Cellular Resistance Mechanisms Research Articles

SnRNA-seq of adipose tissues reveals the potential cellular and molecular mechanisms of cold and disease resistance in Mongolian cattle

BackgroundMongolian cattle are local breeds in northern China with excellent adaptability to harsh environmental conditions. Adipose tissues play essential roles in tolerance to cold and disease, but the associated cellular and molecular mechanisms are unclear.MethodsSingle-nucleus RNA sequencing (snRNA-seq) was performed on the adipose tissues from the subcutaneous (SAT), greater omentum (OAT) and perirenal (PAT) of 3 healthy cattle. The adipogenic trajectory was analyzed, and the functional roles of gene of interest were verified in vitro.ResultsThere were different cell subpopulations in adipose tissues. The lipid-deposition adipocytes identified by the PTGER3 marker exhibited outstanding characteristics in SAT. In PAT and OAT, aldosterone was expressed to provide clues for the differential brown adipocytes. Among the DEGs by comparing OAT with SAT and PAT with OAT, C3 was significantly expressed in most of the cell populations in SAT. G0S2, LIPE, LPIN1, PTGER3 and RGCC took part in the adipogenic trajectory from preadipocyte commitment to mature adipocytes. S100A4 expression affected Ca2+ signaling and the expression of UCP1 ~ 3, FABP4 and PTGER3.ConclusionThe cell heterogeneity and genes expressed in adipose tissues of Mongolian cattle not only determine the endocrine and energy storage, but contribute to adapt to cold and disease resistance.

Research Progress of Plant-Derived Natural Products against Drug-Resistant Cancer.

As one of the malignant diseases globally, cancer seriously endangers human physical and mental health because of its high morbidity and mortality. Conventional cancer treatment strategies, such as surgical resection and chemoradiotherapy, are effective at the early stage of cancer but have limited efficacy for advanced cancer. Along with cancer progress and treatment, resistance develops gradually within the population of tumor cells. As a consequence, drug resistance become the major cause that leads to disease progression and poor clinical prognosis in some patients. The mechanisms of cancer drug resistance are quite complex and involve various molecular and cellular mechanisms. Therefore, exploring the mechanisms and finding specific targets are becoming imperative to overcome drug resistance. In recent years, plant-derived natural products have been evaluated as potential therapeutic candidates against cancer with drug resistance due to low side effects and high anticancer efficacy. A growing number of studies have shown that natural products can achieve superior antitumor effects through multiple signaling pathways. The mechanisms include regulation of multiple drug resistance (MDR)-related genes, inhibition of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, induction of autophagy, and blockade of the cell cycle. This paper reviews the molecular and cellular mechanisms of cancer drug resistance, as well as the therapeutic effects and mechanisms of plant-derived natural products against cancer drug resistance. It provides references for developing therapeutic medication for drug-resistant cancer treatment with high efficacy and low side effects.

Effects of small heat shock proteins from thermotolerant bacteria on the stress resistance of Escherichia coli to temperature, pH, and hyperosmolarity

Small heat shock proteins (HSPs), such as HSP20, represent cellular thermal resistance mechanisms, to avoid protein aggregation at elevated temperatures. Recombinantly expressed HSP20s serve as a molecular tool for improving the tolerance of living cells to various physical and chemical stressors. Here, we aimed to heterologously express 18 HSP20s from 12 thermotolerant bacteria in Escherichia coli and evaluate their effects on various physical and chemical cellular stresses. Seventeen HSP20s were successfully expressed as soluble proteins. Recombinant E. coli cells were subjected to heat, cold, acidic, alkaline, and hyperosmolar stress to evaluate the effects of HSP20 proteins on stress resistance. Notably, the overexpression of 15 HSP20s enhanced the stress resistance of E. coli compared to that of the control strain. In particular, HSPs from Tepidimonas sediminis and Oceanithermus profundus improved the stress tolerance of E. coli under all tested conditions. In addition, E. coli harboring HSP20 from T. sediminis retained cell viability even after heat treatment at 52 °C for 5 days. To our knowledge, this is the first report of E. coli tolerance to prolonged (> 100 h) high-temperature stress. These findings indicate the potential of thermotolerant HSPs as molecular tools for improving stress tolerance in E. coli.

Selinexor Disrupts Epigenetic Programing and Modulates Immunogenicity in Multiple Myeloma

Introduction: Multiple myeloma (MM), remains an incurable, but treatable, malignancy that has benefitted from the integration of novel immunologic agents in clinical use, such as monoclonal antibody, bispecific T cell engager (TCE) and chimeric antigen receptor T (CAR-T) cell and antibody-drug conjugate based therapies. While these agents have shown great promise in treating relapsed/refractory MM patients, there is heterogeneity in response to therapy due to differences in patients' immune tumor microenvironments (iTME) and immunogenicity of the tumor cells. Previously (Sudalagunta et. al., ASH 2021), we used functional genomic screening of 53 MM samples to characterize the transcriptomic profile associated with sensitivity to the selective nuclear export inhibitor, selinexor (SELI), which was shown to be inversely correlated with the transcriptomic profile associated with sensitivity to an anti-CD38 monoclonal antibody in 106 MM samples. In this study, we investigate the cellular mechanisms of SELI resistance leading to an immunogenic cell state. Methods: Primary cells from an additional set of MM patients (n=65) were used for paired bulk RNAseq and an organotypic ex vivo drug sensitivity assay. Cellular viability was assessed using digital analysis of brightfield longitudinal images, and drug sensitivity was assessed as area under the dose-time-response curve (AUC) for each sample. We conducted gene set enrichment analysis (GSEA) on paired RNAseq data and ex vivo AUC of SELI. Individual genes and their respective correlations with ex vivo AUC of SELI were ranked from highest to lowest. We used RNAseq to examine the role of acute exposure to SELI on CD138-selected cells from paired pre/post treatment bone marrow specimens of five MM patients enrolled in the NCT02186834 clinical trial, taken prior to treatment with SELI and dexamethasone (DEX) and one/two weeks post-induction. Paired t-tests were carried out to quantify the changes. Results: In our previous analysis of 844 MM samples, a subset of the genes whose expression is positively correlated with SELI AUC are controlled by H3K27me3, which is catalyzed by nuclear accumulation of EZH2. Tripathi et. al. (AACR Annual Meeting 2022 & 2023) have demonstrated that XPO1 and EZH2 form a complex, and inhibition of XPO1 activity blocks export of EZH2. GSEA of a new cohort of 65 MM patient samples demonstrated increased ex vivo SELI sensitivity (lower AUC, hence negative correlation) in samples with higher expression of known epigenetic regulators of the polycomb repressive complex 2 (PRC2), such as EZH2, SUZ12, and EED; as shown in Figure a, which led to lowering of expression of genes bound by these proteins due to gene silencing via H3K27me3, shown in red in Figure a, according to GSEA of ChIPseq databases. Furthermore, gene expression profiles of paired pre/post SELI/DEX treatment samples showed reduced expression of genes associated with H3K27me3 histone modification (paired t-test p=2.06e-83, 1.1061e-81, 2.56e-80, 5.15e-12, 6.22e-4) in all five patients. These observations suggest that XPO1 inhibition leads to nuclear accumulation of epigenetic modulators like EZH2, SUZ12, and EED, which catalyze H3K27me3 activity leading to silencing of H3K27me3-regulated genes. GSEA of paired ex vivo AUC and RNAseq data for an EZH2 inhibitor (EPZ-6438) resulted in negative correlation between expression of genes in PRC2 and ex vivo sensitivity, similar to SELI. Interestingly, we observed that this epigenetic mechanism of resistance to SELI is inversely associated with key immunological targets in MM such as CD74, SLAMF7, CD38, CD46, TNFRSF13B, and LY9 as shown in Figure a. In Figure b, we present a graphical visualization of intracellular changes due to exposure to SELI in a naïve and a SELI-resistant MM cell. Conclusions: Here we propose that SELI disrupts EZH2 subcellular localization, dysregulating H3K27me3-gene suppression. Sequential BM biopsies from SELI treated patients demonstrate decreased expression of H3K27me3-regulated genes, suggesting increased EZH2 activity by nuclear accumulation. Finally, SELI-resistant primary cells have increased expression of EZH2 regulated immunotherapy target genes (e.g. CD38, SLAMF7), suggesting a possible therapeutic strategy of sequential treatment of SELI followed by immunotherapy. Using multiparameter flow cytometry of samples pre/post SELI/DEX induction, we will study SELI's effect on iTME.

GITR and TIGIT immunotherapy provokes divergent multicellular responses in the tumor microenvironment of gastrointestinal cancers

BackgroundUnderstanding the mechanistic effects of novel immunotherapy agents is critical to improving their successful clinical translation. These effects need to be studied in preclinical models that maintain the heterogenous tumor microenvironment (TME) and dysfunctional cell states found in a patient’s tumor. We investigated immunotherapy perturbations targeting co-stimulatory molecule GITR and co-inhibitory immune checkpoint TIGIT in a patient-derived ex vivo system that maintains the TME in its near-native state. Leveraging single-cell genomics, we identified cell type-specific transcriptional reprogramming in response to immunotherapy perturbations.MethodsWe generated ex vivo tumor slice cultures from fresh surgical resections of gastric and colon cancer and treated them with GITR agonist or TIGIT antagonist antibodies. We applied paired single-cell RNA and TCR sequencing to the original surgical resections, control, and treated ex vivo tumor slice cultures. We additionally confirmed target expression using multiplex immunofluorescence and validated our findings with RNA in situ hybridization.ResultsWe confirmed that tumor slice cultures maintained the cell types, transcriptional cell states and proportions of the original surgical resection. The GITR agonist was limited to increasing effector gene expression only in cytotoxic CD8 T cells. Dysfunctional exhausted CD8 T cells did not respond to GITR agonist. In contrast, the TIGIT antagonist increased TCR signaling and activated both cytotoxic and dysfunctional CD8 T cells. This included cells corresponding to TCR clonotypes with features indicative of potential tumor antigen reactivity. The TIGIT antagonist also activated T follicular helper-like cells and dendritic cells, and reduced markers of immunosuppression in regulatory T cells.ConclusionsWe identified novel cellular mechanisms of action of GITR and TIGIT immunotherapy in the patients’ TME. Unlike the GITR agonist that generated a limited transcriptional response, TIGIT antagonist orchestrated a multicellular response involving CD8 T cells, T follicular helper-like cells, dendritic cells, and regulatory T cells. Our experimental strategy combining single-cell genomics with preclinical models can successfully identify mechanisms of action of novel immunotherapy agents. Understanding the cellular and transcriptional mechanisms of response or resistance will aid in prioritization of targets and their clinical translation.

Valproic acid reprograms the metabolic aberration of cisplatin treatment via ALDH modulation in triple-negative breast cancer cells.

We recently demonstrated that the histone deacetylase inhibitor valproic acid (VPA) reprograms the cisplatin-induced metabolome of triple-negative breast cancer (TNBC) cells, including a shift in hexose levels. Accordingly, here, we tested the hypothesis that VPA alters glucose metabolism in correlation with cisplatin sensitivity. Two TNBC cell lines, MDA-MB-231 (a cisplatin-resistant line) and MDA-MB-436 (a cisplatin-sensitive line), were analyzed. The glycolysis and oxidative metabolism were measured using the Glycolysis Stress Test kit. The expression of aldehyde dehydrogenases (ALDHs), enzymes linked to drug resistance, was investigated by Western blot and real-time PCR analyses. We additionally studied the influence of ALDH inhibition by disulfiram on the viability of MDA-MB-231 cells and on a TNBC patient-derived organoid system. Cisplatin treatment reduced the extracellular acidification rate in MDA-MB-436 cells but not MDA-MB-231 cells, whereas VPA addition increased the extracellular acidification rate in both cell lines. VPA further reduced the oxygen consumption rate of cisplatin-treated MDA-MB-436 cells, which correlated with cell cycle alterations. However, in MDA-MB-231 cells, the cell cycle distribution did not change between cisplatin/VPA-cisplatin treatments. In both cell lines, VPA increased the expression of ALDH isoform and ALDH1A1 expression. However, only in MDA-MB-231 cells, VPA synergized with cisplatin to augment this effect. Disulfiram sensitized the cells to the cytotoxic effects of the VPA-cisplatin combination. Furthermore, the disulfiram-VPA-chemotherapy combination was most effective in TNBC organoids. Our results show that ALDH overexpression may act as one mechanism of cellular resistance to VPA in TNBC and that its inhibition may enhance the therapeutic efficacy of VPA-chemotherapeutic drug combinations.

OS07.5.A GLIOBLASTOMA VESSEL CO-OPTION AND TRANSITION TO A RESISTANT CELL STATE ARE INDUCED BY CHEMORADIATION

Abstract BACKGROUND Glioblastoma (GB) is one of the deadliest types of human cancer. Despite a very aggressive treatment regime, including resection of the tumor, radiation, and chemotherapy, the recurrence rate is more than 90%. Recurrence is mostly caused by the regrowth of highly invasive and resistant cells that have spread from the tumor bulk and are not removed by resection. To develop an effective therapeutic approach, we need to better understand the underlying molecular and cellular mechanisms of GB chemoradiation resistance and tumor spreading. MATERIAL AND METHODS To dynamically follow the changes occurring in GB post-therapy and investigate its relationship with vascular microenvironment, we employed multiple bulk and single-cell RNA-Seq analyses, phosphoproteome, in vitro and in vivo real-time imaging, organotypic cultures and functional assays, digital pathology, and spatial transcriptomics on patient material or preclinical models of GB. RESULTS We demonstrated that chemoradiation and the brain vasculature induce a transition to a functional cell state, which we named VC-Resist. This cell state is midway through the transcriptomic axis between proneural and mesenchymal GB cells and is closer to the AC/MES-like state. Better cell survival, G2M-arrest, activation of senescence/stemness pathways make this GB cell state more resistant to therapy. Notably, these persister GB cells are highly vessel co-opting, allowing homing to the perivascular niche, which, in turn, increases their transition to this cell state and resistance to therapy. Molecularly, the transition to the VC-Resist cell state is driven by FGF-FGFR1 signaling, which leads to the activation of DNA damage repair and YAP1 pathways. CONCLUSION These findings demonstrate that the perivascular niche and GB cell plasticity jointly generate a vicious loop that leads to resistance to therapy and brain infiltration during GB recurrence. SUPPORT This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 (Grant Agreement No. 805225), the INSERM-CNRS ATIP-Avenir grant, the NanoTheRad grant from Paris-Saclay University, Fondation ARC pour la recherche sur le cancer, Campus France and Canceropole Ile-de-France (2022-1-EMERG-06-ICR-1).

Colorectal cancer: epidemiology, carcinogenesis, molecular subtypes and cellular mechanisms of therapy resistance (analytical review)

This article analyzes the statistical data on colorectal cancer in Russia and in the world, including incidence, mortality and survival. The main pathways of colorectal cancer carcinogenesis, molecular subtypes and their influence on the difference in lesions of the proximal and distal large intestine are presented. The paper provides an overview of the leading chemotherapy agents and targeted therapy in colorectal cancer, as well as the main reasons for the development of therapeutic resistance, including changes in the cellular microenvironment of the tumor.

Cellular resistance mechanisms in cancer and the new approaches to overcome resistance mechanisms chemotherapy.

Despite major advancements in cancer healing approaches over the last few decades, chemotherapy remains the most popular malignancy treatment. Chemotherapeutic drugs are classified into many kinds based on their mechanism of action. Multidrug resistance (MDR) is responsible for approximately 90% of fatalities in malignancy cases treated with standard chemotherapeutics or innovative targeted medicines. Many innovative prospective anti-cancer medicines displayed high anti-cancer efficacy in a single application. However, combining them with other medications improves cancer treatment efficacy. This supports the belief that a combination of drugs is significantly more effective than a single medicine. Due to the intricacy of MDR processes and the diversity of tumor illnesses, there will rarely be a single medicine that can be utilized to treat all types of cancer. Finding new medications that can reverse MDR in malignancy cells will augment efficacy of chemotherapeutic agents and allow us to treat cancers that are now incurable.

Abstract OT3-05-01: Adaptive Multi-Drug Treatment of Evolving Cancers (AMTEC): A Phase II, Open-Label, Study of Olaparib in Combination with either Durvalumab, Selumetinib or Capivasertib, or Ceralasertib Monotherapy in Patients with Metastatic TNBC

Abstract BACKGROUND PARP inhibitors (PARPi) afford a rational therapeutic strategy in metastatic TNBC (mTNBC) due to the high incidence of dysregulated DNA damage repair mechanisms and high-level genomic instability that resemble tumors originating in germline BRCA-mutated carriers. However, PARPi monotherapy has limited efficacy in BRCA wild-type mTNBC; in BRCA mutant disease following initial response, compensatory mechanisms inevitably restore replication fork protection. AMTEC leverages pre- and on-therapy biopsies from a 4-week PARPi monotherapy run-in period for personalized biomarker-driven patient selection to interdict adaptive resistance to the PARPi. Data from our pilot study (NCT03544125) and from Arm 1 of AMTEC (olaparib + durvalumab) identified PI3K-AKT, RAS-MEK, and ATR/CHK1/WEE1 as targetable pathways contributing to PARPi adaptive resistance in individual participants. Clinically validated assays (DNA, RNA, and protein) enable the identification of cellular mechanisms of PARPi sensitivity and resistance in individual patients and further reveal combined drug treatments that could prevent emergence of PARPi resistance. METHODS AMTEC is a non-comparative, multi-arm, open-label, phase II study to assess the efficacy of combining olaparib (ola) with durvalumab (dur), or MEKi, selumetinib (sel), or AKTi, capivasertib (cap), or monotherapy with ATRi, ceralasertib, (cer mono) in mTNBC patients. Participants with biopsy proven mTNBC (ER< 10%, PR< 10%, and HER-2 non-amplified), AR< 80% are eligible. - Participants undergo a pre-treatment biopsy, then start a 28-day induction with ola (300 mg PO BID, D1-28). On C1D14, patients undergo a repeat, on-treatment biopsy. Clinically validated assays (DNA, RNA, protein) from both biopsies inform patient assignment to a specific ola combination arm starting on C2D1: - Arm 1 tumor immune activated: ola + dur (1500 mg IV Q4W) - Arm 2 RAS-MEK-ERK pathway activation: ola + sel (BSA-based BID D1-28) - Arm 3 PI3K-AKT pathway activation: ola + cap (400 mg PO BID, 4 days on/3 days off) - Arm 4: If not eligible for Arms 1-3 (per biomarker selection criteria): Cer mono (240 mg PO BID D1-14) Endpoints: The primary endpoint is objective response rate (ORR per RECIST 1.1). Secondary endpoints include safety and toxicity, clinical benefit rate, duration of response, and survival. Statistical Methods: - Arm 1 will enroll 28 patients to detect an ORR difference of 20% (H0: π = 0.15 and Ha: π =0.35). Arm 1 will continue on to stage 2 if ORR ≥3 of the first 15 patients. The null hypothesis for Arm 1 is rejected if ≥ 7/28 patient achieve a response. - Arms 2, 3, and 4, will each enroll 22 patients to detect an ORR difference of 25% (H0: π = 0.15 and Ha: π =0.40). Arms 2, 3, and 4 will each continue on to stage 2 if ORR ≥2 of first 11 patients in each arm, respectively. The null hypothesis for Arm 2, 3, and 4 is rejected if ≥ 7/22 patients achieve a response in each arm, respectively. For arms 2 and 3, if there are ≥5/11 responses, the trial will open a biomarker negative expansion cohort for each arm (N = 19 patients/arm). ENROLLMENT The study was activated on 1/7/2019. Arm 1 met pre-specified interim analysis criteria in 12/2020, and accrual to stage 2 began in 1/2021. Arms 2, 3, and 4 start enrolling in Q4 of 2022. Up to 132 patients will be enrolled. Clinical trial information: NCT03801369 Contact information: For more information or to refer a patient, email hobbev@ohsu.edu Citation Format: Evthokia Hobbs, Gordon Mills, Jeong Lim, Marlana Klinger, Kiara Siex, Sidney Huszti, Annie Yang, Danielle Galipeau, Christina Zheng, Lauren Murray, Becky Goodford, Nicholas Marter-Sanders, Anastasiya Olson, Jayne Stommel, Brett Johnson, Jamie Keck, Ben Kong, Allison Solanki, Shaun Goodyear, Christopher Corless, Joe Gray, Mitri Zahi. Adaptive Multi-Drug Treatment of Evolving Cancers (AMTEC): A Phase II, Open-Label, Study of Olaparib in Combination with either Durvalumab, Selumetinib or Capivasertib, or Ceralasertib Monotherapy in Patients with Metastatic TNBC [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-05-01.

Pathogenic Drug Resistant Fungi: A Review of Mitigation Strategies.

Fungal pathogens cause significant human morbidity and mortality globally, where there is a propensity to infect vulnerable people such as the immunocompromised ones. There is increasing evidence of resistance to antifungal drugs, which has significant implications for cutaneous, invasive and bloodstream infections. The World Health Organization (WHO) published a priority list of fungal pathogens in October 2022, thus, highlighting that a crisis point has been reached where there is a pressing need to address the solutions. This review provides a timely insight into the challenges and implications on the topic of antifungal drug resistance along with discussing the effectiveness of established disease mitigation modalities and approaches. There is also a need to elucidate the cellular and molecular mechanisms of fungal resistance to inform effective solutions. The established fungal decontamination approaches are effective for medical device processing and sterilization, but the presence of pathogenic fungi in recalcitrant biofilms can lead to challenges, particularly during cleaning. Future design ideas for implantable and reusable medical devices should consider antifungal materials and appropriates for disinfection, and where it is relevant, sterilization. Preventing the growth of mycotoxin-producing fungi on foods through the use of appropriate end-to-end processes is advisable, as mycotoxins are recalcitrant and challenging to eliminate once they have formed.

A monoadduct generating Ru(ii) complex induces ribosome biogenesis stress and is a molecular mimic of phenanthriplatin.

Ruthenium complexes are often investigated as potential replacements for platinum-based chemotherapeutics in hopes of identifying systems with improved tolerability in vivo and reduced susceptibility to cellular resistance mechanisms. Inspired by phenanthriplatin, a non-traditional platinum agent that contains only one labile ligand, monofunctional ruthenium polypyridyl agents have been developed, but until now, few demonstrated promising anticancer activity. Here we introduce a potent new scaffold, based on [Ru(tpy)(dip)Cl]Cl (tpy = 2,2':6',2''-terpyridine and dip = 4,7-diphenyl-1,10-phenanthroline) in pursuit of effective Ru(ii)-based monofunctional agents. Notably, the extension of the terpyridine at the 4' position with an aromatic ring resulted in a molecule that was cytotoxic in several cancer cell lines with sub-micromolar IC50 values, induced ribosome biogenesis stress, and exhibited minimal zebrafish embryo toxicity. This study demonstrates the successful design of a Ru(ii) agent that mimics many of the biological effects and phenotypes seen with phenanthriplatin, despite numerous differences in both the ligands and metal center structure.

Structure-function analyses of coiled-coil immune receptors define a hydrophobic module for improving plant virus resistance.

Plant immunity relies on nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) that detect microbial patterns released by pathogens, and activate localized cell death to prevent the spread of pathogens. Tsw is the only identified resistance (R) gene encoding an NLR, conferring resistance to tomato spotted wilt orthotospovirus (TSWV) in pepper species (Capsicum, Solanaceae). However, molecular and cellular mechanisms of Tsw-mediated resistance are still elusive. Here, we analysed the structural and cellular functional features of Tsw protein, and defined a hydrophobic module to improve NLR-mediated virus resistance. The plasma membrane associated N-terminal 137 amino acid in the coiled-coil (CC) domain of Tsw is the minimum fragment sufficient to trigger cell death in Nicotiana benthamiana plants. Transient and transgenic expression assays in plants indicated that the amino acids of the hydrophobic groove (134th-137th amino acid) in the CC domain is critical for its full function and can be modified for enhanced disease resistance. Based on the structural features of Tsw, a super-hydrophobic funnel-like mutant, TswY137W, was identified to confer higher resistance to TSWV in a SGT1 (Suppressor of G-two allele of Skp1)-dependent manner. The same point mutation in a tomato Tsw-like NLR protein also improved resistance to pathogens, suggesting a feasible way of structure-assisted improvement of NLRs.

Euphorbia tirucalli latex loaded polymer nanoparticles: Synthesis, characterization, in vitro release and in vivo antinociceptive action.

The encapsulation of drugs in micro and nanocarriers has helped to resolve mechanisms of cellular resistance and decrease drug side effects as well. In this study, poly(D,L-lactide-co-glycolide) (PLGA) was used to encapsulate the Euphol active substance-containing latex from Euphorbia tirucalli (E-latex). The nanoparticles (NP) were prepared using the solvent evaporation method and the physical and chemical properties were evaluated using spectrophotometric techniques. FTIR was used to prove the formation of the ester bond between the E-latex and PLGA-NP. The UV-Vis spectroscopic technique was used to show that more than 75% of the latex was encapsulated; the same technique was used to determine the release profile of the compound at different pH values, as well as determining the speed with which the process occurs through kinetic models, and it was observed that the best adjustments occurred for the Korsmeyer-Peppas model and the Higuchi model. The DLS technique was used to determine the diameter of the particles produced as well as their zeta potential (ZP). The sizes of the particles varied from 497 to 764 nm, and it was observed that the increase in E-latex concentration causes a reduction in the diameter of the NP and an increase in the ZP (-1.44 to -22.7 mV), due to more functional groups from latex film being adsorbed to the NPs surfaces. The thermogravimetric experiments exhibit the glass transition temperatures (Tg) that is appropriate for the use of formulated NPs as a stable drug delivery device before use. The in vivo activity of E-NPs (30 and 100 mg/Kg/p.o.) was tested against carrageenan-induced mechanical hypernociception. The data demonstrated a significantly antinociceptive effect for E-NPs, suggesting that E-latex nanoencapsulation preserved its desired properties.

Corrigendum: Coupling next-generation sequencing to dominant positive screens for finding antibiotic cellular targets and resistance mechanisms in Escherichia coli.

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Resistance to Trastuzumab.

Simple SummaryTrastuzumab is a humanized antibody that has significantly improved the management and treatment outcomes of patients with cancers that overexpress HER2. Many research groups, both in academia and industry, have contributed towards understanding the various mechanisms engaged by trastuzumab to mediate its anti-tumor effects. Nevertheless, data from several clinical studies have indicated that a significant proportion of patients exhibit primary or acquired resistance to trastuzumab therapy. In this article, we discuss underlying mechanisms that contribute towards to resistance. Furthermore, we discuss the potential strategies to overcome some of the mechanisms of resistance to enhance the therapeutic efficacy of trastuzumab and other therapies based on it.One of the most impactful biologics for the treatment of breast cancer is the humanized monoclonal antibody, trastuzumab, which specifically recognizes the HER2/neu (HER2) protein encoded by the ERBB2 gene. Useful for both advanced and early breast cancers, trastuzumab has multiple mechanisms of action. Classical mechanisms attributed to trastuzumab action include cell cycle arrest, induction of apoptosis, and antibody-dependent cell-mediated cytotoxicity (ADCC). Recent studies have identified the role of the adaptive immune system in the clinical actions of trastuzumab. Despite the multiple mechanisms of action, many patients demonstrate resistance, primary or adaptive. Newly identified molecular and cellular mechanisms of trastuzumab resistance include induction of immune suppression, vascular mimicry, generation of breast cancer stem cells, deregulation of long non-coding RNAs, and metabolic escape. These newly identified mechanisms of resistance are discussed in detail in this review, particularly considering how they may lead to the development of well-rationalized, patient-tailored combinations that improve patient survival.

P1246: DIFFERENTIAL ROLE OF MICROENVIRONMENT AND STROMAL-CELL INDUCED VENETOCLAX RESISTANCE IN MANTLE CELL LYMPHOMA

Background: Venetoclax is an inhibitor of the anti-apoptotic protein BCL-2 and a promising drug for MCL treatment. However, resistance towards venetoclax has already been reported in several MCL studies. There is growing evidence suggesting that interactions between lymphoma cells and their surrounding microenvironment might play a key role in not only promoting tumor cell survival but also facilitating resistance to certain chemotherapeutic agents, but little is known about the role of microenvironment in drug resistance in MCL. Aims: To understand the cellular and molecular resistance mechanisms mediated by the lymphoma microenvironment to prevent MCL patients from developing resistance to venetoclax treatment. To investigate the interactions of MCL cell lines with Mesenchymal Stromal Cells (MSCs), a key component of the microenvironment, in an in vitro co-culture system. Methods: MCL cell lines (MAVER-1, MINO) were cultured alone or in co-culture with HS-5, a human MSC cell line, or MS-5, a murine MSC cell line and exposed to different drugs, including venetoclax. Flow cytometry was used to assess cell viability. RNA sequencing of suspension and adherent MCL cells was used to identify differentially expressed genes. ELISA multiplex analysis was used to identify human or murine pro-inflammatory cytokines in co-culture supernatant. Results: Co-culture of MCL cell lines with both HS-5 and MS-5 induced resistance in MCL cells to doxorubicin and vincristine. However, only co-culture with MS-5 induced venetoclax resistance in MCL cells. Interestingly, conditioned media alone from MS-5 cultures was sufficient to induce venetoclax resistance, suggesting that soluble factors (such as cytokines and microvesicles) might mediate the venetoclax resistance mechanisms in MCL cells. Induction of apoptosis through MCL-1 inhibition with drug S6338 was also abrogated by co-culture with both HS-5 and MS-5, while BCL-xL inhibition using drug WEHI-549 was only ineffective in MINO cells in MS-5 co-culture. MCL cells in HS-5 co-cultures exhibited upregulation of genes involved in inflammatory response, cytokine signaling, and apoptosis, compared to MS-5 co-cultures. Accordingly, we identified unique sets of genes that were significantly upregulated in adherent MCL cells in HS-5 co-cultures, mainly involved in cell adhesion and cytokine signaling. Moreover, HS-5 co-cultures contained high levels of IL-6 and IL-8, corresponding with the increased gene expression levels of genes involved in the IL-6 signaling pathway in MCL cells. On the contrary, MS-5 co-cultures contained only high levels of murine KC/GRO, corresponding to human chemokine CXCL1. Image:Summary/Conclusion: We show that HS-5 provides MCL cells resistance to drugs, such as vincristine and doxorubicin, but not venetoclax, reflecting the different mechanisms of action of these therapies as well as their differential effects in the MCL microenvironment. MCL cells were resistant to venetoclax only in murine MSCs co-cultures, and further transcriptome and cytokine analysis supported this difference. Among others, our data questions how reliable murine MSCs are for obtaining translatable results, as MS-5 is the most used MSC cell line for co-cultures studies. These findings together provide a rationale to further investigate the role of MSCs in MCL drug resistance, as understanding the role of microenvironment in venetoclax sensitivity in MCL will potentially allow for more effective and targeted therapy.

Abstract 1068: Anti-DR5 agonist IgM antibody IGM-8444 combined with SMAC mimetic birinapant induces strong synergistic tumor cytotoxicity

Abstract Apoptosis is induced through extrinsic and intrinsic signaling pathways. Extrinsic apoptosis can be activated through multimerization of death receptor 5 (DR5), a tumor necrosis factor (TNF) receptor family member highly expressed in many cancers. However, cellular resistance mechanisms within the intrinsic pathway may limit DR5 activity, including inhibitor of apoptosis proteins (IAPs) that block caspase activity or promote pro-survival NFκB signaling. Second mitochondria-derived activator of caspases (SMAC) is an endogenous bivalent IAP antagonist. Birinapant, a bivalent SMAC mimetic that binds and degrades IAPs, has been evaluated through Phase 2, demonstrating good safety and on target activity but minimal efficacy as a monotherapy. We hypothesized that simultaneously targeting the extrinsic apoptotic pathway with IGM-8444, an anti-DR5 multivalent IgM agonist, and the intrinsic apoptotic pathway with birinapant could enhance tumor cell apoptosis. Human cancer cell lines including triple negative breast cancer (TNBC), head and neck, ovarian, colorectal, lung, and sarcomas, were screened for sensitivity to IGM-8444 and birinapant combination. Strong synergistic cytotoxicity was observed in vitro in 36/45 (80%) cancer cell lines, as measured by both Bliss synergy score and maximal killing. IGM-8444 and birinapant combination also induced synergistic cytotoxicity in cells with acquired resistance to DR5 agonist antibodies. By contrast, IGM-8444 and birinapant did not kill primary human hepatocytes in vitro, demonstrating the potential clinical safety for this combination. In vivo, the IGM-8444 and birinapant combination dose-dependently reduced tumor growth in a MDA-MB-231 TNBC model, with 8/10 tumor-free mice at the highest dose of birinapant tested. By comparison, birinapant combination with an anti-DR5 IgG agonist showed a modest response. IGM-8444 and birinapant also showed significant anti-tumor responses in additional cell line and patient-derived xenograft models, including 7/9 tumor-free animals in a HT-1080 fibrosarcoma model and 9/10 complete responses in a EBC-1 lung squamous cell lung carcinoma model. Lastly, pharmacodynamic biomarkers including cIAP1 degradation, caspase activation, and caspase-cleaved cytokeratin 18 in tumor and serum correlated with anti-tumor response. In summary, combined targeting of the extrinsic and intrinsic apoptotic pathways with IGM-8444 and birinapant respectively enhances tumor cytotoxicity in multiple preclinical models. The combination of IGM-8444 with birinapant is currently under evaluation in a Phase 1 study in patients with relapsed and/or refractory solid cancers (NCT04553692). Citation Format: Beatrice T. Wang, Melanie Desbois, Susan E. Calhoun, Thomas J. Matthew, Poonam Yakkundi, Ling Wang, Xingjie Chen, Tasnim Kothambawala, Miho Oyasu, Maya F. Kotturi, Genevive Hernandez, Xiaohan Liu, Marvin S. Peterson, Eric W. Humke, Bruce A. Keyt, Angus M. Sinclair. Anti-DR5 agonist IgM antibody IGM-8444 combined with SMAC mimetic birinapant induces strong synergistic tumor cytotoxicity [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 1068.

Resistance to BRAF inhibitors: A lesson from clinical observations

The MAPK/ERK pathway is an essential intracellular signaling pathway. Its deregulation is involved in tumor transformation and progression. The discovery of activating mutations of BRAF in various cancers has opened new therapeutic avenues with BRAF protein kinase inhibitors. Depending on the type of cancers, these inhibitors have shown either insufficient efficacy due to primary resistance of tumor cells or transient efficacy due to the development of acquired resistance. In this review, we revisit the discoveries that led to the development of BRAF inhibitors and detail the molecular and cellular mechanisms of resistance in cancers treated with these inhibitors. Understanding these mechanisms is crucial for developing more efficient therapeutic strategies.

Drugging the undruggable-targeting P53: A comparative analysis of APR-246 and coti-2 in human tumor primary culture explants.

e15092 Background: The TP53 tumor suppressor is the most commonly mutated gene in human cancer. P53 protein comprised of 393 amino acids functions as a tetramer with the loss of function mutations concentrated in “hotspots” that often affect DNA binding domains. APR-246 and COTI-2 are novel small molecules that are under clinical investigation for the treatment of P53 mutated cancers. APR, a quinuclidine binds to P53 cysteine residues altering the functional conformation while COTI is a thio-semicarbazone, found active in P53 mutant tumors using a novel computational platform. As the mechanisms of action may differ we undertook a comparative analysis of these compounds in 3D human tumor explants derived from surgical specimens. Methods: Ex Vivo Analyses of Programmed Cell Death (EVA/PCD) (D’Amora, P et al Gyn. Oncol. 2021) was applied to 236 human tumor primary culture explants isolated from surgical specimens. After disaggregation, micro-spheroids, isolated by precise density centrifugation were exposed to APR, COTI and other agents for 72-96 hours. Drug-induced cell death was measured by delayed-loss-of-membrane integrity and ATP content (luciferase). Dose response curves were interpolated to LC50 values. Synergy analysis used the method of Chou & Talalay. Correlation coefficients used Pearson moment. Results: APR and COTI reveal the highest activity in hematologic neoplasms. Solid tumor activity patterns diverged with COTI active in gynecologic malignancies and NSCLC and moderately active in breast and colorectal while APR is active in colorectal but less active in breast and NSCLC. Pearson moments for COTI vs APR revealed an R value = 0.146 (NS). COTI activity correlated with Nitrogen Mustard and Alpelisib (PIK3CA) while APR activity correlated with Doxorubicin and Everolimus. COTI combinations focused upon ovarian cancers identified synergy in 25% and antagonism in 18%. Conclusions: P53’s function as the “guardian of the genome” is consistent with our findings that many cytotoxic and targeted agents that induce cell death reveal correlations with APR and COTI. While APR affects the structural conformation of P53, COTI may function downstream via oxidative, metabolic or other stress responses. The correlation coefficient for APR vs COTI at 0.146 (NS) suggests distinct mechanisms of action. This is supported by different disease-specific-profiles and correlations with other drugs. COTI synergy in ovarian cancers, the target of current clinical trials at 25% is below expectations. With differing disease specificities, selecting between these and related agents to optimize clinical outcomes may require a more granular examination of cellular mechanisms of response and resistance. Primary culture analyses offer insights into drug mechanisms of action, combinatorial potential and have the capacity to improve patient selection.