Approaches to identifying drug resistance mechanisms to clinically relevant treatments in childhood rhabdomyosarcoma

Aim: Despite aggressive multiagent protocols, patients with metastatic rhabdomyosarcoma (RMS) have poor prognosis. In a recent high-risk trial (ARST0431), 25% of patients failed within the first year, while on therapy and 80% had tumor progression within 24 months. However, the mechanisms for tumor resistance are essentially unknown. Here we explore the use of preclinical models to develop resistance to complex chemotherapy regimens used in ARST0431. Methods: A Single Mouse Testing (SMT) protocol was used to evaluate the sensitivity of 34 RMS xenograft models to one cycle of vincristine, actinomycin D, cyclophosphamide (VAC) treatment. Tumor response was determined by caliper measurement, and tumor regression and event-free survival (EFS) were used as endpoints for evaluation. Treated tumors at regrowth were transplanted into recipient mice, and the treatment was repeated until tumors progressed during the treatment period (i.e., became resistant). At transplant, tumor tissue was stored for biochemical and omics analysis. Results: The sensitivity to VAC of 34 RMS models was determined. EFS varied from 3 weeks to > 20 weeks. Tumor models were classified as having intrinsic resistance, intermediate sensitivity, or high sensitivity to VAC therapy. Resistance to VAC was developed in multiple models after 2-5 cycles of therapy; however, there were examples where sensitivity remained unchanged after 3 cycles of treatment. Conclusion: The SMT approach allows for in vivo assessment of drug sensitivity and development of drug resistance in a large number of RMS models. As such, it provides a platform for assessing in vivo drug resistance mechanisms at a “population” level, simulating conditions in vivo that lead to clinical resistance. These VAC-resistant models represent “high-risk” tumors that mimic a preclinical phase 2 population and will be valuable for identifying novel agents active against VAC-resistant disease.

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The Tumor Suppressor MTUS1/ATIP1 Modulates Tumor Promotion in Glioma: Association with Epigenetics and DNA Repair

Cancers ◽

10.3390/cancers13061245 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1245

Author(s):

Nikhil Ranjan ◽

Vimal Pandey ◽

Manas Kumar Panigrahi ◽

Lukas Klumpp ◽

Ulrike Naumann ◽

...

Keyword(s):

High Grade Glioma ◽

Therapy Resistance ◽

Resistance Mechanisms ◽

Tumor Resistance ◽

Tumor Irradiation ◽

Damage Repair ◽

Low Passage ◽

Novel Therapeutic Strategies

Glioblastoma (GBM) is a highly aggressive brain tumor. Resistance mechanisms in GBM present an array of challenges to understand its biology and to develop novel therapeutic strategies. We investigated the role of a TSG, MTUS1/ATIP1 in glioma. Glioma specimen, cells and low passage GBM sphere cultures (GSC) were analyzed for MTUS1/ATIP1 expression at the RNA and protein level. Methylation analyses were done by bisulfite sequencing (BSS). The consequence of chemotherapy and irradiation on ATIP1 expression and the influence of different cellular ATIP1 levels on survival was examined in vitro and in vivo. MTUS1/ATIP1 was downregulated in high-grade glioma (HGG), GSC and GBM cells and hypermethylation at the ATIP1 promoter region seems to be at least partially responsible for this downregulation. ATIP1 overexpression significantly reduced glioma progression by mitigating cell motility, proliferation and facilitate cell death. In glioma-bearing mice, elevated MTUS1/ATIP1 expression prolonged their survival. Chemotherapy, as well as irradiation, recovered ATIP1 expression both in vitro and in vivo. Surprisingly, ATIP1 overexpression increased irradiation-induced DNA-damage repair, resulting in radio-resistance. Our findings indicate that MTUS1/ATIP1 serves as TSG-regulating gliomagenesis, progression and therapy resistance. In HGG, higher MTUS1/ATIP1 expression might interfere with tumor irradiation therapy.

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17 Activity sensors for noninvasive monitoring of immune response and tumor resistance during immune checkpoint blockade therapy

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0017 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A17-A17

Author(s):

Quoc Mac ◽

James Bowen ◽

Hathaichanok Phuengkham ◽

Anirudh Sivakumar ◽

Congmin Xu ◽

...

Keyword(s):

T Cell ◽

Treatment Response ◽

Immune Checkpoint ◽

Tumor Regression ◽

Growth Curves ◽

Resistance Mechanisms ◽

Immune Checkpoint Blockade ◽

Tumor Resistance ◽

Activity Sensors ◽

Therapeutic Responses

BackgroundDespite the curative potential of immune checkpoint blockade (ICB) therapy, only small subsets of patients achieve tumor regression while many responders relapse and acquire resistance. Monitoring treatment response and detecting the onset of resistance are critical for improving patient prognoses. Here we engineered ICB antibody-sensor conjugates known as ICB-Dx by coupling peptides sensing the activity of granzyme B (GzmB), a T cell cytotoxic protease, directly on αPD1 antibody to monitor therapeutic responses by producing a fluorescent reporter into urine. To develop biomarkers that indicate mechanisms of resistance to ICB, we generated B2m-/- and Jak1-/- tumor models and performed transcriptomic analyses to identify unique protease signatures of these resistance mechanisms. We then built a multiplexed library of αPD1-Dx capable of detecting early therapeutic response and illuminating resistance mechanisms during ICB therapy.MethodsFITC-labeled GzmB substrates were synthesized (CEM) and conjugated to αPD1 antibody. B2m-/- and Jak1-/- tumors were generated from WT MC38 cells using CRISPR/Cas9. For tumor studies, 106 cells were inoculated s.c. in B6 mice. Tumor mice were treated with αPD1 or IgG1 isotype conjugates (0.1 mg), and urine was collected at 3 hours. Tumor RNA was isolated with RNEasy kit (Qiagen) and prepared for sequencing with TruSeq mRNA kit (Illumina).ResultsTo synthesize αPD1-Dx, we coupled FITC-labeled GzmB substrates to αPD1 antibody (figure 1a). In MC38 tumors, systemic administration of αPD1-Dx lowered tumor burden relative to control treatment while producing significantly elevated urine signals that preceded tumor regression (figure 1b, c). To investigate the ability to monitor tumor resistance to ICB, we developed knockout tumors to model B2m and Jak1 mutations, which are observed in human patients. in vivo, B2m-/- and Jak1-/- MC38 tumors were resistant to αPD1 monotherapy (figure 1d). Tumor RNA sequencing revealed that gene expression was altered during αPD1 treatment only in WT tumors. Importantly, B2m-/- tumors showed very different expression profiles than Jak1-/- tumors during αPD1 treatment, indicative of unique regulation of resistance (figure 1e). We used differential expression analyses to discover unique protease signatures associated with these two resistance mechanisms. Finally, a multiplexed library of αPD1-Dx engineered to monitor both tumor and immune proteases detected early on-treatment responses and stratified B2m-/- from Jak1-/- resistance with high diagnostic validity (figure 1f).Abstract 17 Figure 1Monitoring response and resistance with ICB-Dx(a) αPD1-Dx can reinvigorate T cell response and monitor protease activities in the tumor microenvironment. (b) Growth curves of WT MC38 tumors treated with αPD1- or IgG1-Dx (ANOVA). (c) Urine signals detect treatment response to αPD1 monotherapy (ANOVA). (d) Growth curves of B2m-/- and Jak1-/- tumors treated with αPD1- or IgG1-Dx (ANOVA). (e) TSNE plot showing RNA profiles of WT, B2m-/-, Jak1-/- tumors treated with αPD1 or isotype control. (f) ROC curves of random forest classifiers built from urine signals that differentiate on-treatment response from on-treatment resistance and B2m-/- from Jak1-/- resistance.ConclusionsWe have engineered activity sensors that accurately detect therapeutic responses and stratify resistance mechanisms noninvasively from urine, thereby potentially expanding the precision of ICB therapy to benefit cancer patients.Ethics ApprovalAll animal studies were approved by Georgia Tech IACUC (A100193)

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Exploiting vulnerabilities in cancer signalling networks to combat targeted therapy resistance

Essays in Biochemistry ◽

10.1042/ebc20180016 ◽

2018 ◽

Vol 62 (4) ◽

pp. 583-593 ◽

Cited By ~ 9

Author(s):

Peter T. Harrison ◽

Paul H. Huang

Keyword(s):

Drug Resistance ◽

Targeted Therapy ◽

Kinase Inhibitors ◽

Effective Means ◽

Deep Understanding ◽

Therapy Resistance ◽

Resistance Mechanisms ◽

Precision Oncology ◽

First Line ◽

Clinical Responses

Drug resistance remains one of the greatest challenges facing precision oncology today. Despite the vast array of resistance mechanisms that cancer cells employ to subvert the effects of targeted therapy, a deep understanding of cancer signalling networks has led to the development of novel strategies to tackle resistance both in the first-line and salvage therapy settings. In this review, we provide a brief overview of the major classes of resistance mechanisms to targeted therapy, including signalling reprogramming and tumour evolution; our discussion also focuses on the use of different forms of polytherapies (such as inhibitor combinations, multi-target kinase inhibitors and HSP90 inhibitors) as a means of combating resistance. The promise and challenges facing each of these polytherapies are elaborated with a perspective on how to effectively deploy such therapies in patients. We highlight efforts to harness computational approaches to predict effective polytherapies and the emerging view that exceptional responders may hold the key to better understanding drug resistance. This review underscores the importance of polytherapies as an effective means of targeting resistance signalling networks and achieving durable clinical responses in the era of personalised cancer medicine.

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Understanding the Evolutionary Games in NSCLC Microenvironment

10.1101/2020.11.30.404350 ◽

2020 ◽

Author(s):

Ranjini Bhattacharya ◽

Robert Vander Velde ◽

Viktoriya Marusyk ◽

Bina Desai ◽

Artem Kaznatcheev ◽

...

Keyword(s):

Tumor Cells ◽

Targeted Therapies ◽

Evolutionary Dynamics ◽

Fusion Gene ◽

Evolutionary Game ◽

Therapy Resistance ◽

Resistance Mechanisms ◽

Intrinsic Resistance ◽

Frequency Changes

AbstractWhile initially highly successful, targeted therapies eventually fail as populations of tumor cells evolve mechanisms of resistance, leading to resumption of tumor growth. Historically, cell-intrinsic mutational changes have been the major focus of experimental and clinical studies to decipher origins of therapy resistance. While the importance of these mutational changes is undeniable, a growing body of evidence suggests that non-cell autonomous interactions between sub-populations of tumor cells, as well as with non-tumor cells within tumor microenvironment, might have a profound impact on both short term sensitivity of cancer cells to therapies, as well as on the evolutionary dynamics of emergent resistance. In contrast to well established tools to interrogate the functional impact of cell-intrinsic mutational changes, methodologies to understand non-cell autonomous interactions are largely lacking.Evolutionary Game Theory (EGT) is one of the main frameworks to understand the dynamics that drive frequency changes in interacting competing populations with different phenotypic strategies. However, despite a few notable exceptions, the use of EGT to understand evolutionary dynamics in the context of evolving tumors has been largely confined to theoretical studies. In order to apply EGT towards advancing our understanding of evolving tumor populations, we decided to focus on the context of the emergence of resistance to targeted therapies, directed against EML4-ALK fusion gene in lung cancers, as clinical responses to ALK inhibitors represent a poster child of limitations, posed by evolving resistance. To this end, we have examined competitive dynamics between differentially labelled therapy-naïve tumor cells, cells with cell-intrinsic resistance mechanisms, and cells with cell-extrinsic resistance, mediated by paracrine action of hepatocyte growth factor (HGF), within in vitro game assays in the presence or absence of front-line ALK inhibitor alectinib. We found that producers of HGF were the fittest in every pairwise game, while also supporting the proliferation of therapy-naïve cells. Both selective advantage of these producer cells and their impact on total population growth was a linearly increasing function of the initial frequency of producers until eventually reaching a plateau. Resistant cells did not significantly interact with the other two phenotypes. These results provide insights on reconciling selection driven emergence of subpopulations with cell non-cell autonomous resistance mechanisms, with lack of evidence of clonal dominance of these subpopulations. Further, our studies elucidate mechanisms for co-existence of multiple resistance strategies within evolving tumors. This manuscript serves as a technical report and will be followed up with a research paper in a different journal.

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An Optimized 3D Coculture Assay for Preclinical Testing of Pro- and Antiangiogenic Drugs

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555216686529 ◽

2017 ◽

Vol 22 (5) ◽

pp. 602-613 ◽

Cited By ~ 1

Author(s):

Daniela Unterleuthner ◽

Nina Kramer ◽

Karoline Pudelko ◽

Alexandra Burian ◽

Markus Hengstschläger ◽

...

Keyword(s):

Basal Membrane ◽

Cell Interaction ◽

Resistance Mechanisms ◽

Culture Conditions ◽

Angiogenic Factors ◽

Branch Points ◽

Intrinsic Resistance ◽

Antiangiogenic Drugs

Angiogenesis is a promising target for anticancer therapies, but also for treating other diseases with pathologic vessel development. Targeting the vascular endothelial growth factor (VEGF) pathway did not proof as effective as expected due to emerging intrinsic resistance mechanisms, as well as stromal contributions leading to drug insensitivity. Therefore, alternative strategies affecting the interaction of endothelial cells (ECs) with other stromal cells seem to be more promising. Human preclinical in vitro angiogenesis models successfully recapitulating these interactions are rare, and two-dimensional (2D) cell cultures cannot mimic tissue architecture in vivo. Consequently, models combining three-dimensionality with heterotypic cell interaction seem to be better suited. Here, we report on an improved human fibroblast–EC coculture assay mimicking sprouting angiogenesis from EC-covered microbeads resembling existing endothelial structures. Culture conditions were optimized to assess pro- and antiangiogenic compounds. Important characteristics of angiogenesis, that is, the number of sprouts and branch points, sprout length protrusion, and overall vessel structure areas, were quantified. Notably, the endothelial sprouts display lumen formation and basal membrane establishment. In this model, angiogenesis can be inhibited by genetic interference of pro-angiogenic factors expressed in the fibroblasts. Moreover, bona fide antiangiogenic drugs decreased, whereas pro-angiogenic factors increased vessel formation in 24-well and 96-well settings, demonstrating the applicability for screening approaches.

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Enhancing the Therapeutic Efficacy of KRASG12C Inhibitors in Lung Adenocarcinoma Cell Models by Cotargeting the MAPK Pathway or HSP90

Journal of Oncology ◽

10.1155/2021/2721466 ◽

2021 ◽

Vol 2021 ◽

pp. 1-13

Author(s):

Ying Liu ◽

Lei Wu ◽

Hong Lu ◽

En Wu ◽

Jun Ni ◽

...

Keyword(s):

Drug Resistance ◽

Lung Adenocarcinoma ◽

Tyrosine Kinases ◽

Tumor Regression ◽

Mapk Pathway ◽

Transcriptional Profiling ◽

Multidrug Resistant ◽

Mitogen Activated Protein Kinase

Background. KRASG12C inhibitors have shown promising efficacy in early clinical trials, but drug resistance compromises their long-term benefits. Therefore, it is critical to understand the mechanisms of drug resistance and to design appropriate combinatory treatments to improve efficacy. Methods. To understand the comprehensive mechanisms of drug resistance, we treated lung cancer cells with KRASG12C inhibitors for different periods and performed transcriptional profiling and signaling analysis to identify critical factors and pathways that drive drug tolerance and resistance. We also evaluated several drug combinations in vitro and in vivo to identify potentially effective therapeutics. Results. We found that the feedback activation of multiple receptor tyrosine kinases (RTKs) may have cooperatively induced intrinsic and adaptive resistance to KRASG12C inhibitors. Notably, continuous KRAS inhibition induced a multidrug-resistant phenotype, implying that upfront combinatory treatment might be required to treat this group of patients. We also demonstrated that concurrently targeting multiple nodes in the RTK/RAS/RAF/MEK/ERK axis improved the efficacy of KRASG12C inhibitors, mainly by suppressing the reactivation of the mitogen-activated protein kinase (MAPK) pathway. Moreover, the combined use of HSP90 and KRASG12C inhibitors effectively induced tumor regression in lung adenocarcinoma models in vitro and in vivo. Conclusion. Together, our findings revealed mechanisms underlying KRASG12C inhibitors resistance and provided novel candidate combinatory strategies to improve their anticancer activity.

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Three-dimensional tumor cell growth models in vivo drug resistance mechanisms

Klinische Pädiatrie ◽

10.1055/s-0036-1593549 ◽

2016 ◽

Vol 228 (06/07) ◽

Author(s):

C Bingel ◽

E Koeneke ◽

A Bittmann ◽

M Sill ◽

I Rettig ◽

...

Keyword(s):

Drug Resistance ◽

Cell Growth ◽

Tumor Cell ◽

Growth Models ◽

Three Dimensional ◽

Resistance Mechanisms ◽

Tumor Cell Growth

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Suppression of poised oncogenes by ZMYND8 promotes chemo-sensitization

Cell Death and Disease ◽

10.1038/s41419-020-03129-x ◽

2020 ◽

Vol 11 (12) ◽

Author(s):

Shravanti Mukherjee ◽

Santanu Adhikary ◽

Shrikanth S. Gadad ◽

Payel Mondal ◽

Sabyasachi Sen ◽

...

Keyword(s):

Breast Cancer ◽

Drug Resistance ◽

Cancer Cells ◽

Tumor Regression ◽

Disease Free Survival ◽

Metastatic Breast ◽

Good Prognosis ◽

Sublethal Dose ◽

Tumorigenic Potential

AbstractThe major challenge in chemotherapy lies in the gain of therapeutic resistance properties of cancer cells. The relatively small fraction of chemo-resistant cancer cells outgrows and are responsible for tumor relapse, with acquired invasiveness and stemness. We demonstrate that zinc-finger MYND type-8 (ZMYND8), a putative chromatin reader, suppresses stemness, drug resistance, and tumor-promoting genes, which are hallmarks of cancer. Reinstating ZMYND8 suppresses chemotherapeutic drug doxorubicin-induced tumorigenic potential (at a sublethal dose) and drug resistance, thereby resetting the transcriptional program of cells to the epithelial state. The ability of ZMYND8 to chemo-sensitize doxorubicin-treated metastatic breast cancer cells by downregulating tumor-associated genes was further confirmed by transcriptome analysis. Interestingly, we observed that ZMYND8 overexpression in doxorubicin-treated cells stimulated those involved in a good prognosis in breast cancer. Consistently, sensitizing the cancer cells with ZMYND8 followed by doxorubicin treatment led to tumor regression in vivo and revert back the phenotypes associated with drug resistance and stemness. Intriguingly, ZMYND8 modulates the bivalent or poised oncogenes through its association with KDM5C and EZH2, thereby chemo-sensitizing the cells to chemotherapy for better disease-free survival. Collectively, our findings indicate that poised chromatin is instrumental for the acquisition of chemo-resistance by cancer cells and propose ZMYND8 as a suitable epigenetic tool that can re-sensitize the chemo-refractory breast carcinoma.

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FOXM1, CDK6 and Rb Dependent Drug Resistance and Senescence in Myeloma

Blood ◽

10.1182/blood.v128.22.4456.4456 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4456-4456 ◽

Cited By ~ 1

Author(s):

Chunyan Gu ◽

Ruini Chen ◽

Xuefang Jing ◽

Siegfried Janz ◽

Ye Yang

Keyword(s):

Drug Resistance ◽

High Risk ◽

Conflicts Of Interest ◽

Expression Profiles ◽

Physical Interaction ◽

Mrna Levels ◽

Agar Culture ◽

Myeloma Cells

Abstract We recently reported that FOXM1 is a promising therapeutic target in multiple myeloma (MM), particularly for the subset of patients with high-risk disease.Because high-risk myeloma exhibits a strong predilection to early drug-resistant relapse following first-line therapy, we here decided to evaluate the role of FOXM1 in the acquisition of drug resistance by myeloma cells. We analyzed gene expression profiles of 88 paired myeloma samples at baseline and relapse from the UAMS Total Therapy 2 cohort and found that FOXM1 mRNA levels were significant upregulated in relapsed myeloma and that this was associated with poor event-free and overall survival. Laboratory studies showed that enforced expression of FOXM1 in human myeloma cell lines (HMCLs) results in decreased sensitivity of cells to widely used myeloma drugs, such as bortezomib and doxorubicin. This was observed in vitro, in both bulk cell and soft-agar culture, and in vivo using xenografting in mice. Biochemical analysis of HMCLs revealed physical interaction of FOXM1 with CDK6 and Rb, key regulators of cell cycle progression and cellular senescence, respectively.Treatment with small-compound CDK6 inhibitor, inhibited myeloma growth, decreased clonogenicity of myeloma, and ameliorated FOXM1-dependent senescence. Genetic and pharmacological targeting of FOXM1 in myeloma cells using shRNA and thiostreptone respectively, led to growth arrest and senescence, while elevated expression of FOXM1 reversed these phenotypes. In sum, our findings implicating the FOXM1-CDK6-Rb network in drug resistance and senescence of high-risk myeloma point to new treatment opportunities for this difficult-to-cure neoplasm. Disclosures No relevant conflicts of interest to declare.

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Emerging Nano-Based Strategies Against Drug Resistance in Tumor Chemotherapy

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.798882 ◽

2021 ◽

Vol 9 ◽

Author(s):

Lei Cao ◽

Yuqin Zhu ◽

Weiju Wang ◽

Gaoxiong Wang ◽

Shuaishuai Zhang ◽

...

Keyword(s):

Drug Delivery ◽

Drug Resistance ◽

Resistance Mechanisms ◽

Patient Treatment ◽

Tumor Resistance ◽

New Horizons ◽

Normal Tissues ◽

Alternative Drug ◽

Delivery Strategies ◽

Tumor Types

Drug resistance is the most significant causes of cancer chemotherapy failure. Various mechanisms of drug resistance include tumor heterogeneity, tumor microenvironment, changes at cellular levels, genetic factors, and other mechanisms. In recent years, more attention has been paid to tumor resistance mechanisms and countermeasures. Nanomedicine is an emerging treatment platform, focusing on alternative drug delivery and improved therapeutic effectiveness while reducing side effects on normal tissues. Here, we reviewed the principal forms of drug resistance and the new possibilities that nanomaterials offer for overcoming these therapeutic barriers. Novel nanomaterials based on tumor types are an excellent modality to equalize drug resistance that enables gain more rational and flexible drug selectivity for individual patient treatment. With the emergence of advanced designs and alternative drug delivery strategies with different nanomaterials, overcome of multidrug resistance shows promising and opens new horizons for cancer therapy. This review discussed different mechanisms of drug resistance and recent advances in nanotechnology-based therapeutic strategies to improve the sensitivity and effectiveness of chemotherapeutic drugs, aiming to show the advantages of nanomaterials in overcoming of drug resistance for tumor chemotherapy, which could accelerate the development of personalized medicine.

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