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Abstract Background Retroviral vectors are valuable tools for gene transfer. Particularly convenient are IRES-containing retroviral vectors expressing both the protein of interest and a marker protein from a single bicistronic mRNA. This coupled expression increases the relevance of tracking and/or selection of transduced cells based on the detection of a marker protein. pAP2 is a retroviral vector containing eGFP downstream of a modified IRES element of EMCV origin, and a CMV enhancer-promoter instead of the U3 region of the 5'LTR, which increases its efficiency in transient transfection. However, pAP2 contains a limited multicloning site (MCS) and shows weak eGFP expression, which previously led us to engineer an improved version, termed pPRIG, harboring: i) the wild-type ECMV IRES sequence, thereby restoring its full activity; ii) an optimized MCS flanked by T7 and SP6 sequences; and iii) a HA tag encoding sequence 5' of the MCS (pPRIG HAa/b/c). Results The convenience of pPRIG makes it a good basic vector to generate additional derivatives for an extended range of use. Here we present several novel pPRIG-based vectors (collectively referred to as PRIGs) in which : i) the HA tag sequence was inserted in the three reading frames 3' of the MCS (3'HA PRIGs); ii) a functional domain (ER, VP16 or KRAB) was inserted either 5' or 3' of the MCS (« modular » PRIGs); iii) eGFP was replaced by either eCFP, eYFP, mCherry or puro-R (« single color/resistance » PRIGs); and iv) mCherry, eYFP or eGFP was inserted 5' of the MCS of the IRES-eGFP, IRES-eCFP or IRES-Puro-R containing PRIGs, respectively (« dual color/selection » PRIGs). Additionally, some of these PRIGs were also constructed in a pMigR MSCV background which has been widely used in pluripotent cells. Conclusion These novel vectors allow for straightforward detection of any expressed protein (3'HA PRIGs), for functional studies of chimeric proteins (« modular » PRIGs), for multiple transductions and fluorescence analyses of transduced cells (« single color/resistance » PRIGs), or for quantitative detection of studied proteins in independently identified/selected transduced cells (« dual color/selection » PRIGs). They maintain the original advantages of pPRIG and provide suitable tools for either transient or stable expression and functional studies in a large range of experimental settings.

A correction to the article "Imatinib Enhances Human Melanoma Cell Susceptibility to TRAIL-Induced Cell Death: Relationship to Bcl-2 Family and Caspase Activation," that was published in the September 18, 2006 issue is presented.

Transcriptional cofactors of the ETO family are recurrent fusion partners in acute leukemia. We characterized the ETO2 regulome by integrating transcriptomic and chromatin binding analyses in human erythroleukemia xenografts and controlled ETO2 depletion models. We demonstrate that beyond its well-established repressive activity, ETO2 directly activates transcription of MYB, among other genes. The ETO2-activated signature is associated with a poorer prognosis in erythroleukemia but also in other acute myeloid and lymphoid leukemia subtypes. Mechanistically, ETO2 colocalizes with EP300 and MYB at enhancers supporting the existence of an ETO2/MYB feedforward transcription activation loop (e.g., on MYB itself). Both small-molecule and PROTAC-mediated inhibition of EP300 acetyltransferases strongly reduced ETO2 protein, chromatin binding, and ETO2-activated transcripts. Taken together, our data show that ETO2 positively enforces a leukemia maintenance program that is mediated in part by the MYB transcription factor and that relies on acetyltransferase cofactors to stabilize ETO2 scaffolding activity., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). HemaSphere published by John Wiley & Sons Ltd on behalf of European Hematology Association.)

Background: Resistance mechanisms to combination therapy with dabrafenib plus trametinib remain poorly understood in patients with BRAF V600E -mutant advanced non-small-cell lung cancer (NSCLC). We examined resistance to BRAF inhibition by single CTC sequencing in BRAF V600E -mutant NSCLC., Methods: CTCs and cfDNA were examined in seven BRAF V600E -mutant NSCLC patients at failure to treatment. Matched tumour tissue was available for four patients. Single CTCs were isolated by fluorescence-activated cell sorting following enrichment and immunofluorescence (Hoechst 33342/CD45/pan-cytokeratins) and sequenced for mutation and copy number-alteration (CNA) analyses., Results: BRAF V600E was found in 4/4 tumour biopsies and 5/7 cfDNA samples. CTC mutations were mostly found in MAPK-independent pathways and only 1/26 CTCs were BRAF V600E mutated. CTC profiles encompassed the majority of matched tumour biopsy CNAs but 72.5% to 84.5% of CTC CNAs were exclusive to CTCs. Extensive diversity, involving MAPK, MAPK-related, cell cycle, DNA repair and immune response pathways, was observed in CTCs and missed by analyses on tumour biopsies and cfDNA. Driver alterations in clinically relevant genes were recurrent in CTCs., Conclusions: Resistance was not driven by BRAF V600E -mutant CTCs. Extensive tumour genomic heterogeneity was found in CTCs compared to tumour biopsies and cfDNA at failure to BRAF inhibition, in BRAF V600E -mutant NSCLC, including relevant alterations that may represent potential treatment opportunities., (© 2024. The Author(s).)

Context: There is a lack of data on pesticide exposure levels during spraying with a knapsack, while it could have important implications for their users' health., Methods: We assessed levels and determinants of exposure in 24 male private landscapers/gardeners and municipal workers in France in 2011. Actual dermal exposure to glyphosate was assessed with cotton undergarments and gloves, and a cotton coverall changed between mixing and spraying to assess the contribution of each phase and body area to overall contamination. A field monitor observed the whole workshift and filled in a standardized observation grid., Results: The median actual contamination was 5,256 µg for the body, and 4,620 µg for hands. Spraying was more exposing than mixing/loading for all body parts except hands, which contributed to nearly 90% of body exposure during mixing/loading, and 30% during spraying, followed by back (14%). In the most exposed quartile, levels were close to some observations in agriculture., Conclusion: Our study provides new data on pesticide exposure levels of knapsack sprayer users; it should lead to a reinforced prevention, in order to make exposures as low as possible and lessen the risk of chronic diseases., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Occupational Hygiene Society.)

Pediatric acute myeloid leukemia expressing the ETO2::GLIS2 fusion oncogene is associated with dismal prognosis. Previous studies have shown that ETO2::GLIS2 can efficiently induce leukemia development associated with strong transcriptional changes but those amenable to pharmacological targeting remained to be identified. By studying an inducible ETO2::GLIS2 cellular model, we uncovered that de novo ETO2::GLIS2 expression in human cells led to increased CASP3 transcription, CASP3 activation, and cell death. Patient-derived ETO2::GLIS2 + leukemic cells expressed both high CASP3 and high BCL2. While BCL2 inhibition partly inhibited ETO2::GLIS2 + leukemic cell proliferation, BH3 profiling revealed that it also sensitized these cells to MCL1 inhibition indicating a functional redundancy between BCL2 and MCL1. We further show that combined inhibition of BCL2 and MCL1 is mandatory to abrogate disease progression using in vivo patient-derived xenograft models. These data reveal that a transcriptional consequence of ETO2::GLIS2 expression includes a positive regulation of the pro-apoptotic CASP3 and associates with a vulnerability to combined targeting of two BCL2 family members providing a novel therapeutic perspective for this aggressive pediatric AML subgroup., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Targeting the reprogramming and phagocytic capacities of tumor-associated macrophages (TAMs) has emerged as a therapeutic opportunity for cancer treatment. Here, we demonstrate that tumor cell phagocytosis drives the pro-inflammatory activation of TAMs and identify a key role for the cyclin-dependent kinase inhibitor CDKN1A (p21). Through the transcriptional repression of Signal-Regularity Protein α (SIRPα), p21 promotes leukemia cell phagocytosis and, subsequently, the pro-inflammatory reprogramming of phagocytic macrophages that extends to surrounding macrophages through Interferon γ. In mouse models of human T-cell acute lymphoblastic leukemia (T-ALL), infusion of human monocytes (Mos) engineered to overexpress p21 (p21TD-Mos) leads to Mo differentiation into phagocytosis-proficient TAMs that, after leukemia cell engulfment, undergo pro-inflammatory activation and trigger the reprogramming of bystander TAMs, reducing the leukemic burden and substantially prolonging survival in mice. These results reveal p21 as a trigger of phagocytosis-guided pro-inflammatory TAM reprogramming and highlight the potential for p21TD-Mo-based cellular therapy as a cancer immunotherapy., (© 2022. The Author(s).)

Objective: Although apple trees are heavily sprayed, few studies have assessed the pesticide exposure of operators and workers in apple orchards. However, these data are crucial for assessing the health impact of such exposures. The aim of this study was to measure pesticide exposure in apple growing according to tasks and body parts., Methods: A non-controlled field study was conducted in apple orchards in 4 regions of France during the 2016 and 2017 treatment seasons. Workers' external contamination and their determinants were assessed over 156 working days corresponding to 30 treatment days, 68 re-entry days and 58 harvesting days. We measured pesticide dermal contamination during each task and made detailed observations of work characteristics throughout the day. Captan and dithianon were used as markers of exposure., Results: The median dermal contamination per day was 5.50 mg of captan and 3.33 mg of dithianon for operators, 24.39 mg of captan and 1.84 mg of dithianon for re-entry workers, and 5.82 mg of captan and 0.74 mg of dithianon for harvesters. Thus, workers performing re-entry tasks, especially thinning and anti-hail net opening, presented higher contamination, either equal to or higher than in operators. For these last ones, mixing/loading and equipment cleaning were the most contaminating tasks. Most of the contamination was observed on workers' hands in all tasks, except for net-opening in which their heads accounted for the most daily contamination., Conclusions: This study highlights the importance of taking indirect exposures into account during re-entry work in apple growing., (© 2021. The Author(s).)

Knowledge of pesticide exposure levels in farmers is necessary for epidemiological studies and regulatory purposes. In the European pesticide registration process, operators' exposure is predicted using the Agricultural Operator Exposure Model (AOEM), created in 2014 by the European Food Safety Authority based on studies conducted by the pesticide industry. We compared operators' exposures during treatment days in the apple-growing industry under non-controlled working conditions and AOEM-predicted values. The dermal exposure of thirty French apple-growers from the CANEPA study when applying two fungicides was measured using body patches and cotton gloves. For each observation, the corresponding exposure was calculated by means of the AOEM, using data recorded about the operator, spraying equipment and personal protective equipment (PPE) used. A significant linear correlation was observed between calculated and measured daily exposures. The model overestimated the daily exposure approximately 4-fold and the exposure during application 10-fold. However, exposure was underestimated during mixing/loading for 70% of the observations when the operator wore PPE. The AOEM did not overestimate exposures in all circumstances, especially during mixing/loading, when operators handle concentrated products. The protection provided by PPE appeared to be overestimated. This could be due to the optimal working conditions under which the "industrial" studies are conducted, which may not be representative of real working conditions of operators in fruit-growing.

Gatekeeper mutations are identified in only 50% of the cases at resistance to Anaplastic Lymphoma Kinase (ALK)-tyrosine kinase inhibitors (TKIs). Circulating tumor cells (CTCs) are relevant tools to identify additional resistance mechanisms and can be sequenced at the single-cell level. Here, we provide in-depth investigation of copy number alteration (CNA) heterogeneity in phenotypically characterized CTCs at resistance to ALK-TKIs in ALK-positive non-small cell lung cancer. Single CTC isolation and phenotyping were performed by DEPArray or fluorescence-activated cell sorting following enrichment and immunofluorescence staining (ALK/cytokeratins/CD45/Hoechst). CNA heterogeneity was evaluated in six ALK-rearranged patients harboring ≥ 10 CTCs/20 mL blood at resistance to 1 st and 3 rd ALK-TKIs and one presented gatekeeper mutations. Out of 82 CTCs isolated by FACS, 30 (37%) were ALK + /cytokeratins - , 46 (56%) ALK - /cytokeratins + and 4 (5%) ALK + /cytokeratins + . Sequencing of 43 CTCs showed highly altered CNA profiles and high levels of chromosomal instability (CIN). Half of CTCs displayed a ploidy >2n and 32% experienced whole-genome doubling. Hierarchical clustering showed significant intra-patient and wide inter-patient CTC diversity. Classification of 121 oncogenic drivers revealed the predominant activation of cell cycle and DNA repair pathways and of RTK/RAS and PI3K to a lower frequency. CTCs showed wide CNA heterogeneity and elevated CIN at resistance to ALK-TKIs. The emergence of epithelial ALK-negative CTCs may drive resistance through activation of bypass signaling pathways, while ALK-rearranged CTCs showed epithelial-to-mesenchymal transition characteristics potentially contributing to ALK-TKI resistance. Comprehensive analysis of CTCs could be of great help to clinicians for precision medicine and resistance to ALK-targeted therapies., (© 2021. The Author(s).)

CD103 + CD8 + tumor-resident memory T cells (T RM ) are important components of anti-tumor immunity. However, their role in response to cancer immunotherapy is not fully understood. The protocol describes how to isolate CD8 + T cells and autologous tumor cells from human lung tumors to study the functional activities of CD8 + T cells. Tumors are heterogeneous in terms of the quantity and quality of immune cell types, so the yield of T RM cells depends on the features of the tumor. For complete details on the use and execution of this protocol, please refer to Corgnac et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2020 The Authors.)

Purpose: Children with Down syndrome (constitutive trisomy 21) that develop acute lymphoblastic leukemia (DS-ALL) have a 3-fold increased likelihood of treatment-related mortality coupled with a higher cumulative incidence of relapse, compared with other children with B-cell acute lymphoblastic leukemia (B-ALL). This highlights the lack of suitable treatment for Down syndrome children with B-ALL., Experimental Design: To facilitate the translation of new therapeutic agents into clinical trials, we built the first preclinical cohort of patient-derived xenograft (PDX) models of DS-ALL, comprehensively characterized at the genetic and transcriptomic levels, and have proven its suitability for preclinical studies by assessing the efficacy of drug combination between the MEK inhibitor trametinib and conventional chemotherapy agents., Results: Whole-exome and RNA-sequencing experiments revealed a high incidence of somatic alterations leading to RAS/MAPK pathway activation in our cohort of DS-ALL, as well as in other pediatric B-ALL presenting somatic gain of the chromosome 21 (B-ALL+21). In murine and human B-cell precursors, activated KRAS G12D functionally cooperates with trisomy 21 to deregulate transcriptional networks that promote increased proliferation and self renewal, as well as B-cell differentiation blockade. Moreover, we revealed that inhibition of RAS/MAPK pathway activation using the MEK1/2 inhibitor trametinib decreased leukemia burden in several PDX models of B-ALL+21, and enhanced survival of DS-ALL PDX in combination with conventional chemotherapy agents such as vincristine., Conclusions: Altogether, using novel and suitable PDX models, this study indicates that RAS/MAPK pathway inhibition represents a promising strategy to improve the outcome of Down syndrome children with B-cell precursor leukemia., (©2020 American Association for Cancer Research.)

Aberrant NF-κB activation is a hallmark of most B-cell malignancies. Recurrent inactivating somatic mutations in the NFKBIE gene, which encodes IκBε, an inhibitor of NF-κB-inducible activity, are reported in several B-cell malignancies with highest frequencies in chronic lymphocytic leukemia and primary mediastinal B-cell lymphoma, and account for a fraction of NF-κB pathway activation. The impact of NFKBIE deficiency on B-cell development and function remains, however, largely unknown. Here, we show that Nfkbie-deficient mice exhibit an amplification of marginal zone B cells and an expansion of B1 B-cell subsets. In germinal center (GC)-dependent immune response, Nfkbie deficiency triggers expansion of GC B-cells through increasing cell proliferation in a B-cell autonomous manner. We also show that Nfkbie deficiency results in hyperproliferation of a B1 B-cell subset and leads to increased NF-κB activation in these cells upon Toll-like receptor stimulation. Nfkbie deficiency cooperates with mutant MYD88 signaling and enhances B-cell proliferation in vitro. In aged mice, Nfkbie absence drives the development of an oligoclonal indolent B-cell lymphoproliferative disorders, resembling monoclonal B-cell lymphocytosis. Collectively, these findings shed light on an essential role of IκBε in finely tuning B-cell development and function.

Fusion oncogenes are prevalent in several pediatric cancers, yet little is known about the specific associations between age and phenotype. We observed that fusion oncogenes, such as ETO2-GLIS2 , are associated with acute megakaryoblastic or other myeloid leukemia subtypes in an age-dependent manner. Analysis of a novel inducible transgenic mouse model showed that ETO2-GLIS2 expression in fetal hematopoietic stem cells induced rapid megakaryoblastic leukemia whereas expression in adult bone marrow hematopoietic stem cells resulted in a shift toward myeloid transformation with a strikingly delayed in vivo leukemogenic potential. Chromatin accessibility and single-cell transcriptome analyses indicate ontogeny-dependent intrinsic and ETO2-GLIS2 -induced differences in the activities of key transcription factors, including ERG, SPI1, GATA1, and CEBPA. Importantly, switching off the fusion oncogene restored terminal differentiation of the leukemic blasts. Together, these data show that aggressiveness and phenotypes in pediatric acute myeloid leukemia result from an ontogeny-related differential susceptibility to transformation by fusion oncogenes. SIGNIFICANCE: This work demonstrates that the clinical phenotype of pediatric acute myeloid leukemia is determined by ontogeny-dependent susceptibility for transformation by oncogenic fusion genes. The phenotype is maintained by potentially reversible alteration of key transcription factors, indicating that targeting of the fusions may overcome the differentiation blockage and revert the leukemic state. See related commentary by Cruz Hernandez and Vyas, p. 1653 . This article is highlighted in the In This Issue feature, p. 1631 ., (©2019 American Association for Cancer Research.)

Purpose: Patients with anaplastic lymphoma kinase ( ALK )-rearranged non-small-cell lung cancer (NSCLC) inevitably develop resistance to ALK inhibitors. New diagnostic strategies are needed to assess resistance mechanisms and provide patients with the most effective therapy. We asked whether single circulating tumor cell (CTC) sequencing can inform on resistance mutations to ALK inhibitors and underlying tumor heterogeneity in ALK -rearranged NSCLC., Experimental Design: Resistance mutations were investigated in CTCs isolated at the single-cell level from patients at disease progression on crizotinib ( n = 14) or lorlatinib ( n = 3). Three strategies including filter laser-capture microdissection, fluorescence activated cell sorting, and the DEPArray were used. One hundred twenty-six CTC pools and 56 single CTCs were isolated and sequenced. Hotspot regions over 48 cancer-related genes and 14 ALK mutations were examined to identify ALK -independent and ALK -dependent resistance mechanisms., Results: Multiple mutations in various genes in ALK-independent pathways were predominantly identified in CTCs of crizotinib-resistant patients. The RTK-KRAS ( EGFR, KRAS, BRAF genes) and TP53 pathways were recurrently mutated. In one lorlatinib-resistant patient, two single CTCs out of 12 harbored ALK compound mutations. CTC-1 harbored the ALK G1202R/F1174C compound mutation virtually similar to ALK G1202R/F1174L present in the corresponding tumor biopsy. CTC-10 harbored a second ALK G1202R/T1151M compound mutation not detected in the tumor biopsy. By copy-number analysis, CTC-1 and the tumor biopsy had similar profiles, whereas CTC-10 harbored multiple copy-number alterations and whole-genome duplication., Conclusions: Our results highlight the genetic heterogeneity and clinical utility of CTCs to identify therapeutic resistance mutations in ALK -rearranged patients. Single CTC sequencing may be a unique tool to assess heterogeneous resistance mechanisms and help clinicians for treatment personalization and resistance options to ALK-targeted therapies., (©2019 American Association for Cancer Research.)