Mechanisms Of Therapy Resistance Research Articles

Abstract 5226: The PI3K inhibitor Paxalisib combines synergistically with the pan-Raf inhibitor TAK580 (DAY 101) to extend survival in orthotopic xenograft models of atypical teratoid/rhabdoid tumors

Abstract Atypical teratoid/rhabdoid tumors (AT/RT) are aggressive pediatric brain tumors and the most common malignant brain tumors of infancy. The four-year event-free survival rate is only 37%. For patients with relapsed AT/RT, there are limited treatment options, but novel precision therapies may help improve survival for patients with this deadly disease. We have previously identified strong activation of the PI3K-AKT-mTOR (mTOR) signaling pathways in AT/RT. Paxalisib is a highly brain-penetrant PI3K inhibitor acting upstream of mTOR. We find that Paxalisib slows tumor growth in orthotopic xenograft models of AT/RT and extends median survival from 40 to 54 days (p=0.001, log-rank test). RNASeq after mTOR pathway inhibition identifies reflexive activation of the MAPK pathway as a possible mechanism of therapy resistance (KEGG pathway analysis). TAK580 (DAY 101) is a highly brain penetrant, pan-RAF kinase inhibitor, currently in clinical trials in pediatric low grade gliomas (NCT03429803, NCT04775485). Paxalisib combines synergistically with TAK580 to reduce AT/RT growth and viability (Bliss synergy score 24.22). A pilot study suggests combination therapy is well tolerated in mice bearing AT/RT orthotopic xenografts and slows tumor growth compared to Paxalisib alone and vehicle controls. This novel combination of precision therapies can translate into a clinical trial aimed at improving survival in this deadly disease. Citation Format: Kristen Malebranche, Tyler Findlay, Charles Eberhart, Eric Raabe, Jeffrey Rubens. The PI3K inhibitor Paxalisib combines synergistically with the pan-Raf inhibitor TAK580 (DAY 101) to extend survival in orthotopic xenograft models of atypical teratoid/rhabdoid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5226.

Abstract 6291: Integrative single-cell tracking of genome evolution and tumor cell plasticity in small cell lung cancer (SCLC)

Abstract SCLC is the deadliest type of lung cancer, which is mostly diagnosed at late stages and accounts for 15% of pulmonary tumors. Even though chemotherapy is initially effective, SCLC patients relapse quickly with therapy resistant tumors. Genome studies revealed universal loss of TP53 and RB1; recent transcriptional studies categorized SCLC tumors based on the expression of 4 lineage transcription factors (TFs). We aimed to dissect how genomic cues impact transcriptional phenotypes, tumor cell plasticity and the dynamics of tumor and immune cell interactions, to thus decipher molecular mechanisms of phenotypic divergence and therapy resistance in SCLC. We performed genome sequencing and single cell transcriptome profiling on 61 SCLC patient tumors, including primary tumors, metastases and paired relapse tumors acquired throughout therapy. Single cell RNA-seq was performed with the 10x Genomics platform for patient tumors to study cancer cells and the tumor micro-environment (TME)(n=17), and on xenograft models to study tumor cell intrinsic heterogeneity (n=44). Single cell and genome sequencing data were processed with an in-house pipeline. To further validate our findings on the transcriptional level, we performed imaging mass cytometry (IMC) of matched tissue samples to delineate the spatial context of cellular subsets. Single cell transcriptome profiles were determined for 211,446 cells from 61 tumors. Transcriptome profiling at single cell levels revealed co-expression of at least two of the lineage TFs ASCL1, NEUROD1 or POU2F3 for each SCLC tumor. Furthermore, tumor subsets without any expression of the four described TFs were found. Instead of distinct subgroups, our data points to a concerted regulation of a multitude of lineage factors and transcriptional programs in each SCLC tumor. To specifically investigate how underlying genetic alterations affect molecular phenotypes, integrative studies across all patients were performed. This revealed shared transcriptional programs in tumors with MYC family member amplifications; trajectory inference for cases with low and high-level copy gains of MYCN pointed to distinct transcriptional states. We performed multi-regional and longitudinal studies of matched patient cases. We reconstructed genomic patterns of clonal evolution from bulk genome sequencing data, and projected genomic subsets to transcriptional trajectories at single cell level, thus analyzing the effect of genome diversity on the transcriptional landscape. Additionally, tumor intrinsic profiles of heterogeneity were mapped to single cell data of patient tumors to elucidate the effects on the TME, focusing on tumor-immune cell interactions. All together our data provides a first comprehensive framework for the study of underlying genetic alterations that shape the transcriptional landscape and phenotypic plasticity in SCLC. Citation Format: Marcel Schmiel, Roman K. Thomas, Julie George. Integrative single-cell tracking of genome evolution and tumor cell plasticity in small cell lung cancer (SCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6291.

Abstract LB030: Mechanisms of ERK action in MEK inhibitor-insensitive lung cancers

Abstract Small cell lung cancer (SCLC) and other neuroendocrine (NE) lung tumors account for 25% of all lung cancers, they are the most aggressive type and have the poorest prognosis. SCLC does not present driver oncogenes involved in the regulation of kinase signaling, which makes these tumors hard to treat. The few available treatments yield responses early, but relapse occurs and they become resistant. Due to the poor efficacy of therapies, research in SCLC had been moved to the background, with basically no progress over the past decades. Mutations in the RAS-ERK pathway, elevating ERK activity and enhancing tumorigenesis, are common in other types of cancer, including NSCLC. Nevertheless, they are rare in SCLC. Indeed, previous studies demonstrated that ERK activation interferes with growth of SCLC. However, the mechanism by which ERK hinders tumor progression has not been defined yet. One of the most important players in SCLC and other NE tumors is the transcription factor ASCL1, essential for their survival. It has been reported that ASCL1 increases DUSP6 transcription with the consequent ERK activity suppression. Its downregulation affects cell cycle progression and survival. Conversely, inhibition of ERK activation by MEK inhibitors increases ASCL1 mRNA levels. Previous studies in our laboratory suggested that DUSP6 has a major role in a subset of SCLC and NSCLC-NE cell lines. Inhibition of DUSP6 enhances ERK signaling and significantly decreases survival of NE lung tumor cells. We have considered DUSP6 a good candidate to try to understand the mechanism by which ERK interferes with survival. However, hurdles in studying DUSP6 in SCLC include off-target effects of inhibitors and difficulty of gene-edited cell lines. To date, we have been able to generate a DUSP6 KO NSCLC-NE cell line which shows a clear drop in proliferation. Also, the overexpression of ERK or a constitutively active MEK1 mutant in a few SCLC, NE-NSCLC and MEK inhibitor-insensitive squamous lung cancer cell lines render a decrease in survival. DUSP6 is a cytoplasmic anchor for ERK and may reduce the ability of ERK to suppress ASCL1 transcription. For this reason, we are working on studying the effect on survival of ERK localization in the nucleus or the cytoplasm. With our ongoing experiments we are defining the conditions to examine transcriptional impact of ERK on SCLC. Discovering molecular events antagonized by the ERK pathway in SCLC will reveal novel targets controlling the inhibitory mechanisms. One of the SCLC major questions of translational relevance is what are the mechanisms upholding the development of chemoresistance, where the ERK pathway seems to be involved. Hence, the definition of the underlying mechanisms of tumorigenesis and therapy resistance will uncover new potential antitumor targets that currently escape our notice. A better understanding of the molecular biology of SCLC will let us find new therapeutic strategies to overcome the impasse in chemotherapy. Citation Format: Ana Martin-Vega, Svetlana Eartnest, John D. Minna, Jane E. Johnson, Melanie H. Cobb. Mechanisms of ERK action in MEK inhibitor-insensitive lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB030.

Abstract 5429: Therapeutic targeting of oncogenic long non-coding RNAs utilizing antisense oligonucleotides

Abstract Non-coding RNAs (ncRNAs) are emerging as potent regulators of biological process including human diseases such as cancer. Long non-coding RNAs (lncRNAs) are a dynamic and versatile class of ncRNAs for which there is extensive evidence of these transcripts modulating epigenetic programs to affect cellular phenotypes. Our initial studies using a model of glioblastoma (GBM) tumor recurrence has revealed a number of oncogenic lncRNAs associated with therapy resistance and/or tumor recurrence. We have validated the expression of these oncogenic lncRNAs in another cancer, pediatric rhabdomyosarcoma (RMS). There have been few effective therapeutic advances made in past decades for either RMS or GBM. Radiation therapy is commonly employed alongside surgery and standard chemotherapy, especially in the case of unresectable disease, advanced disease, or upon tumor recurrence. Tumors may inherently possess or acquire resistance to radiation therapy and other treatment modalities over the course of disease. Tumor therapy resistance and recurrence are mediated by complex epigenetic programs which lead to increased capacity for DNA damage repair or tolerance of double stranded breaks. LncRNAs may regulate mechanisms of tumor recurrence and therapy resistance across multiple cancer types. The functional roles of ZFAS1 modulating epithelial to mesenchymal transition in colon cancers and SAMMSON which drives aberrant mitochondrial function in melanoma have been described. CASC19/PCAT2, ZFAS1, DUXAP9, and SOX2-OT are overexpressed in multiple cancers with CASC19 having a significant negative association with GBM patient survival. In silico analysis identifies DNA-binding capacities harbored by these lncRNA transcripts, and their predicted binding sites are proximal to several cancer hallmark genes. We have verified the expression of isoforms of these lncRNA transcripts that contain DNA-binding domains through sequencing and qPCR. We hypothesize that these lncRNAs regulate transcriptional networks via recruitment of transcriptional or epigenetic machinery to genomic loci to modulate oncogenic programs and/or tumor suppressor pathways. As potent epigenetic regulators, lncRNAs present an attractive class of therapeutic targets that could complement standard treatments to reduce disease burden and improve patient survival. It is possible that acquired radiation resistance mediated by lncRNAs is conserved across multiple malignancies. We are developing chemically modified, novel antisense oligonucleotides to target these transcripts in GBM and RMS patient-derived xenografts. Using these systems, we hope to reveal novel associations with disease progression and oncogenic mechanisms that could be exploited through the direct targeting of lncRNAs. We hope our work will lead to the development of novel clinical agents to treat patients with RMS, GBM, and potentially other intractable cancers. Citation Format: Christian Tyler Stackhouse, Corinne Linardic, Malaika Kimmons, Samantha Weitzel. Therapeutic targeting of oncogenic long non-coding RNAs utilizing antisense oligonucleotides [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5429.

Abstract 4079: Single-cell RNA-seq revealed important pathways in response to neoadjuvant therapy resistance in rectal cancer

Abstract Colorectal cancer (CRC) is the second leading cause of cancer-related death in Europe and United States and is often diagnosed at advanced stage. Rectal cancer (RC) accounts for one third of all CRCs. The treatment options of RC include surgery, chemotherapy, and radiation. Locally advanced RC is treated with neoadjuvant chemoradiotherapy (nCRT) and subsequent surgery. Less than 20% of RC patients have a complete response to nCRT. Our previous work demonstrated that RC subclones respond differently to nCRT. However, the resistance mechanisms remain elusive. To explore the mechanisms underlying therapy resistance in RC, we performed single-cell RNA sequencing (scRNA-Seq) to identify and characterize the therapy-resistant RC cells. We generated patient-derived xenograft model in athymic nude mice with primary tumor cells collected from pre-treatment advanced RC patients. The compete treatment for xenograft animals includes 2 weeks of chemoradiotherapy and 1 week off. Tumor tissues were collected from mice for scRNA-Seq at a series of time points: no treatment, 2 days, 1 week, and 3 weeks after treatment. Single-cell analysis revealed five tumor cellular clusters with distinct gene expression profiles. The marker genes of cluster 1 were related to cell cycle, DNA replication, and p53 signaling pathway based on KEGG pathway enrichment analysis, which suggests that this cluster of cells are actively proliferating. Marker genes of cluster 3 were related to mineral absorption, PPAR signaling pathway, and HIF-1 signaling pathway. We compared the composition of the five cell clusters at each time point and found that while the cellular fractions of cluster 1 and cluster 3 initially dropped (2 days), they later increased (1 week and 3 weeks), suggesting that these two clusters were therapy-resistant and continued proliferating after the nCRT. Consistent with our previous DNA sequencing results, different cell populations also demonstrated tumor heterogeneity based on scRNA-seq clustering analysis of the RC xenograft model. We further compared the gene expression levels before and after treatment and identified 147 differently expressed genes (DEGs). Pathway enrichment analysis of these DEGs highlighted that signaling pathways of IL-17, TNF, MAPK, and ErbB were up-regulated posttreatment, suggesting that they may be responsible for the therapy resistance of the tumor cell clusters. These results shed light on the importance of these signaling pathways on the mechanisms of rectal cancer therapy resistance. Further work is needed to validate this discovery. Citation Format: Yu Chen, Regina K. Irwin, Gregory W. Williams, Mary Smithson, Karin M. Hardiman, Zechen Chong. Single-cell RNA-seq revealed important pathways in response to neoadjuvant therapy resistance in rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4079.

Abstract 1526: Circulatory insulin-like growth factor 1 predicts metastasis in triple-negative breast cancer

Abstract Background and aims: Aberrant insulin-like growth factor (IGFs) signaling is directly implicated in oncogenesis and metastasis in various solid tumors including breast cancer. Targeting the IGF axis may provide an insight into the mechanism of cancer stemness and therapy resistance. The present study aims to explore the association of the circulatory levels of these proteins in aggressive triple-negative breast cancer (TNBC) in Bangladesh. Methods: A total of 165 BC patients were recruited in the present study where 42 were TNBC subjects (selected based on their complete hormonal profile). Data were collected from retrospective information. Anthropometric and biochemical parameters were measured by standard methods. Serum IGF1, IGFBP1, and insulin were estimated by ELISA. Results: TNBC subjects showed significantly higher levels of circulatory IGF1 (185 vs 145, p=0.004), while patients who had distant metastasis showed increased levels of IGF1, IGFBP1, and alkaline phosphatase (ALP) [178 vs 153, p=0.001; 6.44 vs 3.05, and 140 vs 88, p<0.05; metastatic vs non-metastatic BC respectively]. In addition, serum IGF1 was positively associated with serum insulin levels and IGFBP1 was associated with ALP, SGPT, CA15.3, and duration of OCP (p<0.05). In multivariate regression, IGF1 was found to be a significant predictor of breast cancer survival after adjusting confounding variables. Conclusion: High serum levels of IGF1 may contribute to metastasis in basal breast cancer in a Bangladeshi population. These results need to be confirmed in larger TNBC survivor cohorts. Citation Format: Salima Akter, Mithila Faruque, Hajara Akhter, Mousumi Bhowmik, Mohammad Nazmul Hasan, Lipika Sultana, Imran Khan, Mamun Mia, Md. Mustafizur Rahman, Habib Sadat Chaudhury, Islam S. Rafiqul, Kamrun Nahar, Tae Gyu Choi, Sung Soo Kim. Circulatory insulin-like growth factor 1 predicts metastasis in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1526.

Abstract 6051: Longitudinal single-cell sequencing of high-risk neuroblastoma tumors reveals intrinsic and extrinsic mechanisms of therapy resistance

Abstract Neuroblastoma is a childhood cancer originating from embryonic neuronal progenitor cells and is the most common cancer diagnosed in infants. Although the majority of patients respond to initial chemotherapy, most high-risk patients suffer relapse due to therapy resistance. Here, we generated single-cell transcriptome and bulk whole-genome sequencing data of diagnosis and post-therapy samples from 23 patients diagnosed with high-risk neuroblastoma. We found two distinct adrenergic populations in the patient samples. Most tumor cells showed a mature sympathoblast phenotype whereas a small fraction was in an undifferentiated state with activation of protein translation, unfolded protein, and oxidative phosphorylation pathways. We found that therapy-resistant tumor cells in these two subpopulations had distinct characteristics. The more mature resistant subpopulation upregulated genes associated with epithelial-to-mesenchymal transition, including TWIST1, PLCB1 and CD276. The undifferentiated resistant subpopulation upregulated Ras signal transduction and TP53 pathway genes. Analysis of the immune microenvironment revealed that most tumor-associated macrophages became more immunosuppressive post-therapy via multiple newly gained signaling interactions including the complement signaling pathway. We discovered a limited infiltration of T lymphocytes in the tumor microenvironment, and chemotherapy induced an effector state with upregulated mTOR signaling and metabolism. Overall, our study revealed subpopulations of tumor cells in neuroblastoma that responded differently to induction chemotherapy. Our findings uncovered distinct molecular signatures of the resistant cells and their interactions with the immune microenvironment, paving the way for developing novel therapies for high-risk neuroblastoma. Citation Format: Yasin Uzun, Liron D. Grossmann, Chia-Hui Chen, Anusha Thadi, Chi-Yun Wu, Peng Gao, Dinh Diep, Lea Surrey, Daniel Martinez, Tasleema Patel, Qi Qiu, Sarah Johnson, Wenbao Yu, Shane Drabings, Changya Chen, Yuxuan Hu, Gregory Chen, Derek A. Oldridge, Kun Zhang, Hao Wu, Kathrin Bernt, Nancy Zhang, John M. Maris, Kai Tan. Longitudinal single-cell sequencing of high-risk neuroblastoma tumors reveals intrinsic and extrinsic mechanisms of therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6051.

Resistance to DNA repair inhibitors in cancer.

The DNA damage response (DDR) represents a complex network of proteins which detect and repair DNA damage, thereby maintaining the integrity of the genome and preventing the transmission of mutations and rearranged chromosomes to daughter cells. Faults in the DDR are a known driver and hallmark of cancer. Furthermore, inhibition of DDR enzymes can be used to treat the disease. This is exemplified by PARP inhibitors (PARPi) used to treat cancers with defects in the homologous recombination DDR pathway. A series of novel DDR targets are now also under pre-clinical or clinical investigation, including inhibitors of ATR kinase, WRN helicase or the DNA polymerase/helicase Polθ (Pol-Theta). Drug resistance is a common phenomenon that impairs the overall effectiveness of cancer treatments and there is already some understanding of how resistance to PARPi occurs. Here, we discuss how an understanding of PARPi resistance could inform how resistance to new drugs targeting the DDR emerges. We also discuss potential strategies that could limit the impact of these therapy resistance mechanisms in cancer.

Notch Signaling in Breast Tumor Microenvironment as Mediator of Drug Resistance.

Notch signaling dysregulation encourages breast cancer progression through different mechanisms such as stem cell maintenance, cell proliferation and migration/invasion. Furthermore, Notch is a crucial driver regulating juxtracrine and paracrine communications between tumor and stroma. The complex interplay between the abnormal Notch pathway orchestrating the activation of other signals and cellular heterogeneity contribute towards remodeling of the tumor microenvironment. These changes, together with tumor evolution and treatment pressure, drive breast cancer drug resistance. Preclinical studies have shown that targeting the Notch pathway can prevent or reverse resistance, reducing or eliminating breast cancer stem cells. In the present review, we will summarize the current scientific evidence that highlights the involvement of Notch activation within the breast tumor microenvironment, angiogenesis, extracellular matrix remodeling, and tumor/stroma/immune system interplay and its involvement in mechanisms of therapy resistance.

ETMR-05. Single-cell transcriptomics of ETMR reveals developmental cellular programs and tumor-pericyte communications in the microenvironment

BACKGROUND: Embryonal tumors with multilayered rosettes (ETMR) are pediatric brain tumors bearing a grim prognosis, despite intensive multimodal therapeutic approaches. Insights into cellular heterogeneity and cellular communication of tumor cells with cells of the tumor microenvironment (TME), by applying single-cell (sc) techniques, potentially identify mechanisms of therapy resistance and target-directed treatment approaches. MATERIAL AND METHODS: To explore ETMR cell diversity, we used single-cell RNA sequencing (scRNA-seq) in human (n=2) and murine ETMR (transgenic mode; n=4) samples, spatial transcriptomics, 2D and 3D cultures (including co-cultures with TME cells), multiplex immunohistochemistry and drug screens. RESULTS: ETMR microenvironment is composed of tumor and non-tumor cell types. The ETMR malignant compartment harbour cells representing distinct transcriptional metaprograms, (NSC-like, NProg-like and Neuroblast-like), mirroring embryonic neurogenic cell states and fuelled by neurogenic pathways (WNT, SHH, Hippo). The ETMR TME is composed of oligodendrocyte and neuronal progenitor cells, neuroblasts, microglia, and pericytes. Tumor-specific ligand-receptor interaction analysis showed enrichment of intercellular communication between NProg-like ETMR cells and pericytes (PC). Functional network analyses reveal ETMR-PC interactions related to stem-cell signalling and extracellular matrix (ECM) organization, involving factors of the WNT, BMP, and CxCl12 networks. Results from ETMR-PC co-culture and spatial transcriptomics pointed to a pivotal role of pericytes in keeping ETMR in a germinal neurogenic state, enriched in stem-cell signalling. Drug screening considering cellular heterogeneity and cellular communication suggested novel therapeutic approaches. CONCLUSION: ETMR demonstrated diversity in the microenvironment, with enrichment of cell-cell communications with pericytes, supporting stem-cell signalling and interfering in the organization of the tumor extracellular matrix. Targeting ETMR-PC interactions might bring new opportunities for target-directed therapy.

Biomarkers and 3D models predicting response to immune checkpoint blockade in head and neck cancer (Review).

Immunotherapy has evolved into a powerful tool in the fight against a number of types of cancer, including head and neck squamous cell carcinomas (HNSCC). Although checkpoint inhibition (CPI) has definitely enriched the treatment options for advanced stage HNSCC during the past decade, the percentage of patients responding to treatment is widely varying between 14-32% in second-line setting in recurrent or metastatic HNSCC with a sporadic durability. Clinical response and, consecutively, treatment success remain unpredictable in most of the cases. One potential factor is the expression of target molecules of the tumor allowing cancer cells to acquire therapy resistance mechanisms. Accordingly, analyzing and modeling the complexity of the tumor microenvironment (TME) is key to i) stratify subgroups of patients most likely to respond to CPI and ii) to define new combinatorial treatment regimens. Particularly in a heterogeneous disease such as HNSCC, thoroughly studying the interactions and crosstalking between tumor and TME cells is one of the biggest challenges. Sophisticated 3D models are therefore urgently needed to be able to validate such basic science hypotheses and to test novel immuno-oncologic treatment regimens in consideration of the individual biology of each tumor. The present review will first summarize recent findings on immunotherapy, predictive biomarkers, the role of the TME and signaling cascades eliciting during CPI. Second, it will highlight the significance of current promising approaches to establish HNSCC 3D models for new immunotherapies. The results are encouraging and indicate that data obtained from patient-specific tumors in a dish might be finally translated into personalized immuno-oncology.

Immunotherapy and Microbiota for Targeting of Liver Tumor-Initiating Stem-like Cells.

Simple SummaryHepatocellular carcinoma (HCC) remains one of the more incurable diseases. Thus, finding an HCC treatment is urgent for this unmet medical need. Immunotherapy is a break-through treatment that may help 15–20% of HCC patients. In this review, pharmacological and immune-therapeutical targeting of druggable cancer drivers, immune checkpoints, and long non-coding RNAs for HCC and cholangiocarcinoma are discussed.Cancer contains tumor-initiating stem-like cells (TICs) that are resistant to therapies. Hepatocellular carcinoma (HCC) incidence has increased twice over the past few decades, while the incidence of other cancer types has trended downward globally. Therefore, an understanding of HCC development and therapy resistance mechanisms is needed for this incurable malignancy. This review article describes links between immunotherapies and microbiota in tumor-initiating stem-like cells (TICs), which have stem cell characteristics with self-renewal ability and express pluripotency transcription factors such as NANOG, SOX2, and OCT4. This review discusses (1) how immunotherapies fail and (2) how gut dysbiosis inhibits immunotherapy efficacy. Gut dysbiosis promotes resistance to immunotherapies by breaking gut immune tolerance and activating suppressor immune cells. Unfortunately, this leads to incurable recurrence/metastasis development. Personalized medicine approaches targeting these mechanisms of TIC/metastasis-initiating cells are emerging targets for HCC immunotherapy and microbiota modulation therapy.

Advances in Renal Cell Carcinoma Drug Resistance Models.

Renal cell carcinoma (RCC) is the most common form of kidney cancer. Systemic therapy is the preferred method to eliminate residual cancer cells after surgery and prolong the survival of patients with inoperable RCC. A variety of molecular targeted and immunological therapies have been developed to improve the survival rate and prognosis of RCC patients based on their chemotherapy-resistant properties. However, owing to tumor heterogeneity and drug resistance, targeted and immunological therapies lack complete and durable anti-tumor responses; therefore, understanding the mechanisms of systemic therapy resistance and improving clinical curative effects in the treatment of RCC remain challenging. In vitro models with traditional RCC cell lines or primary cell culture, as well as in vivo models with cell or patient-derived xenografts, are used to explore the drug resistance mechanisms of RCC and screen new targeted therapeutic drugs. Here, we review the established methods and applications of in vivo and in vitro RCC drug resistance models, with the aim of improving our understanding of its resistance mechanisms, increasing the efficacy of combination medications, and providing a theoretical foundation for the development and application of new drugs, drug screening, and treatment guidelines for RCC patients.

Comprehensive characterization of pre- and post-treatment samples of breast cancer reveal potential mechanisms of chemotherapy resistance.

When locally advanced breast cancer is treated with neoadjuvant chemotherapy, the recurrence risk is significantly higher if no complete pathologic response is achieved. Identification of the underlying resistance mechanisms is essential to select treatments with maximal efficacy and minimal toxicity. Here we employed gene expression profiles derived from 317 HER2-negative treatment-naïve breast cancer biopsies of patients who underwent neoadjuvant chemotherapy, deep whole exome, and RNA-sequencing profiles of 22 matched pre- and post-treatment tumors, and treatment outcome data to identify biomarkers of response and resistance mechanisms. Molecular profiling of treatment-naïve breast cancer samples revealed that expression levels of proliferation, immune response, and extracellular matrix (ECM) organization combined predict response to chemotherapy. Triple negative patients with high proliferation, high immune response and low ECM expression had a significantly better treatment response and survival benefit (HR 0.29, 95% CI 0.10–0.85; p = 0.02), while in ER+ patients the opposite was seen (HR 4.73, 95% CI 1.51–14.8; p = 0.008). The characterization of paired pre-and post-treatment samples revealed that aberrations of known cancer genes were either only present in the pre-treatment sample (CDKN1B) or in the post-treatment sample (TP53, APC, CTNNB1). Proliferation-associated genes were frequently down-regulated in post-treatment ER+ tumors, but not in triple negative tumors. Genes involved in ECM were upregulated in the majority of post-chemotherapy samples. Genomic and transcriptomic differences between pre- and post-chemotherapy samples are common and may reveal potential mechanisms of therapy resistance. Our results show a wide range of distinct, but related mechanisms, with a prominent role for proliferation- and ECM-related genes.

Manganese Superoxide Dismutase Acetylation and Regulation of Protein Structure in Breast Cancer Biology and Therapy.

The loss and/or dysregulation of several cellular and mitochondrial antioxidants’ expression or enzymatic activity, which leads to the aberrant physiological function of these proteins, has been shown to result in oxidative damage to cellular macromolecules. In this regard, it has been surmised that the disruption of mitochondrial networks responsible for maintaining normal metabolism is an established hallmark of cancer and a novel mechanism of therapy resistance. This altered metabolism leads to aberrant accumulation of reactive oxygen species (ROS), which, under specific physiological conditions, leads to a potential tumor-permissive cellular environment. In this regard, it is becoming increasingly clear that the loss or disruption of mitochondrial oxidant scavenging enzymes may be, in specific tumors, either an early event in transformation or exhibit tumor-promoting properties. One example of such an antioxidant enzyme is manganese superoxide dismutase (MnSOD, also referred to as SOD2), which detoxifies superoxide, a ROS that has been shown, when its normal physiological levels are disrupted, to lead to oncogenicity and therapy resistance. Here, we will also discuss how the acetylation of MnSOD leads to a change in detoxification function that leads to a cellular environment permissive for the development of lineage plasticity-like properties that may be one mechanism leading to tumorigenic and therapy-resistant phenotypes.

HOTTIP Mediated Therapy Resistance in Glioma Cells Involves Regulation of EMT-Related miR-10b.

The advanced grade glioblastomas are characterized by dismal five-year survival rates and are associated with worse outcomes. Additionally, resistance to therapies is an additional burden responsible for glioma associated mortality. We studied the resistance against temozolomide (TMZ) as a surrogate to understand the mechanism of therapy resistance in glioma cancer cells. Screening of three glioma cells lines, A172, LN229 and SF268 revealed that SF268 glioma cells were particularly resistant to TMZ with the IC-50 of this cell line for TMZ ten times higher than for the other two cell lines. A role of lncRNAs in glioma progression has been identified in recent years and, therefore, we focused on lncRNAs for their role in regulating TMZ resistance in glioma cancer cells. lncRNA HOTTIP was found to be particularly elevated in SF268 cells and over-expression of HOTTIP in both A172 and LN229 remarkably increased their TMZ IC-50s, along with increased cell proliferation, migration, clonogenicity and markers of angiogenesis and metastasis. As a mechanism we observed increased expression of miRNA-10b and mesenchymal markers Zeb1/Zeb2 and reduced expression of E-cadherin in SF268 cells indicating a role of EMT in TMZ resistance. A172 and LN229 cells with overexpressed HOTTIP also had similarly induced EMT and the elevated miR-10b levels. Further, silencing of miR-10b in HOTTIP overexpressing cells as well as the SF268 cells reversed EMT with associated sensitization of all the tested cells to TMZ. Our results thus present a case for HOTTIP in native as well as acquired resistance of glioma cells against chemotherapy, with a key mechanistic role of EMT and the miR-10b. Thus, HOTTIP as well as miR-10b are critical targets for glioma therapy, and need to be tested further.

Targeted Therapy and Mechanism of Drug Resistance in Non-small Cell Lung Cancer with Epidermal Growth Factor Receptor Gene Mutation

肺癌是全球第六大死因，也是恶性肿瘤的主要死因之一，非小细胞肺癌（non-small cell lung cancer, NSCLC）是其最常见的类型。表皮生长因子受体（epidermal growth factor receptor, EGFR）基因突变是NSCLC的常见突变之一。针对EGFR基因突变的晚期NSCLC患者，使用EGFR-酪氨酸激酶抑制剂（EGFR-tyrosine kinase inhibitors, EGFR-TKIs）如吉非替尼、阿法替尼、奥希替尼等靶向治疗已经成为许多指南推荐的一线治疗方案，但许多患者在用药约1年左右便发生获得性耐药。出现获得性耐药的患者会比普通患者更早出现疾病进展，对患者的预后造成重大影响。目前对于发生了获得性耐药的患者主要的治疗方式为针对耐药突变进行新的靶点抑制，如发生了T790M突变的患者对第一、第二代药物（如吉非替尼、阿法替尼等）产生耐药，可以使用第三代药物（奥希替尼或阿美替尼）进行治疗，通过这种治疗方式可延缓病情进展。因此，耐药机制的研究及耐药患者的治疗是必不可少的。本文主要就EGFR突变NSCLC患者的靶向治疗及其耐药机制进行综述，以期为EGFR-TKIs的临床应用提供参考。

Stromal Characteristics and Impact on New Therapies for Metastatic Triple-Negative Breast Cancer.

Simple SummaryIn this review, we summarize the recent outcomes from clinical trials with new agents for metastatic triple-negative breast cancer, specifically focusing on immunotherapies targeting the PD-1/PD-L1 pathway and antibody–drug conjugates. In addition to the clinical data supporting these therapies, we review the impact of the tumor microenvironment on the effectiveness of these therapies. The heterogenous nature of triple-negative breast cancer (TNBC) is an underlying factor in therapy resistance, metastasis, and overall poor patient outcome. The lack of hormone and growth factor receptors lends to the use of chemotherapy as the first-line treatment for TNBC. However, the failure of chemotherapy demonstrates the need to develop novel immunotherapies, antibody–drug conjugates (ADCs), and other tumor- and stromal-targeted therapeutics for TNBC patients. The potential for stromal-targeted therapy is driven by studies indicating that the interactions between tumor cells and the stromal extracellular matrix (ECM) activate mechanisms of therapy resistance. Here, we will review recent outcomes from clinical trials targeting metastatic TNBC with immunotherapies aimed at programed death ligand–receptor interactions, and ADCs specifically linked to trophoblast cell surface antigen 2 (Trop-2). We will discuss how biophysical and biochemical cues from the ECM regulate the pathophysiology of tumor and stromal cells toward a pro-tumor immune environment, therapy resistance, and poor TNBC patient outcome. Moreover, we will highlight how ECM-mediated resistance is motivating the development of new stromal-targeted therapeutics with potential to improve therapy for this disease.

Abstract OT1-15-01: SGNTUC-019: Phase 2 basket study of tucatinib and trastuzumab in previously treated solid tumors with HER2 alterations: HER2-mutated breast cancer cohort (ongoing clinical trial)

Abstract Background: Tucatinib (TUC), approved in multiple regions for HER2+ metastatic breast cancer, is a highly selective HER2-directed tyrosine kinase inhibitor with minimal EGFR inhibition. TUC is being developed as a novel therapy for patients with HER2+ metastatic breast cancer, colorectal cancer, and gastric cancer. In xenograft models of HER2+ and HER2-mutated tumors, dual targeting of HER2 with TUC and trastuzumab showed superior activity to either agent alone. Somatic HER2 mutations occur in approximately 3% of breast cancers, mostly in patients with hormone receptor-positive disease and the lobular sub-type. HER2 mutations lead to enhanced tyrosine kinase activity and tumorigenesis in preclinical models and have been postulated as a mechanism of endocrine therapy resistance. The SGNTUC-019 basket study (NCT04579380) is evaluating TUC in combination with trastuzumab in patients with HER2+ or HER2-mutated solid tumors, including a cohort of patients with locally advanced unresectable or metastatic breast cancer that is HER2-mutated and not overexpressed/amplified. Trial Design: SGNTUC-019 is a multi-cohort, open-label, international Phase 2 study evaluating patients with previously treated solid tumors displaying HER2 overexpression/amplification or activating mutations. Eligible breast cancer patients must have HER2-mutated, locally advanced unresectable or metastatic disease with progression on or after ≥1 prior line of therapy (chemotherapy, endocrine or targeted therapy) in the locally advanced or metastatic disease setting. Additionally, an ECOG Performance Status of ≤1; adequate hepatic, hematological, renal, and cardiac functions; and no previous HER2-directed therapy are required for enrollment. For eligibility, HER2 mutations can be demonstrated in a previous or on-study next-generation sequencing (NGS) assay of circulating tumor DNA or a previous tissue NGS assay. The breast cancer cohort will enroll 30 response-evaluable patients with HER2-mutated disease. Patients with HER2+ (overexpression/amplification) breast cancer will not be enrolled. The primary objective in each cohort is antitumor activity. The primary endpoint is a confirmed objective response rate, and secondary endpoints are the disease control rate, duration of response, progression-free survival, and overall survival. Safety and efficacy endpoints will be summarized descriptively. For response rates, the 2-sided exact interval using the Clopper-Pearson method will be calculated. Patients will receive TUC 300 mg orally twice a day and trastuzumab 8 mg/kg intravenously on Cycle 1 Day 1 and 6 mg/kg every 21 days from Cycle 2 Day 1. Hormone receptor-positive HER2-mutated breast cancer patients will also receive fulvestrant 500 mg intramuscularly every 4 weeks and on Cycle 1 Day 15. Disease assessments per RECIST v1.1 are every 6 weeks for 24 weeks, then every 12 weeks. Patients in the breast cancer cohort will undergo baseline brain magnetic resonance imaging, and those patients with brain metastases may be eligible for the study. Quality of life will be evaluated using EQ-5D-5L every 2 cycles. Enrollment began in December 2020 and is ongoing globally. Citation Format: Alicia Okines, Paula R. Pohlmann, Jorge Ramos, Luke Walker, Erika Hamilton. SGNTUC-019: Phase 2 basket study of tucatinib and trastuzumab in previously treated solid tumors with HER2 alterations: HER2-mutated breast cancer cohort (ongoing clinical trial) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT1-15-01.

Adoptive Cellular Therapy for Multiple Myeloma Using CAR- and TCR-Transgenic T Cells: Response and Resistance.

Despite the substantial improvement of therapeutic approaches, multiple myeloma (MM) remains mostly incurable. However, immunotherapeutic and especially T cell-based approaches pioneered the therapeutic landscape for relapsed and refractory disease recently. Targeting B-cell maturation antigen (BCMA) on myeloma cells has been demonstrated to be highly effective not only by antibody-derived constructs but also by adoptive cellular therapies. Chimeric antigen receptor (CAR)-transgenic T cells lead to deep, albeit mostly not durable responses with manageable side-effects in intensively pretreated patients. The spectrum of adoptive T cell-transfer covers synthetic CARs with diverse specificities as well as currently less well-established T cell receptor (TCR)-based personalized strategies. In this review, we want to focus on treatment characteristics including efficacy and safety of CAR- and TCR-transgenic T cells in MM as well as the future potential these novel therapies may have. ACT with transgenic T cells has only entered clinical trials and various engineering strategies for optimization of T cell responses are necessary to overcome therapy resistance mechanisms. We want to outline the current success in engineering CAR- and TCR-T cells, but also discuss challenges including resistance mechanisms of MM for evading T cell therapy and point out possible novel strategies.