Development Of Chemoresistance Research Articles

Metformin as an Enhancer for the Treatment of Chemoresistant CD34+ Acute Myeloid Leukemia Cells.

Acute myeloid leukemia is the second most frequent type of leukemia in adults. Due to a high risk of development of chemoresistance to first-line chemotherapy, the survival rate of patients in a 5-year period is below 30%. One of the reasons is that the AML population is heterogeneous, with cell populations partly composed of very primitive CD34+CD38- hematopoietic stem/progenitor cells, which are often resistant to chemotherapy. First-line treatment with cytarabine and idarubicin fails to inhibit the proliferation of CD34+CD38- cells. In this study, we investigated Metformin's effect with or without first-line conventional chemotherapy, or with other drugs like venetoclax and S63845, on primitive and undifferentiated CD34+ AML cells in order to explore the potential of Metformin or S63845 to serve as adjuvant therapy for AML. We found that first-line conventional chemotherapy treatment inhibited the growth of cells and arrested the cells in the S phase of the cell cycle; however, metformin affected the accumulation of cells in the G2/M phase. We observed that CD34+ KG1a cells respond better to lower doses of cytarabine or idarubicin in combination with metformin. Also, we determined that treatment with cytarabine, venetoclax, and S63845 downregulated the strong tendency of CD34+ KG1a cells to form cell aggregates in culture due to the downregulation of leukemic stem cell markers like CD34 and CD44, as well as adhesion markers. Also, we found that idarubicin slightly upregulated myeloid differentiation markers, CD11b and CD14. Treatment with cytarabine, idarubicin, venetoclax, metformin, and S63845 upregulated some cell surface markers like HLA-DR expression, and metformin upregulated CD9, CD31, and CD105 cell surface marker expression. In conclusion, we believe that metformin has the potential to be used as an adjuvant in the treatment of resistant-to-first-line-chemotherapy AML cells. Also, we believe that the results of our study will stimulate further research and the potential use of changes in the expression of cell surface markers in the development of new therapeutic strategies.

Enhancement of vincristine sensitivity in retinoblastoma through Janus kinase inhibition by ruxolitinib.

Chemotherapy remains the main approach conserving vision during the treatment of retinoblastoma, the most prevalent eye cancer in children. Unfortunately, the development of chemoresistance stands as the primary reason for treatment failure. Within this study, we showed that prolonged exposure to vincristine led to heightened expression of JAK1 and JAK2 in retinoblastoma cells, while the other members of the JAK family exhibited no such changes. Employing a genetic intervention, we demonstrated the efficacy of depleting either JAK1 or JAK2 in countering vincristine-resistant retinoblastoma cells. In addition, the dual depletion of both JAK1 and JAK2 produced a more potent inhibitory outcome compared to the depletion of either gene alone. We further demonstrated that ruxolitinib, a small molecular inhibitor of JAK1/2, effectively reduced viability and colony formation in vincristine-resistant retinoblastoma cells. It also acts synergistically with vincristine in retinoblastoma cells regardless of inherent cellular and genetic heterogeneity. The effectiveness of ruxolitinib as standalone treatment against chemoresistant retinoblastoma, as well as its combination with vincristine, was validated in multiple retinoblastoma mouse models. Importantly, mice exhibited favorable tolerance to ruxolitinib administration. We confirmed that the underlying mechanism of ruxolitinib's action in chemoresistant retinoblastoma cells is the inhibition of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling. Our study reveals that the underlying mechanism driving ruxolitinib's impact on chemoresistant retinoblastoma cells is the inhibition of JAK/STAT signaling. This study reveals the contribution of JAK1/2 to the development of chemoresistance in retinoblastoma and underscores the effectiveness of targeting JAK1/2 as a strategy to sensitize retinoblastoma to chemotherapy.

Abstract PO1-23-08: Exosome-based delivery of microRNAs confers adriamycin-resistance to sensitive cells through modulating the immune and metabolism-related gene PTEN in HER2-negative breast cancer

Abstract Background: Anthracycline-based chemotherapy is widely used to treat breast cancer. However, acquired drug resistance remains a challenge to successful treatment. Recently, increasing evidence has shown that changes in the tumour immune microenvironment (TIME), in addition to increasing drug resistance of tumour cells, consistently contribute to the development of chemoresistance. Methods: TIME scores and tumor-infiltrating immune cells (TICs) scores were used to investigate the prognosis, clinicopathological characteristics and gene transcriptome profiling of HER2-negative breast cancer patients who received anthracycline-based chemotherapy. Exosomes isolated from MCF/7-ADR (ADR-exos) and MCF/7-S (S-exos) cell lines were characterized. We identified dysregulated miRNAs using miRNA microarray analysis on MCF/7-ADR cells and their exosomes compared to MCF/7-S cells and their exosomes. Protein profiling was performed to identify differentially expressed proteins in up- and down-regulated miR-222 cells. The ability of ADR-exos to transfer drug-resistance mediated by miR-222 was assessed by immunofluorescence assay, flow cytometry and qRT-PCR. We optimized the conditions to load miR-222 mimic (or inhibitor) into exosomes produced by HBL-100 cells (H-exos). The influence of miR-222 inhibitor-containing exosomes (inhibitor-exos) on the downstream pathway of miR-222 and the potential therapeutic effects both in vitro and in vivo were evaluated. We also evaluated the relationship between miR-222/PTEN and drug resistance in circulating exosomes from mice after adriamycin-based chemotherapy. Results: Firstly, we discovered the complicated and heterogenous TICs subtypes and their unique biological behaviors in HER2-negative breast cancer. Then, we identified 85 differentially expressed immunologic signature gene sets and 7202 corresponding immune-related genes in three subtypes quantified by gene set variation analysis (GSVA). We identified 12 up-regulated and 13 down-regulated genes using miRNA microarray analysis in adriamycin-resistant cells and their exosomes. The TMT mass spectrometry found 45 differentially expressed proteins affected by the miR-222 level. Moreover, we successfully established miRNA mimic/inhibitor-containing exosomes to explore the solo function of exosomal miR-222 in adriamycin-resistance both in vitro and in vivo. More importantly, for the first time, we detected the levels of miR-222 and its target gene PTEN in circulating exosomes from mice after adriamycin-based chemotherapy and explored the potential clinical implications of miR-222/PTEN-containing exosomes in chemotherapy and immune suppression of breast cancer patients. Conclusions: HER2-negative breast cancer patients own unique TIME subtypes, leading to different outcomes of chemotherapy. We exhibit the potential applications of miR-222-mimic/inhibitor-containing exosomes for reversing chemo-resistance. Adriamycin-resistant cells can transmit drug-resistance to sensitive cells via delivering miR-222 by modulating the immune- and metabolism-related target gene PTEN both in vitro and in vivo without interference from other relevant drug-resistance factors in exosomes. Therefore, miR-222-containing exosomes as well as their target gene PTEN may be a promising biomarker for predicting the efficacy of chemo-resistance and tumor immunity regulation in breast cancer patients receiving anthracycline-based chemotherapy. Keywords: exosomes, breast cancer, tumor immune microenvironment, chemo-resistance, microRNAs Citation Format: Sujin Yang, Tian-cheng Cheng, Jia-hao Wu, Wei-xian Chen. Exosome-based delivery of microRNAs confers adriamycin-resistance to sensitive cells through modulating the immune and metabolism-related gene PTEN in HER2-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-23-08.

Analysis of chemoresistance characteristics and prognostic relevance of postoperative gemcitabine adjuvant chemotherapy in pancreatic cancer.

To investigate the relationship between chemoresistance in pancreatic cancer patients receiving postoperative gemcitabine adjuvant therapy and specific clinical/pathological characteristics, as well as its impact on patient prognosis. From June 2018 to June 2021, clinical and pathological data of 148 pancreatic cancer patients were collected, and 101 patients were followed up for tumor recurrence/metastasis and survival status. The correlation between chemoresistance and specific clinical/pathological characteristics or patient prognosis was retrospectively analyzed. Of the 148 patients, 78 were in the chemoresistance group and 70 in the non-chemoresistance group. Univariate analysis showed that the development of chemoresistance may be related to patient age, combined diabetes, preoperative CA19-9 level, tumor size, AJCC stage, vascular invasion, and positive lymph node ratio. Furthermore, subsequent multivariate analysis incorporating these variables indicated that tumor size may be a key factor influencing chemoresistance (p < 0.001, OR = 1.584). Log-rank test showed patients in the chemoresistance group had worse overall survival (OS) (HR = 2.102, p = 0.018) and progression free survival (PFS) (HR = 3.208, p = 0.002) than patients in the non-chemoresistance group; and patients with smaller size tumors (diameter ≤3 cm) had significantly better OS (HR = 2.923, p < 0.001) and PFS (HR = 2.930, p = 0.003) than those with larger size tumors (diameter >3 cm). Patients with pancreatic cancer receiving postoperative gemcitabine adjuvant therapy are more likely to develop chemoresistance when their tumor sizes are larger (diameter >3 cm). Development of chemoresistance exacerbates the prognosis of patients with pancreatic cancer, and larger tumor size is also a risk factor for poor prognosis in these patients.

CircPDIA3/miR-449a/XBP1 feedback loop curbs pyroptosis by inhibiting palmitoylation of the GSDME-C domain to induce chemoresistance of colorectal cancer

Although oxaliplatin (OXA) is widely used in the frontline treatment of colorectal cancer (CRC), CRC recurrence is commonly observed due to OXA resistance. OXA resistance is associated with a number of factors, including abnormal regulation of pyroptosis. It is therefore important to elucidate the abnormal regulatory mechanism underlying pyroptosis. Here, we identified that the circular RNA circPDIA3 played an important role in chemoresistance in CRC. CircPDIA3 could induce chemoresistance in CRC by inhibiting pyroptosis both in vitro and in vivo. Mechanistically, RIP, RNA pull-down and co-IP assays revealed that circPDIA3 directly bonded to the GSDME-C domain, subsequently enhanced the autoinhibitory effect of the GSDME-C domain through blocking the GSDME-C domain palmitoylation by ZDHHC3 and ZDHHC17, thereby restraining pyroptosis. Additionally, it was found that the circPDIA3/miR-449a/XBP1 positive feedback loop increased the expression of circPDIA3 to induce chemoresistance. Furthermore, our clinical data and patient-derived tumor xenograft (PDX) models supported the positive association of circPDIA3 with development of chemoresistance in CRC patients. Taken together, our findings demonstrated that circPDIA3 could promote chemoresistance by amplifying the autoinhibitory effect of the GSDME-C domain through inhibition of the GSDME-C domain palmitoylation in CRC. This study provides novel insights into the mechanism of circRNA in regulating pyroptosis and providing a potential therapeutic target for reversing chemoresistance of CRC.

PH-responsive targeted nanoparticles release ERK-inhibitor in the hypoxic zone and sensitize free gemcitabine in mutant K-Ras-addicted pancreatic cancer cells and mouse model.

Therapeutic options for managing Pancreatic ductal adenocarcinoma (PDAC), one of the deadliest types of aggressive malignancies, are limited and disappointing. Therefore, despite suboptimal clinical effects, gemcitabine (GEM) remains the first-line chemotherapeutic drug in the clinic for PDAC treatment. The therapeutic limitations of GEM are primarily due to poor bioavailability and the development of chemoresistance resulting from the addiction of mutant-K-RAS/AKT/ERK signaling-mediated desmoplastic barriers with a hypoxic microenvironment. Several new therapeutic approaches, including nanoparticle-assisted drug delivery, are being investigated by us and others. This study used pH-responsive nanoparticles encapsulated ERK inhibitor (SCH772984) and surface functionalized with tumor-penetrating peptide, iRGD, to target PDAC tumors. We used a small molecule, SCH772984, to target ERK1 and ERK2 in PDAC and other cancer cells. This nanocarrier efficiently released ERKi in hypoxic and low-pH environments. We also found that the free-GEM, which is functionally weak when combined with nanoencapsulated ERKi, led to significant synergistic treatment outcomes in vitro and in vivo. In particular, the combination approaches significantly enhanced the GEM effect in PDAC growth inhibition and prolonged survival of the animals in a genetically engineered KPC (LSL-KrasG12D/+/LSL-Trp53R172H/+/Pdx-1-Cre) pancreatic cancer mouse model, which is not observed in a single therapy. Mechanistically, we anticipate that the GEM efficacy was increased as ERKi blocks desmoplasia by impairing the production of desmoplastic regulatory factors in PDAC cells and KPC mouse tumors. Therefore, 2nd generation ERKi (SCH 772984)-iRGD-pHNPs are vital for the cellular response to GEM and denote a promising therapeutic target in PDAC with mutant K-RAS.

Knowledge mapping and research trends of exosomes in pancreatic cancer: a bibliometric analysis and review (2013-2023).

This review aims to provide a quantitative and qualitative bibliometric analysis of literature from 2013 to 2023 on the role of exosomes in PC, with the goal of identifying current trends and predicting future hotspots. We retrieved relevant publications concerning exosomes in PC, published between 2013 and 2023, from the Web of Science Core Collection. Bibliometric analyses were conducted using VOSviewer(1.6.19), CiteSpace(6.2.R4), and Microsoft Excel (2019). A total of 624 papers were analyzed, authored by 4017 researchers from 55 countries/regions and 855 institutions, published in 258 academic journals. China (n=285, 34.42%) and the United States (n=183, 24.87%) were the most frequent contributors and collaborated closely. However, publications from China had a relatively low average number of citations (41.45 times per paper). The output of Shanghai Jiao Tong University ranked first, with 28 papers (accounting for 4.5% of the total publications). Cancers (n=31, 4.9%); published the most papers in this field. Researcher Margot Zoeller published the most papers (n=12) on this topic. Research hotspots mainly focused on the mechanisms of exosomes in PC onset and progression, the role of exosomes in PC early diagnosis and prognosis, exosomes promote the development of PC chemoresistance, and potential applications of exosomes as drug carriers for PC therapies. We observed a shift in research trends, from mechanistic studies toward clinical trials, suggesting that clinical applications will be the focus of future attention. Emerging topics were pancreatic stellate cells, diagnostic biomarkers, mesenchymal stem cells, extracellular vesicles. Our scientometric and visual analysis provides a comprehensive overview of the literature on the role of exosomes in PC published during 2013-2023. This review identifies the frontiers and future directions in this area over the past decade, and is expected to provide a useful reference for researchers in this field.

Plasmonic laser-responsive BioDissolve 3D-printed graphene@cisplatin-implant for prevention of post-surgical relapse of oral cancer

The development of chemoresistance is a major obstacle in post-surgical adjuvant therapy of cancer, leading to cancer cell survival, recurrence, and metastasis. This study reports a 3D-printed plasmonic implant developed for the post-surgical adjuvant therapy of cisplatin-resistant cancer cells to prevent relapse. The implant was printed using optimized biomaterial ink containing biodegradable polymers [poly(L-lactide) and hydroxypropyl methylcellulose] blended suitably with laser-responsive graphene and chemo drug (Cisplatin). The irradiation of scar-driven 3D-printed implant with a laser stimulates graphene to generate a series of hyperthermia events leading to photothermolysis of cisplatin-resistant cancer cells under the combined influence of sustained cisplatin release. The developed personalized implant offers pH-responsive sustained drug release for 28 days. The implant exhibited acceptable biophysical properties (Tensile strength: 3.99 ± 0.15 MPa; modulus: 81 ± 9.58 MPa; thickness: 110 μm). The 3D-printed implant effectively reverses the chemoresistance in cisplatin-resistant 3D spheroid tumor models. Cytotoxicity assay performed using cisplatin-resistant (CisR) cell line revealed that the cell viability was reduced to 39.80 ± 0.68 % from 61.37 ± 0.98 % in CisR tumor spheroids on combined chemo-photothermal therapy. The combination therapy reduced the IC50 value from 71.05 μM to 48.73 μM in CisR spheroids. Apoptosis assay revealed an increase in the population of apoptotic cells (35.45 ± 1.56 % →52.53 ± 2.30 %) on combination therapy. A similar trend was observed in gene expression analysis, where the expression of pro-apoptotic genes Caspase 3 (3.73 ± 0.04 fold) and Bcl-2-associated X protein (BAX) (3.35 ± 0.02 fold) was increased on combination therapy. This 3D-printed, biodegradable implant with chemo-combined thermal ablating potential may provide a promising approach for the adjuvant treatment of resistant cancer.

Abstract LB317: TNBC cell co-culture with primary human lung fibroblasts identifies a paracrine-juxtacrine signaling loop promoting fibroblast activation and autophagy-dependent breast cancer cell therapy resistance

Abstract Triple negative breast cancer (TNBC) carries a poorer prognosis than other breast cancer subtypes due to high metastatic incidence, lack of molecular drug targets, and rapid development of chemoresistance. Cancer-associated fibroblasts (CAFs) are key extrinsic drivers of chemoresistance in primary TNBC tumors, but it is unknown whether resident fibroblasts in distant metastatic tissues also influence TNBC drug sensitivity. Since the lung is a top metastatic site in TNBC, we developed a breast cancer cell (BCC) - primary human donor-derived lung fibroblast (LF) co-culture model to identify therapeutic vulnerabilities and mechanisms of fibroblast-mediated extrinsic chemoresistance in lung-metastatic TNBC. We characterized the growth and chemoresistance phenotypes of BCC co-cultured with primary LFs via bioluminescence and fluorescence imaging and evaluated their transcriptional and secretory profiles using bulk and scRNAseq and multiplex cytokine analysis. Co-culture of human BCC and primary LFs resulted in BCC molecular subtype-dependent changes in cell growth and drug resistance compared with conventional monoculture. Moreover, we also observed increased LF secretion of the immunomodulatory cytokines IL-6 and CCL2 in response to both BCC co-culture and BCC-conditioned media. Transcriptomic analysis of LFs sorted from co-culture identified upregulation of pro-inflammatory and pro-fibrotic iCAF and myCAF-like gene signatures, while co-cultured TNBC cells upregulated interferon response-mediated multi-therapy resistance genes and cellular dormancy signatures. We applied this model in a high throughput screen of 846 kinase inhibitor compounds, which identified reproducible differential BCC responses to kinase inhibitors between monoculture and co-culture conditions. When co-cultured with LFs, BCC were broadly more resistant to kinase inhibitor compounds but showed increased sensitivity to inhibitors of the lipid kinase VPS34, an important initiator of autophagy. We subsequently established that autophagy is increased in co-cultured TNBC cells and contributes to LF-mediated resistance to kinase inhibitors. Moreover, we determined that while paracrine signals from BCC can promote LF activation indicated by enhanced cytokine secretion, cell-cell contact is required for LF modulation of TNBC therapy responses in co-culture. Thus, we conclude that breast cancer cell co-culture with primary lung fibroblasts is a scalable in vitro model that can facilitate therapeutic discovery and mechanistic evaluation of extrinsic modulation of therapy responses in metastatic breast cancer, and further demonstrate that BCC can promote paracrine activation of co-cultured LFs in association with juxtacrine induction of BCC autophagy and extrinsic therapy resistance. Citation Format: Marika L. Klosowski, Kathryn Cronise, Claire Stratton, Qiong Zhou, Hector Esquer, Daniel LaBarbera, Daniel Regan. TNBC cell co-culture with primary human lung fibroblasts identifies a paracrine-juxtacrine signaling loop promoting fibroblast activation and autophagy-dependent breast cancer cell therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB317.

Efficient CAR T cell targeting of the CA125 extracellular repeat domain of MUC16

BackgroundOvarian cancer (OC) is the leading cause of death from gynecologic malignancies in the Western world. Contributing factors include a high frequency of late-stage diagnosis, the development of chemoresistance, and...

Deep-targeted gene sequencing reveals ARID1A mutation as an important driver of glioblastoma.

To investigate the key factors influencing glioma progression and the emergence of treatment resistance by examining the intrinsic connection between mutations in DNA damage and repair-related genes and the development of chemoresistance in gliomas. We conducted a comprehensive analysis of deep-targeted gene sequencing data from 228 glioma samples. This involved identifying differentially mutated genes across various glioma grades, assessing their functions, and employing I-TASSER for homology modeling. We elucidated the functional changes induced by high-frequency site mutations in these genes and investigated their impact on glioma progression. The analysis of sequencing mutation results of deep targeted genes in integration revealed that ARID1A gene mutation occurs frequently in glioblastoma and alteration of ARID1A could affect the tolerance of glioma cells to temozolomide treatment. The deletion of proline at position 16 in the ARID1A protein affected the stability of binding of the SWI/SNF core subunit BRG1, which in turn affected the stability of the SWI/SNF complex and led to altered histone modifications in the CDKN1A promoter region, thereby affecting the biological activity of glioma cells, as inferred from modeling and protein interaction analysis. The ARID1A gene is a critical predictive biomarker for glioma. Mutations at the ARID1A locus alter the stability of the SWI/SNF complex, leading to changes in transcriptional regulation in glioma cells. This contributes to an increased malignant phenotype of GBM and plays a pivotal role in mediating chemoresistance.

Role and mechanism of COL3A1 in regulating the growth, metastasis, and drug sensitivity in cisplatin-resistant non-small cell lung cancer cells

ABSTRACT Non-small cell lung cancer (NSCLC) is among the most difficult malignancies to treat. Type III collagen (COL3A1) can affect the progression and chemoresistance development of NSCLC. We herein explored the mechanism that drives COL3A1 dysregulation in NSCLC. Potential RNA-binding proteins (RBPs) and transcription factors (TFs) that could bind to COL3A1 were searched by bioinformatics. mRNA expression was detected by quantitative PCR. Protein expression was evaluated using immunoblotting and immunohistochemistry. The effects of the variables were assessed by gauging cell growth, invasiveness, migratory capacity, apoptosis, and cisplatin (DDP) sensitivity. The direct YY1/COL3A1 relationship was confirmed by ChIP and luciferase reporter experiments. Xenograft experiments were done to examine COL3A1’s function in DDP efficacy. COL3A1 showed enhanced expression in DDP-resistant NSCLC. In H460/DDP and A549/DDP cells, downregulation of COL3A1 exerted inhibitory functions in cell growth, invasiveness, and migration, as well as promoting effects on cell DDP sensitivity and apoptosis. Mechanistically, ELAV-like RNA binding protein 1 (ELAVL1) enhanced the mRNA stability and expression of COL3A1, and Yin Yang 1 (YY1) promoted the transcription and expression of COL3A1. Furthermore, upregulation of COL3A1 reversed ELAVL1 inhibition- or YY1 deficiency-mediated functions in DDP-resistant NSCLC cells. Additionally, COL3A1 downregulation enhanced the anti-tumor efficacy of DDP in vivo. Our investigation demonstrates that COL3A1 upregulation, induced by both RBP ELAVL1 and TF YY1, exerts important functions in phenotypes of NSCLC cells with DDP resistance, offering an innovative opportunity in the treatment of drug-resistant NSCLC.

Nanosystem Delivers Senescence Activators and Immunomodulators to Combat Liver Cancer.

CD47 blockade has emerged as a promising immunotherapy against liver cancer. However, the optimization of its antitumor effectiveness using efficient drug delivery systems or combinations of therapeutic agents remains largely incomplete. Here, patients with liver cancer co-expressing CD47 and CDC7 (cell division cycle 7, a negative senescence-related gene) are found to have the worst prognosis. Moreover, CD47 is highly expressed, and senescence is inhibited after the development of chemoresistance, suggesting that combination therapy targeting CD47 and CDC7 to inhibit CD47 and induce senescence may be a promising strategy for liver cancer. The efficacy of intravenously administered CDC7 and CD47 inhibitors is limited by low uptake and short circulation times. Here, inhibitors are coloaded into a dual-targeted nanosystem. The sequential release of the inhibitors from the nanosystem under acidic conditions first induces cellular senescence and then promotes immune responses. In an in situ liver cancer mouse model and a chemotherapy-resistant mouse model, the nanosystem effectively inhibited tumor growth by 90.33% and 85.15%, respectively. Overall, the nanosystem in this work achieved the sequential release of CDC7 and CD47 inhibitors in situ to trigger senescence and induce immunotherapy, effectively combating liver cancer and overcoming chemoresistance.

Abstract 3951: Timing the development of chemoresistance in relapsed pediatric cancer

Abstract Survivors of pediatric cancer face lifelong battles with severe morbidities, which includes a significant risk of recurrence. Pre-existing genetic variation within primary tumors may offer certain cell populations an evolutionary advantage, increasing the likelihood that some will be resistant to treatment. But the inherent complexity of the tumor means finding these markers of resistance has yet to be fully explored across childhood cancer. To address this, we developed a pipeline that combines clonal evolution reconstruction with mutational signature extraction at the subclonal level to elucidate changes in mutational processes. Using this pipeline to analyze 1,743 pediatric tumor genomes we found a common pattern of parallel evolution in most pediatric cancers, irrespective of their tissue of origin. Further, we detected therapy-associated mutational signatures linked to specific treatments such as temozolomide, platinum, fluorouracil, and thiopurine in post-treatment tumor subpopulations. Of 235 samples with known treatment exposure, 37.9% displayed one or more treatment-associated signatures. In the context of the phylogenetic tree, we identified specific subpopulations with therapy signatures. In multi-sample cases, resistant subpopulations had ancestors present in primary tumors at the time of diagnosis, suggesting certain subpopulations possessed the ability to withstand chemotherapy-induced pressures from an early stage and then expanded following treatment. Finally, we observed differences in the timing of mutational signatures between antimetabolite and alkylating agent treatments, reflecting the mechanisms of these drugs. Antimetabolite agents showed subclonal mutational signatures occurring in later branches, likely due to dependence on cell cycling. In contrast, alkylating agents displayed signatures in both early and late subpopulations, as they are unaffected by cell cycle state. Thus, investigating mutational signatures at the subclonal level unveils new insights into the clonal dynamics of pediatric cancers and the development of chemoresistance. This research offers valuable insights for identifying pediatric cancer patients at greater risk of recurrence, enabling the development of more effective treatment plans to enhance their quality of life and overall survival rates. Citation Format: Sasha Blay, Mehdi Layeghifard, Scott Davidson, David Chen, Astra Schwertschkow, Vijay Ramaswamy, Michael Taylor, Elli Papaemmanuil, Anita Villani, David Malkin, Ludmil Alexandrov, Mark Cowley, Adam Shlien. Timing the development of chemoresistance in relapsed pediatric 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 3951.

Abstract 7155: Dichloroacetate reverses cisplatin resistance in ovarian cancer through promoting ROS production

Abstract Chemotherapy resistance and the high rate of ovarian cancer relapse pose major challenges in the treatment of ovarian cancer. The development of chemoresistance contributes to poor overall prognosis associated with ovarian cancer and high cancer-related mortality in women worldwide. Herein, we found that Dichloroacetate (DCA), a pan-pyruvate dehydrogenase kinase (PDK) inhibitor, is a potent agent to overcome Cisplatin (CP) resistance in ovarian cancer treatment. Firstly, we elucidated that phosphorylation of pyruvate dehydrogenase (pPDH) was elevated in CP-resistant ovarian cancer cells. Then, blockage of pPDH by DCA treatment significantly inhibited cell migratory and invasive capacity in CP-resistant ovarian cancer cells through the transition of motile mesenchymal phenotypes into epithelial characteristics. DCA markedly decreased the number of sphere formations and attenuated the stem cell-like properties in CP-resistant ovarian cancer cells. In combination with CP treatment, DCA effectively inhibited cell proliferation in chemoresistant ovarian cancer cells by triggering apoptosis. Collectively, we suggested that DCA is a promising drug to reverse CP resistance for ovarian cancer treatment. We further tested the underlying mechanisms of DCA to restore CP sensitivity in ovarian cancer. We found that CP treatment promoted reactive oxygen species (ROS) production to induce cell death in CP-sensitive ovarian cancer. However, CP-resistant ovarian cancer cells exhibited higher basal levels of ROS compared with sensitive cancer cells, and CP treatment failed to generate ROS production. That means ovarian cancer cells steadily adapted to elevated ROS levels, then becoming ROS-adapted ovarian cancer cells to survive. Interestingly, DCA treatment results in the buildup of ROS production, causing cell death in CP-resistant ovarian cancer. Therefore, our study suggests that ROS production can be an attractive target in DCA treatment to restore CP sensitivity in ovarian cancer. Citation Format: Yen Thi Do, Seungmee Lee, Changmin Shin, Hyewon Chung, Jin Young Kim, Eunyoung Ha, Sojin Shin, Ji Hae Seo. Dichloroacetate reverses cisplatin resistance in ovarian cancer through promoting ROS production [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 7155.

Abstract 278: Investigating the functional role of cancer-associated fibroblasts in the emergence of drug-tolerant persisters in ovarian cancer

Abstract Chemoresistance remains a major clinical challenge for the treatment of high-grade serous ovarian cancer (HGSC), the most lethal gynecologic malignancy. The majority of patients who initially respond to first-line platinum/taxane-based chemotherapy eventually relapse with chemoresistant disease, leading to poor survival outcomes. It is increasingly recognized that cancer cells can enter a reversible drug-tolerant persister (DTP) state through non-genetic mechanisms to evade chemotherapy-induced cytotoxicity prior to the development of chemoresistance. However, how environmental signals and extracellular factors from the tumor microenvironment drive the process remains poorly understood. Cancer-associated fibroblasts (CAFs) are a functionally heterogeneous population of activated fibroblasts. CAFs play a critical role in shaping the tumor microenvironment and tumor behaviors, including promoting tumor growth and mediating therapy resistance. Here, we demonstrated that HGSC cell lines co-cultured with patient-derived FAP-high CAFs, a subpopulation of CAFs previously identified in our lab to be tumor-promoting, significantly increase the formation of DTPs upon prolonged carboplatin treatment compared to the mono-cultured condition. Interestingly, the extracellular matrix (ECM) organization signature is enriched in FAP-high CAFs compared to their FAP-low counterparts, and we found that ECM deposited by FAP-high CAFs also facilitates the emergence of DTPs in HGSC cells. Future work will focus on elucidating the mechanisms by which FAP-high CAFs and the associated ECM promote DTP formation in HGSC cells through transcriptomic and proteomic profiling. This will aid the identification of potential therapeutic vulnerabilities to target cancer cells in the DTP state in FAP-high HGSC patients. Citation Format: Angel S. Ng, Ngoc Bui, Laurie Ailles. Investigating the functional role of cancer-associated fibroblasts in the emergence of drug-tolerant persisters in ovarian 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 278.

Competitive endogenous RNA networks: Decoding the role of long non-coding RNAs and circular RNAs in colorectal cancer chemoresistance.

Colorectal cancer (CRC) is recognized as one of the most common gastrointestinal malignancies across the globe. Despite significant progress in designing novel treatments for CRC, there is a pressing need for more effective therapeutic approaches. Unfortunately, many patients undergoing chemotherapy develop drug resistance, posing a significant challenge for cancer treatment. Non-coding RNAs (ncRNAs) have been found to play crucial roles in CRC development and its response to chemotherapy. However, there are still gaps in our understanding of interactions among various ncRNAs, such as long non-coding RNAs (lncRNAs), circular RNAs (circRNAs) and microRNAs (miRNAs). These ncRNAs can act as either oncogenes or tumour suppressors, affecting numerous biological functions in different cancers including CRC. A class of ncRNA molecules known as competitive endogenous RNAs (ceRNAs) has emerged as a key player in various cellular processes. These molecules form networks through lncRNA/miRNA/mRNA and circRNA/miRNA/mRNA interactions. In CRC, dysregulation of ceRNA networks has been observed across various cellular processes, including proliferation, apoptosis and angiogenesis. These dysregulations are believed to play a significant role in the progression of CRC and, in certain instances, may contribute to the development of chemoresistance. Enriching our knowledge of these dysregulations holds promise for advancing the field of diagnostic and therapeutic modalities for CRC. In this review, we discuss lncRNA- and circRNA-associated ceRNA networks implicated in the emergence and advancement of drug resistance in colorectal carcinogenesis.

Trilobolide-6-O-isobutyrate from Sphagneticola trilobata acts by inducing oxidative stress, metabolic changes and apoptosis-like processes by caspase 3/7 activation of human lung cancer cell lines

BackgroundLung cancer, a chronic and heterogeneous disease, is the leading cause of cancer-related death on a global scale. Presently, despite a variety of available treatments, their effectiveness is limited, often resulting in considerable toxicity and adverse effects. Additionally, the development of chemoresistance in cancer cells poses a challenge. Trilobolide-6-O-isobutyrate (TBB), a natural sesquiterpene lactone extracted from Sphagneticola trilobata, has exhibited antitumor effects. Its pharmacological properties in NSCLC lung cancer, however, have not been explored. PurposeThis study evaluated the impact of TBB on the A549 and NCI-H460 tumor cell lines in vitro, examining its antiproliferative properties and initial mechanisms of cell death. MethodsTBB, obtained at 98 % purity from S. trilobata leaves, was characterized using chromatographic techniques. Subsequently, its impact on inhibiting tumor cell proliferation in vitro, TBB-induced cytotoxicity in LLC-MK2, THP-1, AMJ2-C11 cells, as well as its effects on sheep erythrocytes, and the underlying mechanisms of cell death, were assessed. ResultsIn silico predictions have shown promising drug-likeness potential for TBB, indicating high oral bioavailability and intestinal absorption. Treatment of A549 and NCI-H460 human tumor cells with TBB demonstrated a direct impact, inducing significant morphological and structural alterations. TBB also reduced migratory capacity without causing toxicity at lower concentrations to LLC-MK2, THP-1 and AMJ2-C11 cell lines. This antiproliferative effect correlated with elevated oxidative stress, characterized by increased levels of ROS, superoxide anion radicals and NO, accompanied by a decrease in antioxidant markers: SOD and GSH. TBB-stress-induced led to changes in cell metabolism, fostering the accumulation of lipid droplets and autophagic vacuoles. Stress also resulted in compromised mitochondrial integrity, a crucial aspect of cellular function. Additionally, TBB prompted apoptosis-like cell death through activation of caspase 3/7 stressors. ConclusionThese findings underscore the potential of TBB as a promising candidate for future studies and suggest its viability as an additional component in the development of novel anticancer drugs prototypes.

Assessment of the Efficacy of the Combination of RNAi of lncRNA DANCR with Chemotherapy to Treat Triple Negative Breast Cancer Using Magnetic Resonance Molecular Imaging.

Long noncoding RNA (lncRNA) differentiation antagonizing noncoding RNA (DANCR) is overexpressed in human triple-negative breast cancer (TNBC) and promotes cell migration and proliferation. TNBC is limited in treatment options relative to hormone-receptor-positive breast cancer and is commonly treated with chemotherapy, which is often compromised by acquired resistance. DANCR has been implicated in the development of chemoresistance across multiple cancer types. Here, we applied magnetic resonance molecular imaging (MRMI) with a targeted contrast agent, MT218, specific to extradomain-B fibronectin (EDB-FN), a marker for epithelial-to-mesenchymal transition, to assess the therapeutic efficacy of the combination of paclitaxel and ZD2-PEG-ECO/siDANCR nanoparticles (ZD2-siDANCR-ELNP) to treat TNBC. The treatment of orthotopic MDA-MB-231 TNBC in mice with paclitaxel significantly suppressed tumor growth but with a significant increase of EDB-FN in the tumor, as revealed by MRMI and immunohistochemistry. Combining ZD2-siDANCR-ELNP with paclitaxel further reduced tumor sizes, along with reduced EDB-FN expression. Interestingly, MT218-MRMI revealed a lower reduction of tumor signal enhancement with the combination treatment than that with the siDANCR treatment alone, which was supported by higher cell density in the tumors treated with the combination therapy, as shown by histochemical analysis. MT218-MRMI clearly revealed the changes of the tumor microenvironment in response to various therapies and is effective to noninvasively assess the response of TNBC tumors to the therapies. Regulating oncogenic lncRNA DANCR is an effective strategy for improving the outcomes of chemotherapy in TNBC.

AurkA/TPX2 co-overexpression in nontransformed cells promotes genome instability through induction of chromosome mis-segregation and attenuation of the p53 signalling pathway

The Aurora-A kinase (AurkA) and its major regulator TPX2 (Targeting Protein for Xklp2) are key mitotic players frequently co-overexpressed in human cancers, and the link between deregulation of the AurkA/TPX2 complex and tumourigenesis is actively investigated. Chromosomal instability, one of the hallmarks of cancer related to the development of intra-tumour heterogeneity, metastasis and chemo-resistance, has been frequently associated with TPX2-overexpressing tumours. In this study we aimed to investigate the actual contribution to chromosomal instability of deregulating the AurkA/TPX2 complex, by overexpressing it in nontransformed hTERT RPE-1 cells. Our results show that overexpression of both AurkA and TPX2 results in increased AurkA activation and severe mitotic defects, compared to AurkA overexpression alone. We also show that AurkA/TPX2 co-overexpression yields increased aneuploidy in daughter cells and the generation of micronucleated cells. Interestingly, the p53/p21 axis response is impaired in AurkA/TPX2 overexpressing cells subjected to different stimuli; consistently, cells acquire increased ability to proliferate after independent induction of mitotic errors, i.e. following nocodazole treatment. Based on our observation that increased levels of the AurkA/TPX2 complex affect chromosome segregation fidelity and interfere with the activation of a pivotal surveillance mechanism in response to altered cell division, we propose that co-overexpression of AurkA and TPX2 per se represents a condition promoting the generation of a genetically unstable context in nontransformed human cells.