Explore Resistance Mechanisms Research Articles

EstroGene2.0: A multi-omic database of response to estrogens, ER-modulators, and resistance to endocrine therapies in breast cancer.

Endocrine therapies targeting the estrogen receptor (ER/ESR1) are the cornerstone to treat ER-positive breast cancers patients, but resistance often limits their effectiveness. Understanding the molecular mechanisms is thus key to optimize the existing drugs and to develop new ER-modulators. Notable progress has been made although the fragmented way data is reported has reduced their potential impact. Here, we introduce EstroGene2.0, an expanded database of its precursor 1.0 version. EstroGene2.0 focusses on response and resistance to endocrine therapies in breast cancer models. Incorporating multi-omic profiling of 361 experiments from 212 studies across 28 cell lines, a user-friendly browser offers comprehensive data visualization and metadata mining capabilities (https://estrogeneii.web.app/). Taking advantage of the harmonized data collection, our follow-up meta-analysis revealed substantial diversity in response to different classes of ER-modulators including SERMs, SERDs, SERCA and LDD/PROTAC. Notably, endocrine resistant models exhibit a spectrum of transcriptomic alterations including a contra-directional shift in ER and interferon signaling, which is recapitulated clinically. Furthermore, dissecting multiple ESR1-mutant cell models revealed the different clinical relevance of genome-edited versus ectopic overexpression model engineering and identified high-confidence mutant-ER targets, such as NPY1R. These examples demonstrate how EstroGene2.0 helps investigate breast cancer's response to endocrine therapies and explore resistance mechanisms.

Temporary Persistence of Bedaquiline-resistant Mycobacterium tuberculosis Isolates under Bedaquiline Pressure as a Model to Explore Resistance Mechanisms

Temporary Persistence of Bedaquiline-resistant Mycobacterium tuberculosis Isolates under Bedaquiline Pressure as a Model to Explore Resistance Mechanisms

MicroRNA-3037 targeting CYP6CY2 confers imidacloprid resistance to Sitobion miscanthi (Takahashi)

The wheat aphid Sitobion miscanthi is a dominant and destructive pest in agricultural production. Insecticides are the main substances used for effective control of wheat aphids. However, their extensive application has caused severe resistance of wheat aphids to some insecticides; therefore, exploring resistance mechanisms is essential for wheat aphid management. In the present study, CYP6CY2, a new P450 gene, was isolated and overexpressed in the imidacloprid-resistant strain (SM-R) compared to the imidacloprid-susceptible strain (SM-S). The increased sensitivity of S. miscanthi to imidacloprid after knockdown of CYP6CY2 indicates that it could be associated with imidacloprid resistance. Subsequently, the posttranscriptional regulation of CYP6CY2 in the 3′ UTR by miR-3037 was confirmed, and CYP6CY2 participated in imidacloprid resistance. This finding is critical for determining the role of P450 in relation to the resistance of S. miscanthi to imidacloprid. It is of great significance to understand this regulatory mechanism of P450 expression in the resistance of S. miscanthi to neonicotinoids.

Advancing CAR T-cell therapy for chronic lymphocytic leukemia: exploring resistance mechanisms and the innovative strategies to overcome them.

Chimeric antigen receptor (CAR) T-cell therapy has ushered in substantial advancements in the management of various B-cell malignancies. However, its integration into chronic lymphocytic leukemia (CLL) treatment has been challenging, attributed largely to the development of very effective chemo-free alternatives. Additionally, CAR T-cell responses in CLL have not been as high as in other B-cell lymphomas or leukemias. However, a critical void exists in therapeutic options for patients with high-risk diseases who are resistant to the current CLL therapies, underscoring the urgency for adoptive immunotherapies in these patients. The diminished CAR T-cell efficacy within CLL can be traced to factors such as compromised T-cell fitness due to persistent antigenic stimulation inherent to CLL. Resistance mechanisms encompass tumor-related factors like antigen escape, CAR T-cell-intrinsic factors like T-cell exhaustion, and a suppressive tumor microenvironment (TME). New strategies to combat CAR T-cell resistance include the concurrent administration of therapies that augment CAR T-cell endurance and function, as well as the engineering of novel CAR T-cells targeting different antigens. Moreover, the concept of "armored" CAR T-cells, armed with transgenic modulators to modify both CAR T-cell function and the tumor milieu, is gaining traction. Beyond this, the development of readily available, allogeneic CAR T-cells and natural killer (NK) cells presents a promising countermeasure to innate T-cell defects in CLL patients. In this review, we explore the role of CAR T-cell therapy in CLL, the intricate tapestry of resistance mechanisms, and the pioneering methods studied to overcome resistance.

Abstract 5844: Landscapes of acquired EGFR and VEGF resistance alterations in colorectal cancer: findings from the PARADIGM biomarker study

Abstract Introduction: Resistance mechanisms to anti-epidermal growth factor receptor (EGFR) treatment, such as panitumumab (PAN), in patients (pts) with metastatic colorectal cancer (mCRC) depend on the diversity of acquired gene alterations and clonal variation. To explore resistance mechanisms and emerging genomic alterations during PAN treatment, we analyzed the circulating cell-free DNA (cfDNA) samples from the PARADIGM trial (NCT02394795), which demonstrated survival benefits of PAN over bevacizumab (BEV), an anti-vascular endothelial growth factor (VEGF) antibody, in combination with first-line chemotherapy in pts with RAS wild-type (WT) mCRC. Methods: cfDNA from blood samples were obtained pre- and post-treatment and analyzed for gene alterations using the custom PlasmaSELECT-R 91 PGDx panel (NCT02394834). We analyzed the landscapes of acquired alterations and their association with functional pathways in mCRC according to The Cancer Genome Atlas signaling classification. Result: Among the enrolled 802 pts, 390 discontinued treatment due to progressive disease, of which 276 (70.8%) had evaluable pre- and post-treatment cfDNA samples (PAN, n=126; BEV, n=150). Though the proportion of pts with acquired alterations in any of 30 RTK-RAS-related genes was similar in PAN vs BEV (44.4% vs 42.7%), known EGFR resistance-related genes were observed more frequently in PAN compared with BEV: KRAS (19.1 vs 2.7%), NRAS (9.5 vs 2.7%), BRAF (11.1 vs 0.7%), MAP2K1 (5.6 vs 0.7%), and EGFR (5.6 vs 3.3%). These acquired mutations clustered at hotspots within those genes in the PAN group, unlike in the BEV group. Furthermore, the co-occurrence rates of acquired alterations among the RTK-RAS pathway of the individual cases were higher in PAN (25.4%) vs BEV (9.3%), suggesting an increased subclonal complexity in PAN. There were few acquired alterations predominantly observed in BEV, and their biological significance remains unclear. Conclusion: These results suggest that PAN treatment, compared with BEV, more commonly induces multiple resistant subclones in specific pathways under selective treatment pressure. Our findings provide valuable insight for further investigations into the mechanisms of acquired and resistance associated with PAN and BEV. Citation Format: Katsuya Tsuchihara, Riu Yamashita, Takayuki Yoshino, Kohei Shitara, Jun Watanabe, Hirofumi Yasui, Hisatsugu Ohori, Manabu Shiozawa, Kei Muro, Kentaro Yamazaki, Eiji Oki, Takeo Sato, Takeshi Naitoh, Yoshito Komatsu, Takeshi Kato, Kazunori Yamanaka, Ikuo Mori, Masamitsu Hihara, Junpei Soeda, Kouji Yamamoto, Kiwamu Akagi, Atsushi Ochiai, Victor E. Velculescu, Hiroyuki Uetake. Landscapes of acquired EGFR and VEGF resistance alterations in colorectal cancer: findings from the PARADIGM biomarker study [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 5844.

Clinical practice recommendations for the use of next-generation sequencing in patients with solid cancer: a joint report from KSMO and KSP.

In recent years, next-generation sequencing (NGS)-based genetic testing has become crucial in cancer care. While its primary objective is to identify actionable genetic alterations to guide treatment decisions, its scope has broadened to encompass aiding in pathological diagnosis and exploring resistance mechanisms. With the ongoing expansion in NGS application and reliance, a compelling necessity arises for expert consensus on its application in solid cancers. To address this demand, the forthcoming recommendations not only provide pragmatic guidance for the clinical use of NGS but also systematically classify actionable genes based on specific cancer types. Additionally, these recommendations will incorporate expert perspectives on crucial biomarkers, ensuring informed decisions regarding circulating tumor DNA panel testing.

Clinical Practice Recommendations for the Use of Next-Generation Sequencing in Patients with Solid Cancer: A Joint Report from KSMO and KSP.

In recent years, next-generation sequencing (NGS)-based genetic testing has become crucial in cancer care. While its primary objective is to identify actionable genetic alterations to guide treatment decisions, its scope has broadened to encompass aiding in pathological diagnosis and exploring resistance mechanisms. With the ongoing expansion in NGS application and reliance, a compelling necessity arises for expert consensus on its application in solid cancers. To address this demand, the forthcoming recommendations not only provide pragmatic guidance for the clinical use of NGS but also systematically classify actionable genes based on specific cancer types. Additionally, these recommendations will incorporate expert perspectives on crucial biomarkers, ensuring informed decisions regarding circulating tumor DNA panel testing.

Autophagy Induction Constitutes a Targetable Vulnerability in FLT3-Mutant Acute Myeloid Leukemia Cells Treated with FLT3-Inhibitors

Recently, we and others identified autophagy induction as a protective mechanism in FLT3-ITD mutant acute myeloid leukemia (AML) cells upon treatment with FLT3-inhibitors (FLT3i), constituting an increasing issue in clinical practice. Here, we investigated the molecular consequences of autophagy induction upon FLT3i treatment, exploring resistance mechanisms and interrogated whether autophagy inhibition can improve FLT3i antileukemic therapy. Drug response of primary FLT3-ITD AML samples ex vivo treated with FLT3i, quizartinib (AC220, n= 59) and midostaurin (PKC412, n=64), was associated with their transcriptional profile to unravel which molecular programs were enriched in poor responders. Both gene set enrichment analysis (GSEA) and single sample GSEA (ssGSEA) revealed enriched molecular signatures compatible with autophagy activation in poor responders to FLT3i, while sensitive samples enriched molecular signatures consonant with mitochondrial function. Enrichment scores for programs associated with “regulation of autophagy” and “autophagosome organization” were positively correlated to ex vivo response area under the curve values for AC220 and PKC412. To functionally address the molecular pathways associated with autophagy activation upon FLT3i treatment, MOLM-13 and MV4-11 AML cells ( FLT3-ITD) and primary AML samples, were treated with AC220 and PKC412 alone or in combination with the autophagy inhibitor chloroquine (CQ, 5-10μM). In FLT3-ITD AML models, treatment with FLT3i significantly induced the formation of acidic vesicular organelles, suggesting autophagy induction. We also observed a reduction of autophagy regulators in a dose- and time-dependent manner, evidenced by the conversion of LC3B-I to LC3B-II, with degradation of LC3B-II and p62. Furthermore, FLT3i treatment resulted in phosphorylation inhibition of AKT Ser473, mTOR Ser2448 and its downstream protein p70S6K Thr421/Ser424, and phosphorylation reduction of ULK1 Ser757. Combination of FLT3i with CQ (10 μM) significantly reduced cell viability and survival for both PKC412 and AC220 inhibitors. To rule out the off target effects associated with CQ treatment, we performed a knockdown (KD) on the key autophagy mediator ATG5 using shRNA-mediated inhibition in FLT3-ITD cells. In MOLM13 cells, FLT3i treatment in ATG5-KD cells significantly reduced the IC50 (control: 31nM vs. ATG5-KD: 17nM) and increased apoptosis induction (control: 41% vs. ATG5-KD: 58%). We validated these observations in ex vivo treated primary AML samples, where we observed that in FLT3-ITD but not in FLT3 wild-type samples, the combination of FLT3i (PKC412 and AC220) plus autophagy inhibitors (CQ) reduced cell proliferation and survival. In summary, we demonstrated that autophagy induction is a biological process intrinsically associated with drug resistance in FLT3-ITD mutant AMLs when treated with FLT3i. Hence, autophagy inhibition can be leveraged to increase the antileukemic effect of FLT3i and highlights the autophagy as a major acquired biological process associated with FLT3i therapy.

In vitro activity of new combinations of β-lactam and β-lactamase inhibitors against the Mycobacterium tuberculosis complex.

As meropenem-clavulanic acid is recommended for the treatment of drug-resistant tuberculosis, the repurposing of new carbapenem combinations may provide new treatment options, including oral alternatives. Therefore, we studied the in vitro activities of meropenem-vaborbactam, meropenem-clavulanic acid, and tebipenem-clavulanic acid. One hundred nine Mycobacterium tuberculosis complex (MTBC) clinical isolates were tested, of which 69 were pan-susceptible and the remaining pyrazinamide- or multidrug-resistant. Broth microdilution MICs were determined using the EUCAST reference method. Meropenem and tebipenem were tested individually and in combination with vaborbactam 8 mg/L and clavulanic-acid 2 and 4 mg/L, respectively. Whole-genome sequencing was performed to explore resistance mechanisms. Clavulanic acid lowered the modal tebipenem MIC approximately 16-fold (from 16 to 1 mg/L). The modal meropenem MIC was reduced twofold by vaborbactam compared with an approximately eightfold decrease by clavulanic acid. The only previously described high-confidence carbapenem resistance mutation, crfA T62A, was shared by a subgroup of lineage 4.3.4.1 isolates and did not correlate with elevated MICs. The presence of a β-lactamase inhibitor reduced the MTBC MICs of tebipenem and meropenem. The resulting MIC distribution was lowest for the orally available drugs tebipenem-clavulanic acid. Whether this in vitro activity translates to similar or greater clinical efficacy of tebipenem-clavulanic acid compared with the currently WHO-endorsed meropenem-clavulanic acid requires clinical studies. IMPORTANCE Repurposing of already approved antibiotics, such as β-lactams in combination with β-lactamase inhibitors, may provide new treatment alternatives for drug-resistant tuberculosis. Meropenem-clavulanic acid was more active in vitro compared to meropenem-vaborbactam. Notably, tebipenem-clavulanic acid showed even better activity, raising the potential of an all-oral treatment option. Clinical data are needed to investigate whether the better in vitro activity of tebipenem-clavulanic acid correlates with greater clinical efficacy compared with the currently WHO-endorsed meropenem-clavulanic acid.

Pemetrexed induces ROS generation and cellular senescence by attenuating TS-mediated thymidylate metabolism to reverse gefitinib resistance in NSCLC.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) are strongly recommended for non-small-cell lung cancer (NSCLC) patients harbouring active EGFR mutations, while drug resistance makes exploring resistance mechanisms and seeking effective therapeutic strategies urgent endeavours. Thymidylate synthetase (TYMS or TS) is a dominant enzyme in thymidylate nucleotide metabolism. In this study, we found a positive correlation between TS expression and overall survival (OS) and disease-free survival (DFS) in lung adenocarcinoma. The examination of gene sets from 140 NSCLC patients received EGFR-TKI therapy demonstrated a negative correlation between high TS expression and the efficacy of EGFR-TKI therapy. 24 tissue specimens from NSCLC patients exhibited upregulated TS mRNA expression in NSCLC patients resistant to gefitinib. The NSCLC cell PC9 and HCC827 sensitive to gefitinib and relatively resistant PC9/GR and HCC827/GR cells were used to demonstrate the knockdown of TS restored the sensitivity of resistant cells to gefitinib. Furthermore, pemetrexed effectively suppressed TS-mediated thymidylate metabolism and induced ROS generation, DNA damage and cellular senescence, thereby hampering cancer progression and restoring sensitivity to gefitinib. Our findings illuminate the potential mechanism of TS-triggered gefitinib resistance and indicate inhibition of TS by pemetrexed can potentiate the effect of gefitinib in NSCLC. Pemetrexed combined with gefitinib has potent anti-progression potential in gefitinib-resistant NSCLC. This study suggests that NSCLC patients with both high TS expression and EGFR-driving mutations might benefit more from a combination strategy of EGFR-TKI and pemetrexed-based chemotherapy than EGFR-TKI monotherapy, which has profound clinical implications and therapeutic value.

Abstract 138: Establishment of monoclonal cell subpopulations as model systems for functionally exploring resistance mechanisms in EGFR mutant non-small cell cancers

Abstract Background: Coexisting subgroups of cells within a tumor are always evolving. Understanding the molecular events that drive this evolution remains a high priority for understanding dynamic response to treatment. However, models that accurately map individual clones’ activity and sensitivity to treatment remain a challenge. To fill this gap, we established monoclonal cell populations (MCP) from a cell line and used this model system to dissect coexisting cellular and molecular events within an individual tumor and modulated response to anti-EGFR treatment in Non-Small Cell Lung Cancers (NSCLC). Materials/Methods: MCPs were established from the commercially available H1975 EGFR-mutant NSCLC cell line. Parental cells were resuspended at a density of 10 cells per mL and 100 μL of cell suspension was added to each well of a 96-well plate. Eight plates were used, and each well was inspected 24 hours post-seeding by two or more independent scientists. Wells with individual cells were monitored every 3-4 days and followed throughout the expansion process. Once MCPs were established, morphological comparison was performed using the opensource QPath software and clones were classified as circular, branched, or mixed. Selected clones were then treated with 0.7 μM of Osimertinib for 72 hours, in line with the IC50 value of the parental line and treatment responses were compared. Results: Of the 120 single cells detected, 25 expanded successfully and 16 were treated with Osimertinib. Rates of clonal expansion varied from 19 to 49 days and the most prominent morphology was branched (68.75%), followed by mixed (18.75%) and circular (12.5%). No direct correlation was observed between rate of expansion and cell morphology. Response to Osimertinib varied significantly across the clones compared to the parental line from which they were established. Cell viability after 72 hours of treatment with Osimertinib was similar between the parental line and 5 of the 25 clones. For the remaining clones, cell viability was significantly lower in the monoclonal cell subpopulations compared to the parental line. Specifically, cell viability decreased by 40% compared to the parental cell in 5 clones. In the most sensitive clones, cell viability was reduced by 63% and 75% compared to the parental. Conclusion: 25 monoclonal cell subpopulations were successfully established and expanded from the H1975 cell line. MCPs showed significant variations in their growth rate and sensitivity to Osimertinib suggesting that this approach may help identify coexisting mechanisms of response to treatment within an individual tumor. Defining molecular profiles of coexisting MCPs may lead to the identification of biomarkers that may assist physicians in predicting response to treatment or design combination regimens that concomitantly target subclones within the tumor microecology. Citation Format: Claire Blanchard, Kyle Avery, Chelsea Ward, Abduljalil M. Alsubaie, Elisa Baldelli, Mariaelena Pierobon. Establishment of monoclonal cell subpopulations as model systems for functionally exploring resistance mechanisms in EGFR mutant non-small cell cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 138.

Transcriptome profile of halofuginone resistant and sensitive strains of Eimeria tenella.

The antiparasitic drug halofuginone is important for controlling apicomplexan parasites. However, the occurrence of halofuginone resistance is a major obstacle for it to the treatment of apicomplexan parasites. Current studies have identified the molecular marker and drug resistance mechanisms of halofuginone in Plasmodium falciparum. In this study, we tried to use transcriptomic data to explore resistance mechanisms of halofuginone in apicomplexan parasites of the genus Eimeria (Apicomplexa: Eimeriidae). After halofuginone treatment of E. tenella parasites, transcriptome analysis was performed using samples derived from both resistant and sensitive strains. In the sensitive group, DEGs associated with enzymes were significantly downregulated, whereas the DNA damaging process was upregulated after halofuginone treatment, revealing the mechanism of halofuginone-induced parasite death. In addition, 1,325 differentially expressed genes (DEGs) were detected between halofuginone resistant and sensitive strains, and the DEGs related to translation were significantly downregulated after halofuginone induction. Overall, our results provide a gene expression profile for further studies on the mechanism of halofuginone resistance in E. tenella.

Abstract 355: Molecular characterization of BRAF V600E CRC in response to Src-targeted therapy

Abstract Oncogenic mutations in B-type Raf Kinase (BRAF) (particularly V600E) are present in 7-10% of metastatic colorectal cancer (mCRC), conferring resistance to conventional therapies and a poor prognosis. Src Family Kinases (SFKs) play a relevant role in CRC, where Src activation is associated with an aggressive phenotype and a worse prognosis; and could be involved in chemoresistance mechanisms. Dasatinib is a potent Src inhibitor with demonstrated anti-proliferative activity, although deeper knowledge is needed. Thus, we aimed to explore the role of Src in BRAF V600E CRC and to investigate this tumor subgroup in response to Dasatinib. SRC activation was evaluated by Western Blot. We generated Knock-Out (KO) Src cell lines by CRISPR/Cas9 system and stable Src overexpression (OE) by lentivirus. Cell viability was analysed by crystal violet and MTS assay. Phospho-kinase proteins screening was performed using the Proteome Profiler Human Phospho-Kinase Array kit. Expression levels of Src and phospho-Src (p-Src) displayed an inverse correlation with IC50 values for Dasatinib in a panel of 7 CRC cell lines including BRAF V600E (HT29, COLO201, RKO, LS411N) and BRAF WT (DIFI, HCA7 and LIM1215), and these doses were able to inhibit Src activation in all cell lines. Focusing on HT29 cells, inhibition of p-Src occurs in a dose-dependent manner and in shortened periods of treatment. Dasatinib showed a significant inhibition of proliferation and migration on HT29. Moreover, stable Src-OE HT29 cells showed higher clonogenicity, migration and proliferation capacities, as well as higher resistance to Dasatinib. Conversely, HT29 Src KO cells were more sensitive. In line with those results, silencing Src in other BRAF V600E cells (RKO and LS411N) also resulted in a remarkable reduction in clonogenicity and migration abilities. Xenograft mouse model of SRC depletion showed that the growth of SRC KO tumors was significantly slower than WT tumors. Phospho-kinase array quantification in basal and Src-OE HT29 cells showed up- and downregulated proteins related to classical tumorigenic signaling pathways such as MAPK (JNK1/2, MSK), PI3K/Akt (Akt, TOR, GSK3), and others. Further studies to explore resistance mechanisms mediated by Src/SFKs are required. To conclude, lower p-Src expression at baseline is related to a higher resistance to Dasatinib in all CRC cell lines. Dasatinib showed a significant anti-proliferative activity in a subset of CRC cell lines and regulates the activity of Src in a time and dose-dependent manner. Modified expression of Src (OE and KO) in BRAF V600E CRC cells significantly regulates clonogenicity, proliferation and migration abilities in vitro. Src overexpression is responsible of the acquired resistance to Dasatinib and regulates several pathways that could be involved in the tumorigenesis of BRAF V600E CRC, although further studies to explore resistance mechanisms mediated by Src/SFKs are required. Citation Format: Beatriz Rubio Cuesta, Beatriz Gil Calderón, Patricia LLamas Granda, Jacinto Sarmentero, Carlos Carretero Puche, María del Carmen Riesco, Rocío García Carbonero, Beatriz Soldevilla. Molecular characterization of BRAF V600E CRC in response to Src-targeted therapy [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 355.

OS06.7A METPlatform identifies brain metastasis vulnerabilities and predicts patient response to therapy

Abstract BACKGROUND The diagnosis of brain metastasis involves high morbidity and mortality and remains an unmet clinical need in spite of being the most common tumor in the brain. Exclusion of these cancer patients from clinical trials is a major cause of their limited therapeutic options. MATERIAL AND METHODS We report a novel drug-screening platform (METPlatform) based on organotypic cultures which allows identifying effective anti-metastasis agents in the presence of the organ microenvironment. We have applied this approach to clinically relevant stages of brain metastasis using both experimental models and human tumor tissue (by performing patient-derived organotypic cultures - PDOCs -). We have also used METPlatform to perform unbiased proteomics of brain metastases in situ to identify potential novel mediators of this disease and explore resistance mechanisms to targeted therapy. Finally, we have exploited METPlatform as “avatars” to predict response to therapy in patients with primary brain tumors. RESULTS We identified heat shock protein 90 (HSP90) as a promising therapeutic target for brain metastasis. DEBIO-0932, a blood-brain barrier permeable HSP90 inhibitor, shows high potency against mouse and human brain metastases from different primary origin and oncogenomic profile at clinically relevant stages of the disease, including a novel model of local relapse after neurosurgery. Furthermore, in situ proteomic analysis of brain metastases treated with the chaperone inhibitor revealed non-canonical clients of HSP90 as potential novel mediators of brain metastasis and actionable mechanisms of resistance driven by autophagy. Combined therapy using HSP90 and autophagy inhibitors showed synergistic effects compared to sublethal concentrations of each monotherapy, demonstrating the potential of METPlatform to design and test rationale combination therapies to target metastasis more effectively. Finally, we show that brain tumor PDOCs predict the response of the corresponding patient to standard of care, thus proving the potential of METPlatform for improving personalized care in cancer. CONCLUSION Our work validates METPlatform as a potent resource for metastasis research integrating drug-screening and unbiased omic approaches that is fully compatible with human samples and questions the rationale of excluding patients with brain metastasis from clinical trials. We envision that METPlatform will be established as a clinically relevant strategy to personalize the management of metastatic disease in the brain and elsewhere.

Exploring mechanisms of tumor resistance to CAR T cell therapy in immunocompetent mouse models

Abstract Chimeric antigen receptor (CAR) T cell therapies have shown striking clinical efficacy in the treatment of hematological malignancies, but limited success so far against solid tumors, suggesting that solid tumors may harbor mechanisms of resistance. To investigate mechanisms of CAR T cell therapy resistance in solid tumors, we developed immunocompetent mouse models to explore immune cell interactions and the effect of an immunosuppressive tumor microenvironment on CAR T cell behavior. We generated murine CAR T cells against a model antigen that contained a CD3z intracellular signaling domain linked to either a CD28 or a 4-1BB signaling domain. We found that murine CAR T cells exhibited a predominantly central memory phenotype together with potent cytotoxic activity and antigen-dependent cytokine release in vitro. Further, we found that murine CAR T cells exhibited strong anti-tumor efficacy against both disseminated (EL4) and subcutaneous (MC38) murine tumors in vivo, whereas non-binding control CAR T cells had no anti-tumor effect. However, in both the disseminated and subcutaneous models, tumors were suppressed but not completely eradicated. Although murine CAR T cells possessing either CD28 or 41BB co-stimulatory domains demonstrated similar in vitro activities, they exhibited strikingly different efficacies and resistance mechanisms in vivo. Collectively, these results highlight the contributions of specific co-stimulatory signaling pathways to CAR T cell therapy in immunocompetent murine models and provide a platform to explore resistance mechanisms to CAR T therapy against solid tumors.

In-Vitro and In-Vivo Establishment and Characterization of Bioluminescent Orthotopic Chemotherapy-Resistant Human Osteosarcoma Models in NSG Mice.

Osteosarcoma, the most common bone malignancy with a peak incidence at adolescence, had no survival improvement since decades. Persistent problems are chemo-resistance and metastatic spread. We developed in-vitro osteosarcoma models resistant to chemotherapy and in-vivo bioluminescent orthotopic cell-derived-xenografts (CDX). Continuous increasing drug concentration cultures in-vitro resulted in five methotrexate (MTX)-resistant and one doxorubicin (DOXO)-resistant cell lines. Resistance persisted after drug removal except for MG-63. Different resistance mechanisms were identified, affecting drug transport and action mechanisms specific to methotrexate (RFC/SCL19A1 decrease, DHFR up-regulation) for MTX-resistant lines, or a multi-drug phenomenon (PgP up-regulation) for HOS-R/DOXO. Differential analysis of copy number abnormalities (aCGH) and gene expression (RNAseq) revealed changes of several chromosomic regions translated at transcriptomic level depending on drug and cell line, as well as different pathways implicated in invasive and metastatic potential (e.g., Fas, Metalloproteinases) and immunity (enrichment in HLA cluster genes in 6p21.3) in HOS-R/DOXO. Resistant-CDX models (HOS-R/MTX, HOS-R/DOXO and Saos-2-B-R/MTX) injected intratibially into NSG mice behaved as their parental counterpart at primary tumor site; however, they exhibited a slower growth rate and lower metastatic spread, although they retained resistance and CGH main characteristics without drug pressure. These models represent valuable tools to explore resistance mechanisms and new therapies in osteosarcoma.

Re-biopsy and liquid biopsy for patients with non-small cell lung cancer after EGFR-tyrosine kinase inhibitor failure.

BackgroundRe‐biopsy is important for exploring resistance mechanisms, especially for non‐small cell lung cancer (NSCLC) patients who develop resistance to EGFR‐tyrosine kinase inhibitors (TKIs). Liquid biopsy using circulating tumor DNA has come into use for this purpose. This retrospective study investigated the status of re‐biopsy and liquid biopsy in NSCLC patients with EGFR mutations and evaluated their effect on clinical strategies and prognosis.MethodsFive hundred fifty‐five NSCLC patients with resistance to EGFR‐TKIs were included and divided into three groups: re‐biopsy, liquid biopsy, and no re‐biopsy. Amplification refractory mutation system (ARMS) PCR or super ARMS PCR was used to detect EGFR mutations.ResultsThree hundred eight (55.5%) patients underwent re‐biopsy; 45.5% (140/308) were positive for T790M. The most common re‐biopsy procedure was computed tomography‐guided percutaneous core needle biopsy (60.1%), followed by effusion drainage (29.5%) and superficial lymph node biopsy (6.5%). One hundred eighteen (21.3%) patients underwent liquid biopsy; the T790M detection rate was 41.5% (49/118.) Of the 308 patients who underwent re‐biopsy, 69 were examined for EGFR mutations with plasma. The concordance rate of T790M detection between tissue and plasma was 66.7%. A statistical difference in further treatment after EGFR‐TKI failure was observed among all groups (P = 0.014). Patients in the biopsy groups were more likely to receive third‐generation EGFR‐TKIs. Multivariate analysis showed that re‐biopsy had a significant impact on overall survival (P < 0.001).ConclusionRe‐biopsy plays a pivotal role in the management of patients with NSCLC and resistance to EGFR‐TKIs. Liquid biopsy may be an alternative if difficulties performing re‐biopsy exist.

Genome-wide characterization of protein phosphatase 2C genes in Populus euphratica and their expression profiling under multiple abiotic stresses

The protein phosphatase 2Cs (PP2Cs) have been demonstrated to act as negative modulators of protein kinase and to participate in stress signal transduction, as well as plant growth and productivity processes. Populus euphratica is so extraordinarily adaptable to abiotic stresses that it is regarded as a potential model plant for exploring resistance mechanisms of woody plants. To gain insight into the functional characteristics of PP2C genes in P. euphratica, 117 non-redundant PeuPP2C-encoding genes were identified from the whole genome. These members were classified into 13 groups (A–M), each of which was relatively conserved in gene structure and protein domain. A total of 39 paralogous pairs were found to be generated by whole genome duplication events, and Ka/Ks analysis indicated that these paralogous pairs had evolved mainly from purifying selection. The cis-acting elements and expression patterns showed that all the PeuPP2Cs were involved in response to single or multiple stresses including drought, salinity, heat, cold, and ABA. Taken together, our results summarized the genome-wide characterization of PeuPP2Cs and their expression profiling across different tissues and under multiple abiotic stresses in P. euphratica. These data provide a foundation to further investigate potential function of PeuPP2Cs in conferring tolerance to various stresses in P. euphratica.

Abstract B33: Expansion study of pegylated arginine deiminase (ADI-PEG20), pemetrexed, and cisplatin in patients with ASS1-deficient non-squamous non-small cell lung cancer (TRAP)

Abstract Background: Loss of the metabolic tumor suppressor argininosuccinate synthetase 1 (ASS1) sensitizes non-squamous non-small cell lung cancer (NSCLC) cells to arginine deprivation with pegylated arginine deiminase (ADI-PEG20), which also disrupts thymidine pools and potentiates pemetrexed (Pem) cytotoxicity. In the phase I dose-escalation TRAP study (NCT02029690) we showed that ADI-PEG20 with first-line PEM and cisplatin (Cis) chemotherapy (ADIPemCis) produced a 100% disease control rate (DCR) (n = 9) of ASS1-deficient thoracic cancers, with no additional toxicity (Beddowes et al., 2017). Here, we present the TRAP expansion cohort experience in non-squamous NSCLC. Methods: Good-performance (ECOG 0-1) NSCLC patients (pts) with nonresectable disease were enrolled in a phase I TRAP expansion cohort at the maximum tolerated dose (MTD) of ADIPemCis, using tumoral ASS1 loss as a selection biomarker. Pem (500mg/m2) and Cis (75mg/m2) were given every 3 weeks with weekly IM ADI-PEG20 (36mg/m2) for a maximum of 4 cycles with maintenance ADI-PEG20 or Pem in responding pts. Primary endpoint was tumor response rate (RECIST 1.1), with secondary endpoints including progression-free survival (PFS), overall survival (OS), and toxicity. We measured plasma arginine and citrulline concentrations, ADI-PEG20 antibodies, early molecular responses by 18-FLT-PET/CT, and biopsied pts on progression to explore resistance mechanisms. Results: 21 ASS1-deficient non-squamous NSCLC pts (median age 60) were enrolled out of 70 screened pts; 14% (3/21) had stable brain metastases at entry. Plasma arginine decreased with a reciprocal increase in plasma citrulline. The partial response rate was 48% (n=10/21) with a DCR of 85.7% (n=18/21). Median PFS was 2.6 months and median OS was 5.3 months. 23.8% (n=5/21) pts experienced grade 3/4 treatment-related toxicities, the most common being non-febrile neutropenia (14.3%). A partial metabolic response was detected in 57.1% (n=4/7) pts by 24 hrs of arginine deprivation using 18-FLT-PET/CT with EORTC response criteria. Upregulation of ASS1 expression was observed in 1/3 biopsies on progression. Conclusions: The ADIPemCis regimen is active in ASS1-deficient non-squamous NSCLC pts with a higher than expected response rate. However, the short survival (PFS and OS) compared with ASS1-agnostic historical controls indicates that ASS1 loss selects for a biologically more aggressive phenotype of NSCLC. Further studies are warranted exploring rationale combinations with ADIPemCis in patients with ASS1-deficient non-squamous NSCLC. Citation Format: Melissa Phillips, Teresa Szyszko, Peter Hall, Sukaina Rashid, Ramsay Khadeir, Louise Lim, Jeremy Steele, John Conibear, Paula Wells, Stephen Ellis, Xiaoxing Feng, Jim Thomson, Amanda Johnston, Bor-Wen Wu, John Bomalaski, Simon Pacey, Gary Cook, Peter Szlosarek. Expansion study of pegylated arginine deiminase (ADI-PEG20), pemetrexed, and cisplatin in patients with ASS1-deficient non-squamous non-small cell lung cancer (TRAP) [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B33.

Expansion study of ADI-PEG 20, pemetrexed and cisplatin in patients with ASS1-deficient malignant pleural mesothelioma (TRAP).

8553 Background: Argininosuccinate synthetase 1 (ASS1)-deficient malignant pleural mesothelioma (MPM) cells are sensitive to arginine deprivation with pegylated arginine deiminase (ADI-PEG20), which also potentiates the cytotoxic effect of pemetrexed (PEM). In the phase I dose-escalation TRAP study (NCT02029690) we showed that ADI-PEG20 with first-line PEM and cisplatin (CIS) chemotherapy (ADIPEMCIS) produced a 100% disease control rate (DCR) in patients (pts; n = 9) with ASS1-deficient thoracic cancers, with no additional toxicity (Beddowes et al 2017). Here, we present the TRAP expansion cohort experience in MPM. Methods: Good performance (ECOG 0-1) MPM pts with non-resectable disease and measurable by modified RECIST, were enrolled in a phase I TRAP expansion cohort at the maximum tolerated dose (MTD) of ADIPEMCIS, using tumoral ASS1 loss as a selection biomarker. PEM (500mg/m2) and CIS (75mg/m2) were given every 3 weeks with weekly IM ADI-PEG20 (36mg/m2) for a maximum of 6 cycles with maintenance ADI-PEG20 in responding pts. Primary endpoint was tumor response rate (modified RECIST), with secondary endpoints including progression-free survival (PFS), overall survival (OS), and toxicity. We measured plasma arginine and citrulline concentrations, ADI-PEG20 antibodies, and biopsied patients on progression to explore resistance mechanisms. Results: 31 ASS1-deficient MPM pts (median age 67) were enrolled (11 epithelioid, 10 biphasic and 10 sarcomatoid) out of 92 screened pts. Plasma arginine decreased with a reciprocal increase in plasma citrulline. The partial response rate was 35.5% (95% CI 19.2%-54.6%) with a DCR of 93.5% (95% CI 78.6%-99.2%). Median PFS was 5.6 months (95% CI 4-6) and median OS was 10.1 months (95% CI 6.7-17.7). 10/31 pts (32.3%) experienced grade 3/4 treatment-related toxicities, the most common being neutropenia (16.1%). Upregulation of ASS1 expression was observed in 2/3 biopsies on progression. Conclusions: The ADIPEMCIS regimen is active in ASS1-deficient MPM pts, including non-epithelioid disease. Based on these data the ATOMIC-meso phase 2/3 trial has opened comparing ADIPEMCIS versus PEMCISPlacebo, focusing on pts with non-epithelioid MPM. Clinical trial information: NCT02029690.