedly eliminated the inhibitory effect of miR-605-3p on OS progression, suggesting RAF1 as a key mediator of miR-605-3p. Consistent with the decreased level of RAF1, miR-605-3p suppressed the activation of both MEK and ERK in OS cells, which are the targets of RAF1. Moreover, lower levels of miR-605-3p were found in chemoresistant OS patients, and downregulated miR-605-3p increased the resistance of OS cells to therapeutic agents. Our data revealed that miR-605-3p serves as a tumor suppressor gene by regulating RAF1 and increasing the chemosensitivity of OS cells, which provided the novel working mechanism of miR-605-3p in OS. Engineering stable nanovesicles that could efficiently deliver miR-605-3p with therapeutic activity into tumors could be a promising therapeutic approach for the treatment of OS.

Cell surface CD55 traffics to the nucleus leading to cisplatin resistance and stemness by inducing PRC2 and H3K27 trimethylation on chromatin in ovarian cancer

BackgroundPlatinum resistance is the primary cause of poor survival in ovarian cancer (OC) patients. Targeted therapies and biomarkers of chemoresistance are critical for the treatment of OC patients. Our previous studies identified cell surface CD55, a member of the complement regulatory proteins, drives chemoresistance and maintenance of cancer stem cells (CSCs). CSCs are implicated in tumor recurrence and metastasis in multiple cancers.MethodsProtein localization assays including immunofluorescence and subcellular fractionation were used to identify CD55 at the cell surface and nucleus of cancer cells. Protein half-life determinations were used to compare cell surface and nuclear CD55 stability. CD55 deletion mutants were generated and introduced into cancer cells to identify the nuclear trafficking code, cisplatin sensitivity, and stem cell frequency that were assayed using in vitro and in vivo models. Detection of CD55 binding proteins was analyzed by immunoprecipitation followed by mass spectrometry. Target pathways activated by CD55 were identified by RNA sequencing.ResultsCD55 localizes to the nucleus of a subset of OC specimens, ascites from chemoresistant patients, and enriched in chemoresistant OC cells. We determined that nuclear CD55 is glycosylated and derived from the cell surface pool of CD55. Nuclear localization is driven by a trafficking code containing the serine/threonine (S/T) domain of CD55. Nuclear CD55 is necessary for cisplatin resistance, stemness, and cell proliferation in OC cells. CD55 S/T domain is necessary for nuclear entry and inducing chemoresistance to cisplatin in both in vitro and in vivo models. Deletion of the CD55 S/T domain is sufficient to sensitize chemoresistant OC cells to cisplatin. In the nucleus, CD55 binds and attenuates the epigenetic regulator and tumor suppressor ZMYND8 with a parallel increase in H3K27 trimethylation and members of the Polycomb Repressive Complex 2.ConclusionsFor the first time, we show CD55 localizes to the nucleus in OC and promotes CSC and chemoresistance. Our studies identify a therapeutic mechanism for treating platinum resistant ovarian cancer by blocking CD55 nuclear entry.

Adrenergic microenvironment driven by cancer-associated Schwann cells contributes to chemoresistance in patients with lung cancer.

Doublecortin (DCX)-positive neural progenitor-like cells are purported components of the cancer microenvironment. The number of DCX-positive cells in tissues reportedly correlates with cancer progression; however, little is known about the mechanism by which these cells affect cancer progression. Here we demonstrated that DCX-positive cells, which are found in all major histological subtypes of lung cancer, are cancer-associated Schwann cells (CAS) and contribute to the chemoresistance of lung cancer cells by establishing an adrenergic microenvironment. Mechanistically, the activation of the Hippo transducer YAP/TAZ was involved in the acquisition of new traits of CAS and DCX positivity. We further revealed that CAS express catecholamine-synthesizing enzymes and synthesize adrenaline, which potentiates the chemoresistance of lung cancer cells through the activation of YAP/TAZ. Our findings shed light on CAS, which drive the formation of an adrenergic microenvironment by the reciprocal regulation of YAP/TAZ in lung cancer tissues.

Exosome secretion related gene signature predicts chemoresistance in patients with colorectal cancer

BackgroundColorectal cancer (CRC) is a highly heterogeneous malignancy, and patients often have different responses to treatment. In this study, the genetic characteristics related to exosome formation and secretion procedure were used to predict chemoresistance and guide the individualized treatment of patients. MethodsFirstly, seven microarray datasets in Gene Expression Omnibus (GEO) and RNA-Seq dataset from the Cancer Genome Atlas (TCGA) were used to analysis the transcriptome profiles and associated characteristics of CRC patients. Then, a predictive model based on gene features linked to exosome formation and secretion was created and validated using Least Absolute Shrinkage and Selection Operator (LASSO) regression analysis and Support Vector Machine-Recursive Feature Elimination (SVM-RFE) machine learning. Finally, we evaluated the model using chemoresistant/chemosensitive cells and tissues by immunofluorescence (IF), western blot (WB), quantitative real-time PCR (qRT-PCR) and immunocytochemistry (IHC) experiments, and the predictive value of integrated model in the clinical validation cohort were performed by Receiver Operating Characteristic (ROC) and Kaplan-Meier (K-M) curves analyses. ResultsWe established a risk score signature based on three genes related to exosome secretion in CRC. Better Overall Survival (OS) and greater chemosensitivity were seen in the low-risk group, whereas the high-risk group exhibited chemoresistance and a subpar response to immune checkpoint blockade (ICB) therapy. Higher expression of the model genes EXOC2, EXOC3 and STX4 were observed in chemoresistant cells and specimens. The AUC of 5-year disease-free survival (DFS) was 0.804. Compared with that in the low-risk group, patients' DFS was found to be significantly worse in the high-risk group. ConclusionsIn summary, the gene signature related to exosome formation and secretion could reliably predict patients' chemosensitivity and ICB treatment response, which providing new independent biomarkers for the treatment of CRC.

TSPAN8+ myofibroblastic cancer-associated fibroblasts promote chemoresistance in patients with breast cancer.

Cancer-associated fibroblasts (CAFs) are abundant stromal cells in the tumor microenvironment that promote cancer progression and relapse. However, the heterogeneity and regulatory roles of CAFs underlying chemoresistance remain largely unclear. Here, we performed a single-cell analysis using high-dimensional flow cytometry analysis and identified a distinct senescence-like tetraspanin-8 (TSPAN8)+ myofibroblastic CAF (myCAF) subset, which is correlated with therapeutic resistance and poor survival in multiple cohorts of patients with breast cancer (BC). TSPAN8+ myCAFs potentiate the stemness of the surrounding BC cells through secretion of senescence-associated secretory phenotype (SASP)-related factors IL-6 and IL-8 to counteract chemotherapy. NAD-dependent protein deacetylase sirtuin 6 (SIRT6) reduction was responsible for the senescence-like phenotype and tumor-promoting role of TSPAN8+ myCAFs. Mechanistically, TSPAN8 promoted the phosphorylation of ubiquitin E3 ligase retinoblastoma binding protein 6 (RBBP6) at Ser772 by recruiting MAPK11, thereby inducing SIRT6 protein destruction. In turn, SIRT6 down-regulation up-regulated GLS1 and PYCR1, which caused TSPAN8+ myCAFs to secrete aspartate and proline, and therefore proved a nutritional niche to support BC outgrowth. By demonstrating that TSPAN8+SIRT6low myCAFs were tightly associated with unfavorable disease outcomes, we proposed that the combined regimen of anti-TSPAN8 antibody and SIRT6 activator MDL-800 is a promising approach to overcome chemoresistance. These findings highlight that senescence contributes to CAF heterogeneity and chemoresistance and suggest that targeting TSPAN8+ myCAFs is a promising approach to circumvent chemoresistance.

Abstract 2495: CCL3 predicts an exceptional response to TGFβ inhibition in pancreatic ductal adenocarcinoma by sustaining a basal-like ecotype enriched in LIF-producing macrophages

Abstract The TGFβ receptor inhibitor galunisertib (GAL) exhibited promising efficacy in patients with pancreatic ductal adenocarcinoma (PDAC) in the randomized phase 2 H9H-MC-JBAJ study. However, the identification of predictive biomarkers remains of unique importance. We used a 279 multi-analyte panel and confirmed CCL3 as the most significant plasma protein for chemoresistance in patients receiving gemcitabine (GEM) [overall survival (mOS) (95%CI), high vs low, 3.6 (2.5-4.0) vs 10.1 (7.2-17.0) months, HR (95%CI)= 3.92 (2.14-7.20); p<0.001]. GAL dramatically reverted this resistance and CCL3 was the most significant predictive marker for the combination of this agent plus GEM [mOS (95%CI), GAL + GEM vs. placebo + GEM, 9.0 (5.5-12.1) vs 3.6 (2.5-4.0) months, HR (95%CI)= 0.39 (0.23-0.68); p<0.001]. We delved into the mechanisms behind this exceptional response in PDAC with this prognostically negative ecotype sustained by CCL3 in 6 different murine PDAC models. Mice bearing high-Ccl3 tumors had a significantly shorter mOS, a higher infiltration of M2-macrophages (TAMs), and a more mesenchymal/basal-like phenotype than did models with undetectable Ccl3.Ccl3 was stably expressed in DT4313 and PAN610, and silenced in RC416 cells. In co-culture models, DT4313Ccl3 and PAN610Ccl3 had a more mesenchymal phenotype and induced a M2 polarization of RAW264.7 murine TAMs than did controls. Mice bearing DT4313Ccl3 or PAN610Ccl3 tumors had a significantly shorter mOS, an higher infiltration of M2-polarized TAMs and a more mesenchymal/basal-like phenotype than controls. The expression of Tgfβ1 ligand and the phosphorylation of Smad2 were triggered only in TAMs if co-cultured with Ccl3-high RC416scr, DT4313Ccl3 or PAN610Ccl3 cells. This activation of Tgfβ pathway was blocked by maraviroc, a selective inhibitor of Ccl3-receptor Ccr5. In co-culture, GAL reverted the mesenchymal phenotype of PDAC cells and the M2 polarization of TAMs. By using an 111 different cytokine immunoassay, Leukemia Inhibitory Factor (Lif) was the most significantly factor overexpressed by TAMs if stimulated by Ccl3. GAL suppressed this Lif expression. In mice bearing DT4313Ccl3 tumors GEM was ineffective, whereas it prolonged mOS in those with DT4313NTC tumors. Conversely, GAL plus GEM doubled mOS duration vs. GEM, modulated M2- TAMs infiltration, the mesenchymal/basal-like phenotype and plasma Lif only in mice bearing DT4313Ccl3 tumors, but not in those with DT4313NTC controls. In summary, we demonstrated that tumor-derived Ccl3 activates an autocrine Tgfβ signaling in TAMs, triggering their M2-phenotype and secretion of Lif. This Tgfβ-Lif axis sustains paracrinally a mesenchymal/basal-like PDAC ecotype resistant to GEM. Inhibition of Tgfβ signaling modulates this poor prognosis ecotype, and explains the exceptional response to GAL in patients with high-CCL3 PDAC. Citation Format: Silvia Pietrobono, Monica Bertolini, Veronica De Vita, Enza Scarlato, Federica Fazzini, Fabio Sabbadini, Domenico Mangiameli, Alberto Quinzii, Simona Casalino, Davide Melisi. CCL3 predicts an exceptional response to TGFβ inhibition in pancreatic ductal adenocarcinoma by sustaining a basal-like ecotype enriched in LIF-producing macrophages [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 2495.

Abstract 1994: Drug-induced cancer cell secretome promotes resistance in colon cancer

Abstract Colorectal cancer (CRC) is the third most common cancer in the world, with a projection of increased lethality in the upcoming years. Adjuvant chemotherapy with biological agents is the most practiced standard of care today. However, patients with metastatic CRC (mCRC) do not benefit from the wide range of therapies available for CRC, which either owe to unresectability or co-morbidity or more importantly resistance to therapy. 5-fluorouracil (5-FU) combined with leucovorin, oxaliplatin, or irinotecan (FOLFOX or FOLFIRI) has been routinely employed as the first-line therapy in colon cancer, but patients often develop resistance to 5-FU. Additionally, tumor cell dormancy plays a major role in tumor relapse which leads to poor prognosis in the patients. Despite many advances, the understanding of therapy-induced resistance is still in its infancy. Here, we show that 5-FU-induced tumor cell secretome helps in the outgrowth and metastasis of 5-FU-resistant clones in colon cancer. We used parental and 5-FU-resistant colon cancer cell lines, and transgenic zebrafish embryos to test the hypothesis and perform the colon cancer cell-based functional assays. We observed that 5-FU resistant (5-FU-R) colon cancer cells showed an increase in proliferation and survival when cultured with the secretome of 5-FU exposed colon cancer cells compared to the DMSO-induced secretome. Interestingly, we also noted elevated migration and invasion in the 5-FU-R colon cancer cells exposed to 5-FU-induced secretome compared to the DMSO counterpart. Additionally, in zebrafish xenograft, we found that 5-FU-R colon cancer cells cultured with 5-FU-induced secretome showed higher tail-vein metastatic burden compared to the 5-FU-R cells cultured with DMSO-induced secretome. This provides a background to further study in detail the therapy-induced resistance as well as dormancy in colorectal cancer which will help to develop approaches to prevent or reverse chemoresistance in patients who receive systemic therapy for mCRC. Citation Format: Shakti Ranjan Satapathy, Souvik Ghatak, Anita Sjölander. Drug-induced cancer cell secretome promotes resistance in colon 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 1994.

Abstract 4671: Efficacy and mechanism of mebendazole for ovarian cancer therapy

Abstract Introduction: Ovarian cancer (OvCa) is the most lethal gynecological malignancy. Although most OvCa patients initially respond to frontline platinum-based therapies with approximately 80% of patients experiencing remission, approximately 75% of those women experience a recurrence with only approximately 50% of patients alive 5 years following diagnosis. There is a critical need to develop novel therapeutic agents or methods to overcome chemoresistance and toxicities experienced with conventional chemotherapy regimens used for OvCa patients. Repurposing drugs is a promising strategy to safely and more rapidly evaluate potentially effective treatment regimens. Over the past two decades, Mebendazole (MBZ), an anti-parasitic drug, has gained much attention in oncology due to its favorable biosafety profile and potent anti-cancerous activity seen in several human malignancies. Therefore, we examined the efficiency and mechanism of MBZ against OvCa. Methods: The in vitro efficacy of MBZ was evaluated in cisplatin-resistant (CPR) and parent (WT/sensitive) OVCAR-8 cell lines as well as in ascites samples collected from chemo-resistant OvCa patients by using 2D and/or 3D cell viability assay and invasion assay. The in vivo efficacy of MBZ was assessed in orthotopic animal models, utilizing CPR-OVCAR8 spheroids, and patient-derived xenograft (PDX) animal models with specimens from chemo-resistant ovarian cancer patients. Molecular mechanisms were investigated through western blot and immunofluorescence analysis. Result: Our results confirmed that MBZ efficiently decreased cell viability, spheroid size, and the invasion ability of OvCa cell lines and ascites samples. OvCa cells treated with MBZ demonstrated downregulation of proliferation markers and upregulation of apoptosis markers indicating inhibition of cell proliferation and induction of apoptosis. Mechanistically, MBZ inhibited the Wnt/β-catenin signaling pathway by downregulating the expression and nuclear localization of β-catenin and epithelial-mesenchymal transition (EMT) markers. Preclinical studies showed that MBZ at 50 mg/kg/day dose for 28 days significantly reduced tumor growth in PDXs and orthotopic tumor models without any evidence of toxicity. Conclusion: Collectively, our study strongly supports the therapeutic potential of MBZ against OvCa particularly, which warrants further clinical studies. Citation Format: Rajani Rai, Dhanamjai Penta, Debasish Dey, Doris M Benbrook, Lin Wang, Magdalena Bieniasz, Lauren E Dockery. Efficacy and mechanism of mebendazole for ovarian cancer therapy [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 4671.

Abstract 5870: Taxane-based chemotherapy leads to acquired resistance through ecDNA-based amplification of multi-drug resistance genes

Abstract Focal amplification on extrachromosomal DNA (ecDNA) drives high gene copy number and intercellular genomic heterogeneity, which can fuel tumor adaptation and drug resistance. Targeted therapies have largely failed in patients with oncogene amplification, and patients whose tumors harbor ecDNA-based gene amplification have particularly worse survival compared to patients whose cancers harbor other genetic drivers. Due to the lack of approved therapies for patients with oncogene amplified cancers, many patients receive chemotherapy; however, resistance often develops through mechanisms such as ATP-binding cassette (ABC) transporter overexpression, rendering cross-resistance to other forms of chemotherapy. We examined whether ecDNA amplification in tumor cell lines drives ABC transporter overexpression to mediate resistance to paclitaxel, a commonly prescribed taxane chemotherapy for cancer patients. We selected non-amplified (DLD1 and SW48) and amplified cell lines, including COLO320DM and COLO320HSR (MYC amplified on ecDNA or chromosomal loci, respectively); SNU16 and H716, (FGFR2 and MYC amplified ecDNA); and H2170 (ERBB2 and MYC amplified on ecDNA). All cell lines developed paclitaxel resistance, with the majority (5/7) gaining focal amplification of ABCB1, which encodes P-glycoprotein (P-gp), a common multi-drug resistance transporter. Four models–COLO320DM, COLO320HSR, SNU16, and H2170—developed ABCB1 amplification on ecDNA; one non-amplified cell line (DLD1) acquired focal ABCB1 amplification via a breakage-fusion-bridge (BFB) mechanism, a distinct form of amplification from ecDNA. ecDNA-based ABCB1 amplifications exhibited hallmarks of ecDNA, including intercellular copy number heterogeneity and paclitaxel dose-dependent rapid evolution of ABCB1 copy number. We next assessed whether drug resistance was reversible in ABCB1 amplified cell lines following removal of paclitaxel. Reversal of resistance was observed in COLO320DM and COLO320HSR via loss of ABCB1 ecDNA within 4-6 weeks. However, in SNU16 and H2170 resistant cells, ABCB1 amplification was retained up to 12 weeks of drug removal, with apparent integration into MYC ecDNA, demonstrating that ecDNA-enabled plasticity can result in both short- and long-term paclitaxel resistance. Although targeted therapies have revolutionized treatment approaches for patients with actionable oncodriver alterations, most cancer patients do not have actionable drivers and most patients still receive chemotherapy at some point in their course of treatment. We demonstrated that ecDNA-based resistance to paclitaxel is common in cancer cell lines and may also drive resistance to chemotherapy in patients. Thus, ecDNA-directed therapies (ecDTx) may be an important treatment option for chemo-resistant patients, who currently do not have many effective therapeutic options. Citation Format: Kristen Holmes, Dean Perusse, Cory DuPai, Homa Hemmati, Julie Weiss, Claire Kuelbs, John Bibay, Joan Chen, Debbie Liao, Christian Hassig, Shailaja Kasibhatla. Taxane-based chemotherapy leads to acquired resistance through ecDNA-based amplification of multi-drug resistance genes [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 5870.

Abstract 2019: Primary cancer associated fibroblasts increase the chemoresistance of triple negative breast cancer cells by mitochondria metabolic upregulation

Abstract Breast cancers (BCs) are the most prevalent cancers occurring in women. Tumor reoccurrence attributed to chemotherapy resistance remains a main challenge in fighting disease relapse. Cancer associated fibroblasts (CAFs) are the most prominent component of the tumor microenvironment (TME) and have already been implicated in therapy resistances. Although the exact molecular mechanisms behind this resistance remain unclear. In this regard, we recently identified 4 CAF populations (S1-S4) in ovarian and BCs, and showed that only CAF-S1 promote immunotherapy resistance as well as metastasis. Based on these observations, we are now investigating the impact of CAF-S1 on the chemotherapy response of BCs. To study the interactions between CAF-S1 and BC cells, we developed a tumor on chip (ToC) device, mimicking the TME in 3D. Using ToC live imaging, we show that primary CAF-S1 mediate a chemoprotective effect over luminal and triple negative BC (TNBC) cells by significantly decreasing their apoptosis rate as early as 10h after Doxorubicin and 40h after Paclitaxel treatment. Interestingly, CAF-S1 display a highly heterogenous response to Doxorubicin, allowing the classification into chemo-sensitive and -resistant CAF-S1. Regardless of premature CAF-S1 cell death, the chemoprotective effect over BC cells is maintained for at least 3 days. This indicates a crucial CAF-S1 - cancer cell cross talk at early timepoints. Additionally, by applying machine learning algorithms on live imaging videos, we demonstrate that physical proximity of CAF-S1 to BC cells immediately after treatment initiation correlates with BC cell survival, even after several days of treatment. This again implies cancer cell priming by CAF-S1 as a possible mechanism of chemotherapy resistance. To investigate this molecular interplay further, we performed scRNA-seq of ToC extracted and treatment naïve CAF-S1 and TNBC cells as well as bulk RNA-seq of patient derived TNBC tumors before/after chemotherapy treatment. We identify mitochondrial metabolic pathways to be upregulated in TNBC cells after CAF-S1 co-culture inside ToC devices and in TNBC cells of chemo-resistant patients enriched in CAF-S1 at baseline. We are now validating the impact of target gene knock downs on the survival of TNBC cells in CAF-S1 co-cultures after chemotherapy treatment on chip. Overall, these results provide insights into molecular mechanisms by which CAF-S1 promote chemotherapy resistance of breast cancers. Citation Format: Isabella Hofer, Arianna Mencattini, Yann Kieffer, Eugenio Martinelli, Maria Carla Parrini, Fatima Mechta-Grigoriou. Primary cancer associated fibroblasts increase the chemoresistance of triple negative breast cancer cells by mitochondria metabolic upregulation [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 2019.

Histidine re-sensitizes pediatric acute lymphoblastic leukemia to 6-mercaptopurine through tetrahydrofolate consumption and SIRT5-mediated desuccinylation

Despite progressive improvements in the survival rate of pediatric B-cell lineage acute lymphoblastic leukemia (B-ALL), chemoresistance-induced disease progression and recurrence still occur with poor prognosis, thus highlighting the urgent need to eradicate drug resistance in B-ALL. The 6-mercaptopurine (6-MP) is the backbone of ALL combination chemotherapy, and resistance to it is crucially related to relapse. The present study couples chemoresistance in pediatric B-ALL with histidine metabolism deficiency. Evidence was provided that histidine supplementation significantly shifts the 6-MP dose-response in 6-MP-resistant B-ALL. It is revealed that increased tetrahydrofolate consumption via histidine catabolism partially explains the re-sensitization ability of histidine. More importantly, this work provides fresh insights into that desuccinylation mediated by SIRT5 is an indispensable and synergistic requirement for histidine combination therapy against 6-MP resistance, which is undisclosed previously and demonstrates a rational strategy to ameliorate chemoresistance and protect pediatric patients with B-ALL from disease progression or relapse.