Mechanisms Of Tumor Resistance Research Articles

CCL2-CCR2 Axis Inhibition in Osteosarcoma Cell Model: The Impact of Oxygen Level on Cell Phenotype.

Treatment of osteosarcoma is hampered by tumor hypoxia and requires alternative approaches. Although the CCL2-CCR2 axis is indispensable in tumor-induced inflammation and angiogenesis, its blockade has not been effective to date. This study aimed to characterize how CCR2 inhibition affects the crosstalk of osteosarcoma cells with immune cells to better delineate tumor resistance mechanisms that help withstand such treatment. In this study, 143B cells were exposed to healthy donor PBMC supernatants in a transwell assay lacking direct cell-to-cell contact and subjected to different oxygen concentrations. In addition, mice bearing orthotopic 143B tumors were subjected to CCR2 antagonist treatment. Our findings show that hypoxic conditions alter cytokine and cancer- related protein expression on cells and impair CCR2 antagonist effects in the experimental osteosarcoma model. CCL2-CCR2 axis blockade in the 143B xenografts, which are positive for hypoxia marker CAIX, did not slow 143B tumor growth or metastasis but altered tumor microenvironment by VEGFR downregulation and shift in the CD44-positive cell population towards high CD44 expression. This study highlights differential responses of tumor cells to CCR2 antagonists in the presence of different oxygen saturations and expands our knowledge of compensatory mechanisms leading to CCL2-CCR2 treatment resistance.

Necroptosis as a consequence of photodynamic therapy in tumor cells.

Photodynamic therapy (PDT) is an alternative to cancer treatment, demonstrating selectivity and significant cytotoxicity on malignant tissues. Such therapy involves two nontoxic components: photosensitizer (PS) and non-ionizing radiation. In optimal dosage combinations, PDT causes cellular and tissue effects by oxygen-dependent processes, leading tumor cells to regulated cell death pathways. Regulated necrosis, called necroptosis, can be triggered by PDT and is characterized by caspase-8 inhibition and RIPK1, RIPK3, and MLKL activities, leading to plasma membrane pores formation with subsequent cellular content release into the extracellular space. For this review, studies accessed by PubMed describing the relation between necroptosis and PDT were summarized. The results showed that PDT can trigger necroptosis mechanisms in different tumor cells. Moreover, a mix of different cell death types can co-occur. It is also important to highlight that necroptosis triggered by PDT is related to damage-associated molecular patterns (DAMPs) release, involving immunogenic cell death and vaccination. The cell death response is directly related to the photosensitizer chemical characteristics, concentration, incubation time, cellular location, and irradiation parameters. The synergism among all cell death types is an excellent advantage for avowing tumor resistance mechanisms and developing new solutions.

Exploring the Synergy of Immunotherapy and Conventional Treatments in Cancer Therapy

Immunotherapy (cancer immunotherapy) is a promising approach to cancer treatment that recognizes and destroys cancer cells by employing immune-related components or by directing the immune system. Between 2017 and 2020, the R&D pipeline for cancer immunotherapies increased by 233%. Immunotherapy can be broadly divided into five broad categories and is suitable for 20 different types of cancer. With the introduction of immune checkpoint inhibitors and CAR-T cell therapies, this approach has revolutionized the way of cancer treatment, even treating some patients with advanced cancers, while not all cancer types respond well to this approach. The effectiveness of immunotherapy as a stand-alone treatment is often constrained by tumor heterogeneity, immune evasion, and resistance mechanisms. To address these issues, immunotherapy is being investigated in combination with other traditional treatment modalities such as radiotherapy, chemotherapy and targeted drugs as a way to improve treatment outcomes. This review explores the rationale behind these combination therapies and discusses the potential of these combined therapies to improve patient survival and quality of life.

Exploring Piperine: Unleashing the multifaceted potential of a phytochemical in cancer therapy.

Radiotherapy is a cornerstone in the treatment of solid tumors, with extensive Phase III trials confirming its effectiveness. As advancements in treatment technologies and our understanding of tumor resistance mechanisms continue, the role of radiation oncology is set to become even more pivotal. Addressing the global challenge of lethal cancers demands innovative strategies, particularly in minimizing the side effects associated with traditional chemotherapy and ionizing radiation (IR). Recently, there has been growing interest in natural compounds for radioprotection, aiming to prevent tumor development and metastasis. Piperine, a compound found in black and long pepper, has emerged as a promising chemopreventive agent that works effectively without harming normal cells. Mechanistically, piperine modulates key signaling pathways, inhibits cancer cell migration and invasion, and enhances sensitivity to IR. Combining piperine with radiotherapy offers a compelling approach, boosting treatment efficacy while protecting healthy tissues from radiation damage. Piperine's versatile role goes beyond radiosensitization to include radioprotection by inhibiting NF-κB activation, reducing autophagy, and promoting apoptosis in cancer cells. This dual action makes it a promising candidate for personalized cancer care. As research advances, the therapeutic potential of piperine may drive new frontiers in cancer treatment strategies.

Tackling Anticancer Drug Resistance and Endosomal Escape in Aggressive Brain Tumors Using Bioelectronics.

Resistance mechanisms in brain tumors, such as medulloblastoma and glioblastoma, frequently involve the entrapment of chemotherapeutic agents within endosomes and the extracellular expulsion of drugs. These barriers to effective treatment are exacerbated in nanotechnology-based drug delivery systems, where therapeutic nanoparticles often remain confined within endosomes, thus diminishing their therapeutic efficacy. Addressing this challenge necessitates the development of novel strategies to enhance the efficiency of cancer therapies. This study tests the hypothesis that external electrical stimuli can modulate intracellular trafficking of chemotherapeutic drugs in common malignant brain tumors in children (medulloblastoma) and adults (glioblastoma) by using gold nanoparticles (GNPs). In our experiments, alternating current (AC) stimulation ranging from 1 kHz to 5 MHz and at a strength of 1 V/cm significantly reduced cell viability in drug-resistant medulloblastoma and enhanced delivery of GNPs in glioblastoma. Low-frequency AC resulted in a 50% increase in apoptosis compared to controls and an 8-fold increase in cell death in cisplatin-resistant medulloblastoma cells, accompanied by a substantial reduction in EC50 from 2.5 to 0.3 μM. Similarly, vincristine-resistant cells demonstrated a 4-fold enhancement in drug sensitivity. Furthermore, high-frequency AC facilitated a significant increase from 20 to 75% in the endosomal escape of GNPs in glioblastoma cells. These findings underscore the potential of AC to selectively disrupt cancer cell resistance mechanisms and bolster the efficacy of nanoparticle-based therapies. The results indicate the effectiveness of AC stimulation in circumventing the limitations inherent in current nanotechnology-based drug delivery systems but also illustrates its transformative potential for treating aggressive, drug-resistant brain tumors.

The molecular mechanisms of chemotherapeutic resistance in tumors (Review).

Chemotherapy remains a prevalent treatment for a wide range of tumors; however, the majority of patients undergoing conventional chemotherapy experience varying levels of chemoresistance, ultimately leading to suboptimal outcomes. The present article provided an in‑depth review of chemotherapy resistance in tumors, emphasizing the underlying factors contributing to this resistance in tumor cells. It also explored recent advancements in the identification of key molecules and molecular mechanisms within the primary chemoresistant pathways.

Advances in Melanoma: From Genetic Insights to Therapeutic Innovations.

Advances in melanoma research have unveiled critical insights into its genetic and molecular landscape, leading to significant therapeutic innovations. This review explores the intricate interplay between genetic alterations, such as mutations in BRAF, NRAS, and KIT, and melanoma pathogenesis. The MAPK and PI3K/Akt/mTOR signaling pathways are highlighted for their roles in tumor growth and resistance mechanisms. Additionally, this review delves into the impact of epigenetic modifications, including DNA methylation and histone changes, on melanoma progression. The tumor microenvironment, characterized by immune cells, stromal cells, and soluble factors, plays a pivotal role in modulating tumor behavior and treatment responses. Emerging technologies like single-cell sequencing, CRISPR-Cas9, and AI-driven diagnostics are transforming melanoma research, offering precise and personalized approaches to treatment. Immunotherapy, particularly immune checkpoint inhibitors and personalized mRNA vaccines, has revolutionized melanoma therapy by enhancing the body's immune response. Despite these advances, resistance mechanisms remain a challenge, underscoring the need for combined therapies and ongoing research to achieve durable therapeutic responses. This comprehensive overview aims to highlight the current state of melanoma research and the transformative impacts of these advancements on clinical practice.

Next-generation sequencing technology in cancer

Next-generation sequencing (NGS) technology has revolutionized cancer research and treatment by enabling comprehensive analysis of genetic mutations, alterations, and expression profiles. It allows for the identification of cancer-driving mutations, helping in the development of targeted therapies. NGS provides detailed insights into tumor heterogeneity, resistance mechanisms, and clonal evolution. Its high-throughput capacity facilitates large-scale studies, improving our understanding of cancer genomics. By enabling personalized treatment plans based on individual genetic profiles, NGS holds promise for more effective and tailored cancer therapies. Early reviews on cancer genomics often lacked comprehensive coverage of emerging technologies. They missed in-depth analysis of NGS advancements, their impact on cancer research, and clinical applications. The review addresses this gap by reviving a thorough examination of NGS methods, their role in identifying genetic mutations, and their potential in personalized cancer treatment, thus providing essential insights into the evolving landscape of cancer genomics. The article covers the advancements in technology and bioinformatic approaches for NGS data analysis. It delves into NGS applications in research and diagnostics, particularly for solid cancer diagnosis. The review highlights specific cancer types, including hereditary breast cancer, melanoma, prostate cancer, thyroid cancer, lung cancer, and colorectal cancer. It explores NGS contribution in understanding the genetic basis of these cancers and its potential for enhancing personalized diagnosis and treatment strategies. This review rectifies early lacunas by providing a comprehensive and updated examination of NGS technology, addressing gaps in previous analyses and emphasizes bioinformatic approaches for NGS data analysis, crucial for interpreting vast genomic data accurately. The review meets the current need for a thorough understanding of NGS’s role in personalized cancer treatment and research.

Unveiling novel targets in melanoma under melanogenesis stimulation and photodynamic therapy by redox proteomics.

Melanogenesis-stimulated B16-F10 cells enter in a quiescent state, present inhibited mitochondrial respiration and increased reactive oxygen species levels. These alterations suggest that these cells may be under redox signaling, allowing tumor survival. The aim of this study was to evaluate redox-modified proteins in B16-F10 cells after melanogenesis stimulation and rose bengal-photodynamic therapy (RB-PDT). A redox proteomics label-free approach based on the biotin switch assay technique with biotin-HPDP and N-ethylmaleimide was used to assess the thiol-oxidized protein profile. Aconitase was oxidized at Cys-448 and Cys-451, citrate synthase was oxidized at Cys-202 and aspartate aminotransferase (Got2) was oxidized at Cys-272 and Cys-274, exclusively after melanogenesis stimulation. After RB-PDT, only guanine nucleotide-binding protein subunit beta-2-like 1 (Gnb2l1) was oxidized (Cys-168). In contrast, melanogenesis stimulation followed by RB-PDT led to the oxidation of different cysteines in Gnb2l1 (Cys-153 and Cys-249). Besides that, glyceraldehyde-3-phosphate dehydrogenase (Gapdh) presented oxidation at Cys-245, peptidyl-prolyl cis-trans isomerase A (Ppia) was oxidized at Cys-161 and 5,6-dihydroxyindole-2-carboxylic acid oxidase (Tyrp1) was oxidized at Cys-65, Cys-30, and Cys-336 after melanogenesis stimulation followed by RB-PDT. The redox alterations observed in murine melanoma cells and identification of possible target proteins are of great importance to further understand tumor resistance mechanisms.

A novel biomimetic nanoplasmonic sensor for rapid and accurate evaluation of checkpoint inhibitor immunotherapy

Immune checkpoint inhibitors (ICIs) emerged as promising immunotherapies for cancer treatment, harnessing the patient’s immune system to fight and eliminate tumor cells. However, despite their potential and proven efficacies, checkpoint inhibitors still face important challenges such as the tumor heterogeneity and resistance mechanisms, and the complex in vitro testing, which limits their widespread applicability and implementation to treat cancer. To address these challenges, we propose a novel analytical technique utilizing biomimetic label-free nanoplasmonic biosensors for rapid and reliable screening and evaluation of checkpoint inhibitors. We have designed and fabricated a low-density nanostructured plasmonic sensor based on gold nanodisks that enables the direct formation of a functional supported lipid bilayer, which acts as an artificial cell membrane for tumor ligand immobilization. With this biomimetic scaffold, our biosensing approach provides real-time, highly sensitive analysis of immune checkpoint pathways and direct assessment of the blocking effects of monoclonal antibodies in less than 20 min/test. We demonstrate the accuracy of our biomimetic sensor for the study of the programmed cell death protein 1 (PD1) checkpoint pathway, achieving a limit of detection of 6.7 ng/mL for direct PD1/PD-L1 interaction monitoring. Besides, we have performed dose–response inhibition curves for an anti-PD1 monoclonal antibody, obtaining a half maximal inhibitory concentration (IC50) of 0.43 nM, within the same range than those obtained with conventional techniques. Our biomimetic sensor platform combines the potential of plasmonic technologies for rapid label-free analysis with the reliability of cell-based assay in terms of ligand mobility. The biosensor is integrated in a compact user-friendly device for the straightforward implementation in biomedical and pharmaceutical laboratories.Graphical

Peroxiporins in Triple-Negative Breast Cancer: Biomarker Potential and Therapeutic Perspectives.

Triple-negative breast cancer (TNBC) remains one of the most challenging subtypes since it is initially characterized by the absence of specific biomarkers and corresponding targeted therapies. Advances in methodology, translational informatics, genomics, and proteomics have significantly contributed to the identification of therapeutic targets. The development of innovative treatments, such as antibody-drug conjugates and immune checkpoint inhibitors, alongside chemotherapy, has now become the standard of care. However, the quest for biomarkers defining therapy outcomes is still ongoing. Peroxiporins, which comprise a subgroup of aquaporins, which are membrane pores facilitating the transport of water, glycerol, and hydrogen peroxide, have emerged as potential biomarkers for therapy response. Research on peroxiporins reveals their involvement beyond traditional channeling activities, which is also reflected in their cellular localization and roles in cellular signaling pathways. This research on peroxiporins provides fresh insights into the mechanisms of therapy resistance in tumors, offering potential avenues for predicting treatment outcomes and tailoring successful TNBC therapies.

Next generation antimitotic β-carboline derivatives modulate microtubule dynamics and downregulate NF-κB, ERK 1/2 and phospho HSP 27

AimExploring the efficacy of β-carboline-based molecular inhibitors in targeting microtubules for the development of novel anticancer therapeutics. Materials and methodsWe synthesized a series of 1-Aryl-N-substituted-β-carboline-3-carboxamide compounds and evaluated their cytotoxicity against human lung carcinoma (A549) cells using the MTT assay. Normal lung fibroblast cells (WI-38) were used to assess compound selectivity. The mechanism of action of MJ-211 was elucidated through Western blot analysis of key pro-apoptotic and cell cycle regulatory proteins. Additionally, the inhibitory effect of MJ-211 on multicellular 3D spheroid growth of A549 cells was evaluated. Key findingsLead compound MJ-211 exhibited remarkable cytotoxicity against A549 cells with an IC50 of 4.075 μM at 24 h treatment and IC50 of 1.7 nM after 72 h of treatment, while demonstrating selectivity towards normal WI-38 cells. MJ-211 activated pro-apoptotic factors Bim and p53, and suppressed Cyclin B1, Phospho HSP 27, BubR1, Mad 2, ERK1/2, and NF-κB, indicating its potent antimitotic and pro-apoptotic effects. MJ-211 significantly suppressed the migration of cells and inhibited the growth of A549 cell-derived multicellular 3D spheroids, highlighting its efficacy in a more physiologically relevant model. SignificanceCytotoxic effect of MJ-211 against cancer cells, selectivity towards normal cells, and ability to modulate key regulatory proteins involved in apoptosis and cell cycle progression underscore its potential as a promising template for further anticancer lead optimization. Moreover, the inhibitory effect of MJ-211 on multicellular spheroid growth suggests its efficacy in combating tumor heterogeneity and resistance mechanisms, thereby offering a promising avenue for future anticancer drug development.

The Role of Nrf2 in the Regulation of Mitochondrial Function and Ferroptosis in Pancreatic Cancer.

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) represents the master regulator of the cellular antioxidant response and plays a critical role in tumorigenesis. This includes a preventive effect of Nrf2 on cell death through ferroptosis, which represents an essential mechanism of therapy resistance in malignant tumors, such as pancreatic ductal adenocarcinoma (PDAC) as one of the most aggressive and still incurable tumors. Addressing this issue, we provide an overview on Nrf2 mediated antioxidant response with particular emphasis on its effect on mitochondria as the organelle responsible for the execution of ferroptosis. We further outline how deregulated Nrf2 adds to the progression and therapy resistance of PDAC, especially with respect to the role of ferroptosis in anti-cancer drug mediated cell killing and how this is impaired by Nrf2 as an essential mechanism of drug resistance. Our review further discusses recent approaches for Nrf2 inhibition by natural and synthetic compounds to overcome drug resistance based on enhanced ferroptosis. Finally, we provide an outlook on therapeutic strategies based on Nrf2 inhibition combined with ferroptosis inducing drugs.

Delineating three-dimensional behavior of uveal melanoma cells under anchorage independent or dependent conditions

BackgroundAlthough rare, uveal melanoma (UM) is a life-threatening malignancy. Understanding its biology is necessary to improve disease outcome. Three-dimensional (3D) in vitro culture methods have emerged as tools that incorporate physical and spatial cues that better mimic tumor biology and in turn deliver more predictive preclinical data. Herein, we comprehensively characterize UM cells under different 3D culture settings as a suitable model to study tumor cell behavior and therapeutic intervention.MethodsSix UM cell lines were tested in two-dimensional (2D) and 3D-culture conditions. For 3D cultures, we used anchorage-dependent (AD) methods where cells were embedded or seeded on top of basement membrane extracts and anchorage-free (AF) methods where cells were seeded on agarose pre-coated plates, ultra-low attachment plates, and on hanging drops, with or without methylcellulose. Cultures were analyzed for multicellular tumor structures (MCTs) development by phase contrast and confocal imaging, and cell wellbeing was assessed based on viability, membrane integrity, vitality, apoptotic features, and DNA synthesis. Vascular endothelial growth factor (VEGF) production was evaluated under hypoxic conditions for cell function analysis.ResultsUM cells cultured following anchorage-free methods developed MCTs shaped as spheres. Regardless of their sizes and degree of compaction, these spheres displayed an outer ring of viable and proliferating cells, and a core with less proliferating and apoptotic cells. In contrast, UM cells maintained under anchorage-dependent conditions established several morphological adaptations. Some remained isolated and rounded, formed multi-size irregular aggregates, or adopted a 2D-like flat appearance. These cells invariably conserved their metabolic activity and conserved melanocytic markers (i.e., expression of Melan A/Mart-1 and HMB45). Notably, under hypoxia, cells maintained under 3D conditions secrete more VEGF compared to cells cultured under 2D conditions.ConclusionsUnder an anchorage-free environment, UM cells form sphere-like MCTs that acquire attributes reminiscent of abnormal vascularized solid tumors. UM cells behavior in anchorage-dependent manner exposed diverse cells populations in response to cues from an enriched extracellular matrix proteins (ECM) environment, highlighting the plasticity of UM cells. This study provides a 3D cell culture platform that is more predictive of the biology of UM. The integration of such platforms to explore mechanisms of ECM-mediated tumor resistance, metastatic abilities, and to test novel therapeutics (i.e., anti-angiogenics and immunomodulators) would benefit UM care.

Circulating tumour DNA analysis and new uses of anti-EGFR monoclonal antibodies in patients with metastatic colorectal cancer

Currently, tumour tissue biopsy to determine RAS/BRAF gene alterations, assess microsatellite instability status, and determine HER‑2/neu gene amplification/hyperexpression is the gold standard of diagnosis and allows the selection of optimal molecularly targeted therapy when considering treatment strategies for patients with metastatic colorectal cancer. However, biopsy does not fully reflect the existing intratumoural heterogeneity and clonal evolution of tumour cells, which can often be the cause of therapeutic failures. In recent years, liquid biopsy has attracted increasing attention as an additional and potentially alternative non-invasive tool for molecular tumour profiling. Assessment of circulating tumour DNA allows changes in the genetic status of the tumour to be monitored and the «burden» of disease to be measured dynamically in real time. Advances in liquid biopsy technology have led to promising new strategies for the management of patients with metastatic colorectal cancer in late-line therapy. The standard drug arsenal in this group of patients is limited to either repeat administration of previously effective therapy or regorafenib and the combination of trifluridine/tipiracil with bevacizumab, which are characterized by limited clinical activity. However, thanks to the discovery of the NeoRAS wild-type phenomenon and the rechallenge strategy of anti-EGFR monoclonal antibodies based on the study of clonal selection and evolution of tumour cells, the administration of epidermal growth factor inhibitors in a molecularly selected by liquid biopsy population is accompanied by good tolerability and efficacy. Numerous clinical studies are ongoing to further understand the mechanisms of tumour resistance and to develop new evidence-based treatment approaches in order to realise the concept of personalised medicine.

Identification of a set of genes potentially responsible for resistance to ferroptosis in lung adenocarcinoma cancer stem cells

Scientific literature supports the evidence that cancer stem cells (CSCs) retain inside low reactive oxygen species (ROS) levels and are, therefore, less susceptible to cell death, including ferroptosis, a type of cell death dependent on iron-driven lipid peroxidation. A collection of lung adenocarcinoma (LUAD) primary cell lines derived from malignant pleural effusions (MPEs) of patients was used to obtain 3D spheroids enriched for stem‐like properties. We observed that the ferroptosis inducer RSL3 triggered lipid peroxidation and cell death in LUAD cells when grown in 2D conditions; however, when grown in 3D conditions, all cell lines underwent a phenotypic switch, exhibiting substantial resistance to RSL3 and, therefore, protection against ferroptotic cell death. Interestingly, this phenomenon was reversed by disrupting 3D cells and growing them back in adherence, supporting the idea of CSCs plasticity, which holds that cancer cells have the dynamic ability to transition between a CSC state and a non-CSC state. Molecular analyses showed that ferroptosis resistance in 3D spheroids correlated with an increased expression of antioxidant genes and high levels of proteins involved in iron storage and export, indicating protection against oxidative stress and low availability of iron for the initiation of ferroptosis. Moreover, transcriptomic analyses highlighted a novel subset of genes commonly modulated in 3D spheroids and potentially capable of driving ferroptosis protection in LUAD-CSCs, thus allowing to better understand the mechanisms of CSC-mediated drug resistance in tumors.

Abstract 11: Development of innovative off-the-shelf cancer immunotherapies with iPSC-derived CD8-positive T-cells

Abstract Chimeric antigen receptor (CAR) based T-cell immunotherapies have transformed the oncology landscape and evolved as part of the standard of care for several hematological malignancies. To generate CAR-T-cell products, autologous patient-derived αβT-cells are engineered with a CAR for tumor cell targeting. However, generating autologous T-cell products fails in a significant number of cases due to the poor quality and quantity of blood-derived T-cells and is associated with manufacturing and logistical complexity as well as high product costs. Therefore, it is crucial to develop strategies for off-the-shelf T-cell products as otherwise many patients are left without this treatment option. Manufacturing allogeneic T-cells from induced pluripotent stem cells (iPSCs) has a great potential to generate cell products with consistently high quality and scalable quantities. In addition, high fidelity gene-editing can be performed in iPSCs to address tumor resistance mechanisms and to prevent exhaustion of the T-cells. We have established a feeder-free differentiation protocol that enables robust production of iPSC-derived αβT-cells (iαβT). Flow cytometry and single cell transcriptome analysis ensure stringent monitoring of all process stages. To demonstrate functional activity of iαβT, cytotoxicity and cytokine release assays were performed. We compared the differentiation potential of iPSC lines derived from T-cells (t-iPSCs) to a hematopoietic stem cell-derived iPSC line with an αβTCR knock-in. Hematopoietic progenitor cells were induced from the different iPSC lines and differentiated into iαβT. During the differentiation process, surface markers CD45, CD5 and CD7 were displayed, and cells started to express αβTCRs. Importantly, iαβT derived from the different iPSC lines expressed CD8α and CD8β which is crucial for the function of cytotoxic T-cells. The CD4/CD8 double positive stage is an important step in T-cell development and was observed only when T-cells were generated from t-iPSC, but not from the TCR knock-in cells. Functional analysis of iαβT-cells confirmed their cytotoxic activity and potential to release cytokines after activation. Our scalable process for iαβT differentiation generates CD8-positive T-cells that secrete cytokines and show cytotoxic activity against tumor cells, demonstrating their potential as a promising cell source for TCR-T or CAR-T cancer immunotherapies. Citation Format: Riga Tawo, Michela Mirenda, Olivia Cypris, Philip Hublitz, Nadja Wagner, Michael Epstein, Michael Esquerre, Audrey Holtzinger, Monika Braun, Markus Dangl, Rodrigo Grandy, Daniel Sommermeyer. Development of innovative off-the-shelf cancer immunotherapies with iPSC-derived CD8-positive T-cells [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 11.

Abstract 3287: Learning to target CDK4/6 inhibitor resistance via a breast cancer-specific atlas of cellular mechanisms

Abstract Development of resistance to targeted, chemotherapeutic, and immune-oncology treatments alike is a major barrier to long-term remission in advanced cancer patients. We developed an integrated computational-experimental platform to mitigate treatment resistance by identifying therapeutics that selectively target clinical resistance mechanisms. The key innovation is the computational framework that comprehensively maps tumor-specific mechanisms of response and resistance to a treatment based on clinicogenomic and/or preclinical pharmacogenomic data. It then screens for drugs or targets that are synthetically lethal with these resistance mechanisms. Here we present the identification of therapeutic options that mitigate resistance to CDK4/6 inhibitors as a case study. Leveraging a breast cancer-specific atlas of cellular architecture, our framework learned genetic mechanisms of CDK4/6 inhibitor resistance from a high-throughput phenotypic screen of ~700 cell lines treated with the clinical CDK4/6 inhibitor palbociclib. The learned mechanisms formed a parsimonious set of hierarchically linked protein complexes that coordinate G1-to-S transition (P=5.6 × 10−12). The mechanisms unified distinct resistant populations such as samples resistant due to CDK4/6-Rb checkpoint bypass and those resistant due to growth factor receptor activation. Applied to 70 ER+ metastatic breast cancer patients in a real-world dataset, the learned resistance mechanisms accurately predicted patient response to palbociclib (P=3 × 10−4, log-rank test). Computationally screening 20K clinical-grade small molecules and gene targets for therapeutic options synthetically lethal with the learned resistance mechanisms identified the known CDK4/6 resistance targets CDK2, CCNE1, and E2F. It also identified a target that, to our knowledge, has not previously been rigorously evaluated for its potential to mitigate CDK4/6 inhibitor resistance. In-vitro, monotherapy inhibition of the target by siRNA selectively decreased growth of breast cancer cell lines resistant to CDK4/6 inhibitors by 52% compared to CDK4/6i sensitive cell lines (P=0.023, Mann-Whitney U-test). As a combination therapy, inhibition of the target by siRNA selectively increased the sensitivity to palbociclib by nearly an order of magnitude in an otherwise resistant breast cancer cell line (expected vs observed IC50: 9.7 uM vs. 1.3 uM) while not having a substantial effect in a palbociclib-sensitive cell line (expected vs observed IC50: 0.57 uM vs. 0.56 uM). Finally, while monotherapy inhibition of the target and CDK4/6 induced cell cycle arrest, the combination strongly induced apoptosis in cell lines resistant to CDK4/6 inhibitors. These results highlight how computational deconvolution of tumor resistance mechanisms enable algorithmic identification of therapeutic options to target treatment-resistant cell populations. Citation Format: Adam Yaari, Eduardo Farias, Francisco Guedes, Oliver Priebe, Lee McDaniel, Tyler Earnest, Trey Ideker, Maxwell A. Sherman. Learning to target CDK4/6 inhibitor resistance via a breast cancer-specific atlas of cellular mechanisms [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 3287.

Chimeric Antigen Receptor (CAR)-T Cell Therapy for Non-Hodgkin's Lymphoma.

This review focuses on the use of chimeric antigen receptor (CAR)-T cell therapy to treat non-Hodgkin's lymphoma (NHL), a classification of heterogeneous malignant neoplasms of the lymphoid tissue. Despite various conventional and multidrug chemotherapies, the poor prognosis for NHL patients remains and has prompted the utilization of groundbreaking personalized therapies such as CAR-T cells. CAR-T cells are T cells engineered to express a CAR that enables T cells to specifically lyse tumor cells with extracellular expression of a tumor antigen of choice. A CAR is composed of an extracellular antibody fragment or target protein binding domain that is conjugated to activating intracellular signaling motifs common to T cells. In general, CAR-T cell therapies for NHL are designed to recognize cellular markers ubiquitously expressed on B cells such as CD19+, CD20+, and CD22+. Clinical trials using CAR-T cells such as ZUMA-7 and TRANSFORM demonstrated promising results compared to standard of care and ultimately led to FDA approval for the treatment of relapsed/refractory NHL. Despite the success of CAR-T therapy for NHL, challenges include adverse side effects as well as extrinsic and intrinsic mechanisms of tumor resistance that lead to suboptimal outcomes. Overall, CAR-T cell therapies have improved clinical outcomes in NHL patients and generated optimism around their future applications.

Abstract B075: Spatial genomics identifies cancer cell cytokines regulating ovarian cancer immunity

Abstract Ovarian cancer (OvCa) is among the most aggressive and lethal cancers with a poor response to immune checkpoint blockade (ICB) despite its modest tumor mutational burden and expression of known cancer antigens. Consequently, it is critical to understand the molecular mechanisms of tumor immune escape and resistance to immunotherapies. Intercellular signaling between cancer cells and the tumor microenvironment (TME) is a crucial determinant of tumor immunity and given the immunosuppressive nature of OvCa TME, it is of utmost importance to dissect how this immunosuppressed TME is established. To begin to determine which cancer-cell genes may function cell externally in controlling of OvCa immunity, we employed a first-of-its-kind spatial functional genomics approach we developed, called Perturb-map, which enables dozens of CRISPR gene knock-out (KO) cells to be resolved in a tissue/tumor by imaging, along with cells in the local TME. We used Perturb-map to determine how 35 different genes, identified through ligand-receptor analysis of ovarian tumors, influenced tumor response to anti-PD-1 treatment in an OvCa mouse model. Strikingly, Il4 KO tumors were unique amongst the 35 gene KOs making the tumors significantly more responsive to anti-PD-1 treatment despite not impacting tumor growth in the absence of immunotherapy. Further experiments with single CRISPR KO tumors validated the Perturb-map finding, where Il4 KO combined with anti-PD-1 treatment resulted in a major reduction in tumor growth and a significant increase in survival. Cyclic immunofluorescence (CyCIF) multiplex imaging revealed that loss of cancer cell-derived IL-4 led to a significant reduction in pro-tumor macrophages and an increase in dendritic cell (DC) subsets as well as both progenitor and terminally exhausted CD8 T cells. Upon anti-PD-1 treatment, Il4 KO tumors had significant enrichment for activated CD8 T cells and B cells. Importantly, spatial analysis of the tumor samples identified enrichment of T follicular helper cells, B cells, and DCs in tertiary lymphoid structures in tumors with combined IL-4 loss and PD-1 inhibition. Further, we analyzed a human OvCa single-cell transcriptomics dataset and demonstrated that an IL-4 response signature is expressed in tumor-associated macrophages, indicating active IL-4 signaling in human ovarian tumors. Importantly, we employed a CRISPR-knock-in expression reporter in mouse tumors as well as immunohistochemistry of both human and mouse tumor samples to confirm that IL-4 is expressed by cancer cells. Thus, we identified cancer cell-secreted IL-4 as a potent regulator of OvCa TME and response to ICB. Given that an anti-IL-4-receptor antibody is already in clinical use to treat immune diseases, our findings have the potential to be translated to the clinical management of this dreadful disease. Citation Format: Gurkan Mollaoglu, Alexander Tepper, Hunter Potak, Luisanna Pia, Angelo Amabile, Chiara Falcomata, Jaime Mateus-Tique, Noam Rabinovich, Rachel Brody, Lindsay Browning, Jia-Ren Lin, Peter Sorger, Sandro Santagata, Miriam Merad, Alessia Baccarini, Brian Brown. Spatial genomics identifies cancer cell cytokines regulating ovarian cancer immunity [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B075.