Direct Reprogramming of Hepatocytes Into JAK/Stat-Dependent LGR5+ Liver Cells Able to Initiate Intrahepatic Cholangiocarcinoma.

Somatic cells that have been partially reprogrammed by the factors Oct4, Sox2, Klf4, and cMyc (OSKM) have been demonstrated to be potentially tumorigenic in vitro and in vivo due to the acquisition of cancer-associated genomic alterations and the absence of OSKM clearance over time. In the present study, we obtained partially reprogrammed, SSEA1-negative cells by transducing murine hepatocytes with Δ1Δ3-deleted adenoviruses that expressed the 4 OSKM factors. We observed that, under long-term 2D and 3D culture conditions, hepatocytes could be converted into LGR5-positive cells with self-renewal capacity that was dependent on 3 cross-signaling pathways: IL6/Jak/Stat3, LGR5/R-spondin, and Wnt/ß-catenin. Following engraftment in syngeneic mice, LGR5-positive cells that expressed the cancer markers CD51, CD166, and CD73 were capable of forming invasive and metastatic tumors reminiscent of intrahepatic cholangiocarcinoma (ICC): they were positive for CK19 and CK7, featured associations of cord-like structures, and contained cuboidal and atypical cells with dissimilar degrees of pleomorphism and mitosis. The LGR5+-derived tumors exhibited a highly vascularized stroma with substantial fibrosis. In addition, we identified pro-angiogenic factors and signaling pathways involved in neo-angiogenesis and vascular development, which represent potential new targets for anti-angiogenic strategies to overcome tumor resistance to current ICC treatments.

Interaction between AIF and CHCHD4 Regulates Respiratory Chain Biogenesis.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, beyond its apoptotic function, is required for the normal expression of major respiratory chain complexes. Here we identified an AIF-interacting protein, CHCHD4, which is the central component of a redox-sensitive mitochondrial intermembrane space import machinery. Depletion or hypomorphic mutation of AIF caused a downregulation of CHCHD4 protein by diminishing its mitochondrial import. CHCHD4 depletion sufficed to induce a respiratory defect that mimicked that observed in AIF-deficient cells. CHCHD4 levels could be restored in AIF-deficient cells by enforcing its AIF-independent mitochondrial localization. This modified CHCHD4 protein reestablished respiratory function in AIF-deficient cells and enabled AIF-deficient embryoid bodies to undergo cavitation, a process of programmed cell death required for embryonic morphogenesis. These findings explain how AIF contributes to the biogenesis of respiratory chain complexes, and they establish an unexpected link between the vital function of AIF and the propensity of cells to undergo apoptosis.

Deciphering Potential Molecular Signatures to Differentiate Acute Myeloid Leukemia (AML) with BCR::ABL1 from Chronic Myeloid Leukemia (CML) in Blast Crisis.

Acute myeloid leukemia (AML) with BCR::ABL1 has recently been recognized as a distinct subtype in international classifications. Distinguishing it from myeloid blast crisis chronic myeloid leukemia (BC-CML) without evidence of a chronic phase (CP), remains challenging. We aimed to better characterize this entity by integrating clonal architecture analysis, mutational landscape assessment, and gene expression profiling. We analyzed a large retrospective cohort study including CML and AML patients. Two AML patients harboring a BCR::ABL1 fusion were included in the study. We identified BCR::ABL1 fusion as a primary event in one patient and a secondary one in the other. AML-specific variants were identified in both. Real-time RT-PCR experiments demonstrated that CD25 mRNA is overexpressed in advanced-phase CML compared to AML. Unsupervised principal component analysis showed that AML harboring a BCR::ABL1 fusion was clustered within AML. An AML vs. myeloid BC-CML differential expression signature was highlighted, and while ID4 (inhibitor of DNA binding 4) mRNA appears undetectable in most myeloid BC-CML samples, low levels are detected in AML samples. Therefore, CD25 and ID4 mRNA expression might differentiate AML with BCR::ABL1 from BC-CML and assign it to the AML group. A method for identifying this new WHO entity is then proposed. Finally, the hypothesis of AML with BCR::ABL1 arising from driver mutations on a BCR::ABL1 background behaving as a clonal hematopoiesis mutation is discussed. Validation of our data in larger cohorts and basic research are needed to better understand the molecular and cellular aspects of AML with a BCR::ABL1 entity.

Long-term development of human iPSC-derived pyramidal neurons quantified after transplantation into the neonatal mouse cortex.

One of the main obstacles for studying the molecular and cellular mechanisms underlying human neurodevelopment in vivo is the scarcity of experimental models. The discovery that neurons can be generated from human induced pluripotent stem cells (hiPSCs) paves the way for novel approaches that are stem cell-based. Here, we developed a technique to follow the development of transplanted hiPSC-derived neuronal precursors in the cortex of mice over time. Using post-mortem immunohistochemistry we quantified the differentiation and maturation of dendritic patterns of the human neurons over a total of six months. In addition, entirely hiPSC-derived neuronal parenchyma was followed over eight months using two-photon in vivo imaging through a cranial window. We found that transplanted hiPSC-derived neuronal precursors exhibit a "protracted" human developmental programme in different cortical areas. This offers novel possibilities for the sequential in vivo study of human cortical development and its alteration, followed in "real time".

HLA-dependent heterogeneity and macrophage immunoproteasome activation during lung COVID-19 disease.

BACKGROUND: The worldwide pandemic caused by the SARS-CoV-2 virus is characterized by significant and unpredictable heterogeneity in symptoms that remains poorly understood. METHODS: Transcriptome and single cell transcriptome of COVID19 lung were integrated with deeplearning analysis of MHC class I immunopeptidome against SARS-COV2 proteome. RESULTS: An analysis of the transcriptomes of lung samples from COVID-19 patients revealed that activation of MHC class I antigen presentation in these tissues was correlated with the amount of SARS-CoV-2 RNA present. Similarly, a positive relationship was detected in these samples between the level of SARS-CoV-2 and the expression of a genomic cluster located in the 6p21.32 region (40 kb long, inside the MHC-II cluster) that encodes constituents of the immunoproteasome. An analysis of single-cell transcriptomes of bronchoalveolar cells highlighted the activation of the immunoproteasome in CD68 + M1 macrophages of COVID-19 patients in addition to a PSMB8-based trajectory in these cells that featured an activation of defense response during mild cases of the disease, and an impairment of alveolar clearance mechanisms during severe COVID-19. By examining the binding affinity of the SARS-CoV-2 immunopeptidome with the most common HLA-A, -B, and -C alleles worldwide, we found higher numbers of stronger presenters in type A alleles and in Asian populations, which could shed light on why this disease is now less widespread in this part of the world. CONCLUSIONS: HLA-dependent heterogeneity in macrophage immunoproteasome activation during lung COVID-19 disease could have implications for efforts to predict the response to HLA-dependent SARS-CoV-2 vaccines in the global population.

iPSC-Derived Hereditary Breast Cancer Model Reveals the BRCA1-Deleted Tumor Niche as a New Culprit in Disease Progression.

Tumor progression begins when cancer cells recruit tumor-associated stromal cells to produce a vascular niche, ultimately resulting in uncontrolled growth, invasion, and metastasis. It is poorly understood, though, how this process might be affected by deletions or mutations in the breast cancer type 1 susceptibility (BRCA1) gene in patients with a lifetime risk of developing breast and/or ovarian cancer. To model the BRCA1-deleted stroma, we first generated induced pluripotent stem cells (iPSCs) from patients carrying a germline deletion of exon 17 of the BRCA1 gene (BRCA1+/- who, based on their family histories, were at a high risk for cancer. Using peripheral blood mononuclear cells (PBMCs) of these two affected family members and two normal (BRCA1+/+) individuals, we established a number of iPSC clones via non-integrating Sendai virus-based delivery of the four OCT4, SOX2, KLF4, and c-MYC factors. Induced mesenchymal stem cells (iMSCs) were generated and used as normal and pathological stromal cells. In transcriptome analyses, BRCA1+/- iMSCs exhibited a unique pro-angiogenic signature: compared to non-mutated iMSCs, they expressed high levels of HIF-1α, angiogenic factors belonging to the VEGF, PDGF, and ANGPT subfamilies showing high angiogenic potential. This was confirmed in vitro through the increased capacity to generate tube-like structures compared to BRCA1+/+ iMSCs and in vivo by a matrigel plug angiogenesis assay where the BRCA1+/- iMSCs promoted the development of an extended and organized vessel network. We also reported a highly increased migration capacity of BRCA1+/- iMSCs through an in vitro wound healing assay that correlated with the upregulation of the periostin (POSTN). Finally, we assessed the ability of both iMSCs to facilitate the engraftment of murine breast cancer cells using a xenogenic 4T1 transplant model. The co-injection of BRCA1+/- iMSCs and 4T1 breast cancer cells into mouse mammary fat pads gave rise to highly aggressive tumor growth (2-fold increase in tumor volume compared to 4T1 alone, p = 0.01283) and a higher prevalence of spontaneous metastatic spread to the lungs. Here, we report for the first time a major effect of BRCA1 haploinsufficiency on tumor-associated stroma in the context of BRCA1-associated cancers. The unique iMSC model used here was generated using patient-specific iPSCs, which opens new therapeutic avenues for the prevention and personalized treatment of BRCA1-associated hereditary breast cancer.

Embryonic Program Activated during Blast Crisis of Chronic Myelogenous Leukemia (CML) Implicates a TCF7L2 and MYC Cooperative Chromatin Binding.

Chronic myeloid leukemia (CML) is characterized by an inherent genetic instability, which contributes to the progression of the disease towards an accelerated phase (AP) and blast crisis (BC). Several cytogenetic and genomic alterations have been reported in the progression towards BC, but the precise molecular mechanisms of this event are undetermined. Transcription Factor 7 like 2 (TFC7L2) is a member of the TCF family of proteins that are known to activate WNT target genes such as Cyclin D1. TCF7L2 has been shown to be overexpressed in acute myeloid leukemia (AML) and represents a druggable target. We report here that TCF7L2 transcription factor expression was found to be correlated to blast cell numbers during the progression of the disease. In these cells, TCF7L2 CHIP-sequencing highlighted distal cis active enhancer, such as elements in SMAD3, ATF5, and PRMT1 genomic regions and a proximal active transcriptional program of 144 genes. The analysis of CHIP-sequencing of MYC revealed a significant overlapping of TCF7L2 epigenetic program with MYC. The ß-catenin activator lithium chloride and the MYC-MAX dimerization inhibitor 10058-F4 significantly modified the expression of three epigenetic targets in the BC cell line K562. These results suggest for the first time the cooperative role of TCF7L2 and MYC during CML-BC and they strengthen previous data showing a possible involvement of embryonic genes in this process.

Global MicroRNA Profiling Uncovers miR-206 as a Negative Regulator of Hematopoietic Commitment in Human Pluripotent Stem Cells.

Although human pluripotent stem cells (hPSCs) can theoretically differentiate into any cell type, their ability to produce hematopoietic cells is highly variable from one cell line to another. The underlying mechanisms of this heterogeneity are not clearly understood. Here, using a whole miRNome analysis approach in hPSCs, we discovered that their hematopoietic competency was associated with the expression of several miRNAs and conversely correlated to that of miR-206 specifically. Lentiviral-based miR-206 ectopic expression in H1 hematopoietic competent embryonic stem (ES) cells markedly impaired their differentiation toward the blood lineage. Integrative bioinformatics identified a potential miR-206 target gene network which included hematopoietic master regulators RUNX1 and TAL1. This work sheds light on the critical role of miR-206 in the generation of blood cells off hPSCs. Our results pave the way for future genetic manipulation of hPSCs aimed at increasing their blood regenerative potential and designing better protocols for the generation of bona fide hPSC-derived hematopoietic stem cells.

iPSC-Derived Embryoid Bodies as Models of c-Met-Mutated Hereditary Papillary Renal Cell Carcinoma.

Hereditary cancers with cancer-predisposing mutations represent unique models of human oncogenesis, as a driving oncogenic event is present in germline. Currently, there are no satisfactory models to study these malignancies. We report the generation of IPSC from the somatic cells of a patient with hereditary c-met-mutated papillary renal cell carcinoma (PRCC). From these cells we have generated spontaneous aggregates organizing in structures which expressed kidney markers such as PODXL and Six2. These structures expressed PRCC markers both in vitro and in vivo in NSG mice. Gene-expression profiling showed striking molecular similarities with signatures found in a large cohort of PRCC tumor samples. This analysis, applied to primary cancers with and without c-met mutation, showed overexpression of the BHLHE40 and KDM4C only in the c-met-mutated PRCC tumors, as predicted by c-met-mutated embryoid bodies transcriptome. These data therefore represent the first proof of concept of "hereditary renal cancer in a dish" model using c-met-mutated iPSC-derived embryoid bodies, opening new perspectives for discovery of novel predictive progression markers and for drug-screening for future precision-medicine strategies.

Altered microtubule dynamics and vesicular transport in mouse and human MeCP2-deficient astrocytes.

Rett syndrome (RTT) is a rare X-linked neurodevelopmental disorder, characterized by normal post-natal development followed by a sudden deceleration in brain growth with progressive loss of acquired motor and language skills, stereotypic hand movements and severe cognitive impairment. Mutations in the methyl-CpG-binding protein 2 (MECP2) cause more than 95% of classic cases. Recently, it has been shown that the loss of Mecp2 from glia negatively influences neurons in a non-cell-autonomous fashion, and that in Mecp2-null mice, re-expression of Mecp2 preferentially in astrocytes significantly improved locomotion and anxiety levels, restored respiratory abnormalities to a normal pattern and greatly prolonged lifespan compared with globally null mice. We now report that microtubule (MT)-dependent vesicle transport is altered in Mecp2-deficient astrocytes from newborn Mecp2-deficient mice compared with control wild-type littermates. Similar observation has been made in human MECP2 p.Arg294* iPSC-derived astrocytes. Importantly, administration of Epothilone D, a brain-penetrant MT-stabilizing natural product, was found to restore MT dynamics in Mecp2-deficient astrocytes and in MECP2 p.Arg294* iPSC-derived astrocytes in vitro. Finally, we report that relatively low weekly doses of Epothilone D also partially reversed the impaired exploratory behavior in Mecp2(308/y) male mice. These findings represent a first step toward the validation of an innovative treatment for RTT.

Direct and rapid identification of T315I-Mutated BCR-ABL expressing leukemic cells using infrared microspectroscopy.

Despite the major success obtained by the use of tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML), resistances to therapies occur due to mutations in the ABL-kinase domain of the BCR-ABL oncogene. Amongst these mutations, the "gatekeeper" T315I is a major concern as it renders leukemic cells resistant to all licenced TKI except Ponatinib. We report here that Fourier transform infrared (FTIR) microspectroscopy is a powerful methodology allowing rapid and direct identification of a spectral signature in single cells expressing T315I-mutated BCR-ABL. The specificity of this spectral signature is confirmed using a Dox-inducible T315I-mutated BCR-ABL-expressing human UT-7 cells as well as in murine embryonic stem cells. Transcriptome analysis of UT-7 cells expressing BCR-ABL as compared to BCR-ABL T315I clearly identified a molecular signature which could be at the origin of the generation of metabolic changes giving rise to the spectral signature. Thus, these results suggest that this new methodology can be applied to the identification of leukemic cells harbouring the T315I mutation at the single cell level and could represent a novel early detection tool of mutant clones. It could also be applied to drug screening strategies to target T315I-mutated leukemic cells.

Superoxide dismutase 2 (SOD2) contributes to genetic stability of native and T315I-mutated BCR-ABL expressing leukemic cells.

Manganese Superoxide dismutase 2 (SOD2) plays a crucial role in antioxidant defense but there are no data suggesting its role in genetic instability in CML. We evaluated the effects of SOD2 silencing in human UT7 cell line expressing either non-mutated or T315I-mutated BCR-ABL. Array-CGH experiments detected in BCR-ABL-expressing cells silenced for SOD2 a major genetic instability within several chromosomal loci, especially in regions carrying the glypican family (duplicated) and ß-defensin genes (deleted). In a large cohort of patients with chronic myeloid leukemia (CML), a significant decrease of SOD2 mRNA was observed. This reduction appeared inversely correlated with leukocytosis and Sokal score, high-risk patients showing lower SOD2 levels. The analysis of anti-oxidant gene expression analysis revealed a specific down-regulation of the expression of PRDX2 in UT7-BCR-ABL and UT7-T315I cells silenced for SOD2 expression. Gene set enrichment analysis performed between the two SOD2-dependent classes of CML patients revealed a significant enrichment of Reactive Oxygen Species (ROS) Pathway. Our data provide the first evidence for a link between SOD2 expression and genetic instability in CML. Consequently, SOD2 mRNA levels should be analyzed in prospective studies as patients with low SOD2 expression could be more prone to develop a mutator phenotype under TKI therapies.

In vitro culture and phenotypic and molecular characterization of gastric stem cells from human stomach.

BACKGROUND: Human gastric mucosa shows continuous self-renewal via differentiation from stem cells that remain poorly characterized. METHODS: We describe an original protocol for culture of gastric stem/progenitor cells from adult human stomach. The molecular characteristics of cells were studied using TaqMan low-density array and qRT-PCR analyses using the well-characterized H1 and H9 embryonic stem cells as reference. Epithelial progenitor cells were challenged with H. pylori to characterize their inflammatory response. RESULTS: Resident gastric stem cells expressed specific molecular markers of embryonic stem cells (SOX2, NANOG, and OCT4), as well as others specific to adult stem cells, particularly LGR5 and CD44. We show that gastric stem cells spontaneously differentiate into epithelial progenitor cells that can be challenged with H. pylori. The epithelial progenitor response to H. pylori showed a cag pathogenicity island-dependent induction of matrix metalloproteinases 1 and 3, chemokine (CXCL1, CXCL5, CXCL8, CCL20) and interleukine 33 expression. CONCLUSION: This study opens new outlooks for investigation of gastric stem cell biology and pathobiology as well as host-H. pylori interactions.

TLR ligands stimulation protects MSC from NK killing.

Mesenchymal stem cells (MSCs) play a fundamental role in allograft rejection and graft-versus-host disease through their immunosuppressive abilities. Recently, Toll-like receptors (TLR) have been shown to modulate MSC functions. The aim of this study was to investigate the effects of several TLR ligands on the interaction between MSC and natural killer (NK) cells. Our results show that TLR-primed adult bone marrow and embryonic MSC are more resistant than unprimed MSC to IL-2-activated NK-induced killing. Such protection can be explained by the modulation of Natural Killer group 2D ligands major histocompatibility complex class I chain A and ULBP3 and DNAM-1 ligands by TLR-primed MSC. These results indicate that MSCs are able to adapt their immuno-behavior in an inflammatory context, decreasing their susceptibility to NK killing. In addition, TLR3 but not TLR4-primed MSC enhance their suppressive functions against NK cells. However, the efficiency of this response is heterogeneous, even if the phenotypes of different analyzed MSC are rather homogeneous. The consequences could be important in MSC-mediated cell therapy, since the heterogeneity of adult MSC responders may be explored in order to select the more efficient responders.

Malignant germ cell-like tumors, expressing Ki-1 antigen (CD30), are revealed during in vivo differentiation of partially reprogrammed human-induced pluripotent stem cells.

Because many of the genes used to produce induced pluripotent stem cells (iPSCs) from somatic cells are either outright established oncogenes, such as c-myc and Klf4, or potentially related to tumorigenesis in various cancers, both the safety and the risks of tumorigenesis linked to iPSC generation require evaluation. In this work, we generated, by lentivirus-mediated gene transfer of Oct4, Sox2, Nanog, and Lin28, two types of iPSCs from human mesenchymal stem cells and human amniotic fluid-derived cells: fully reprogrammed iPSCs with silencing of the four transgenes and partially reprogrammed iPSCs that still express one or several transgenes. We assessed the behavior of these cells during both their differentiation and proliferation using in vivo teratoma assays in nonobese diabetic mice with severe combined immunodeficiency. In contrast to fully reprogrammed iPSCs, 43% of partially reprogrammed iPSC cases (6 of 14 teratomas) generated major dysplasia and malignant tumors, with yolk sac tumors and embryonal carcinomas positive for α-fetoprotein, cytokeratin AE1/AE3, and CD30. This correlated with the expression of one or several transgenes used for the reprogramming, down-regulation of CDK 1A mRNA (p21/CDKN1A), and up-regulation of antiapoptotic Bcl-2 mRNA. Therefore, the oncogenicity of therapeutically valuable patient-specific iPSC-derived cells should be scrupulously evaluated before they are used for any clinical applications.

Human mesenchymal stem cells derived from induced pluripotent stem cells down-regulate NK-cell cytolytic machinery.

A major issue in immunosuppressive biotherapy is the use of mesenchymal stem cells (MSCs) that harbor regulatory capacity. However, currently used bone marrow-derived MSCs (BM-MSCs) are short-lived and cannot assure long lasting immunoregulatory function both in vitro and in vivo. Consequently, we have generated MSCs from human induced pluripotent stem (IPS-MSCs) cells that share similar properties with embryonic stem cells (ES-MSCs). Herein, we compared the immunoregulatory properties of ES/IPS-MSCs with those of BM-MSCs and showed, for the first time, that IPS-derived MSCs display remarkable inhibition of NK-cell proliferation and cytolytic function in a similar way to ES-MSCs. Both MSCs disrupt NK-cell cytolytic machinery in the same fashion that BM-MSCs, by down-regulating the expression of different activation markers and ERK1/2 signaling, leading to an impairment to form immunologic synapses with target cells and, therefore, secretion of cytotoxic granules. In addition, they are more resistant than adult BM-MSCs to preactivated NK cells. IPS-MSCs could represent an attractive alternative source of immunoregulatory cells, and their capacity to impair NK-cell cytotoxicity constitutes a complex mechanism to prevent allograft rejection.

Leukemic stem cell persistence in chronic myeloid leukemia patients with sustained undetectable molecular residual disease.

Sustained undetectable molecular residual disease (UMRD) is obtained in a minority of patients with chronic myeloid leukemia treated with tyrosine kinase inhibitors. It remains unclear whether these patients are definitively cured of their leukemia or whether leukemic stem cells (LSCs) persist in their BM. We have evaluated the presence of BCR-ABL-expressing marrow LSCs in 6 patients with chronic myeloid leukemia with sustained UMRD induced by IFN-α (n = 3), imatinib mesylate after IFN-α failure (n = 2), and dasatinib after imatinib intolerance (n = 1). Purified CD34(+) cells were used for clonogenic and long-term culture-initiating cell assays performed on classic or HOXB4-expressing MS-5 feeders. Using this strategy, we identified BCR-ABL-expressing LSCs in all patients. Interestingly, long-term culture-initiating cell assays with MS-5/HOXB4 stromal feeders increased detected numbers of LSCs in 3 patients. The relation between LSC persistency and a potential risk of disease relapse for patients with durable UMRD (on or off tyrosine kinase inhibitor therapy) warrants further investigation.

Modeling RET-Rearranged Non-Small Cell Lung Cancer (NSCLC): Generation of Lung Progenitor Cells (LPCs) from Patient-Derived Induced Pluripotent Stem Cells (iPSCs).

REarranged during Transfection (RET) oncogenic rearrangements can occur in 1-2% of lung adenocarcinomas. While RET-driven NSCLC models have been developed using various approaches, no model based on patient-derived induced pluripotent stem cells (iPSCs) has yet been described. Patient-derived iPSCs hold great promise for disease modeling and drug screening. However, generating iPSCs with specific oncogenic drivers, like RET rearrangements, presents challenges due to reprogramming efficiency and genotypic variability within tumors. To address this issue, we aimed to generate lung progenitor cells (LPCs) from patient-derived iPSCs carrying the mutation RETC634Y, commonly associated with medullary thyroid carcinoma. Additionally, we established a RETC634Y knock-in iPSC model to validate the effect of this oncogenic mutation during LPC differentiation. We successfully generated LPCs from RETC634Y iPSCs using a 16-day protocol and detected an overexpression of cancer-associated markers as compared to control iPSCs. Transcriptomic analysis revealed a distinct signature of NSCLC tumor repression, suggesting a lung multilineage lung dedifferentiation, along with an upregulated signature associated with RETC634Y mutation, potentially linked to poor NSCLC prognosis. These findings were validated using the RETC634Y knock-in iPSC model, highlighting key cancerous targets such as PROM2 and C1QTNF6, known to be associated with poor prognostic outcomes. Furthermore, the LPCs derived from RETC634Y iPSCs exhibited a positive response to the RET inhibitor pralsetinib, evidenced by the downregulation of the cancer markers. This study provides a novel patient-derived off-the-shelf iPSC model of RET-driven NSCLC, paving the way for exploring the molecular mechanisms involved in RET-driven NSCLC to study disease progression and to uncover potential therapeutic targets.

Case report: Long-term voluntary Tyrosine Kinase Inhibitor (TKI) discontinuation in chronic myeloid leukemia (CML): Molecular evidence of an immune surveillance.

The classical natural history of chronic myeloid leukemia (CML) has been drastically modified by the introduction of tyrosine kinase inhibitor (TKI) therapies. TKI discontinuation is currently possible in patients in deep molecular responses, using strict recommendations of molecular follow-up due to risk of molecular relapse, especially during the first 6 months. We report here the case of a patient who voluntarily interrupted her TKI therapy. She remained in deep molecular remission (MR4) for 18 months followed by detection of a molecular relapse at +20 months. Despite this relapse, she declined therapy until the occurrence of the hematological relapse (+ 4 years and 10 months). Retrospective sequential transcriptome experiments and a single-cell transcriptome RNA-seq analysis were performed. They revealed a molecular network focusing on several genes involved in both activation and inhibition of NK-T cell activity. Interestingly, the single-cell transcriptome analysis showed the presence of cells expressing NKG7, a gene involved in granule exocytosis and highly involved in anti-tumor immunity. Single cells expressing as granzyme H, cathepsin-W, and granulysin were also identified. The study of this case suggests that CML was controlled for a long period of time, potentially via an immune surveillance phenomenon. The role of NKG7 expression in the occurrence of treatment-free remissions (TFR) should be evaluated in future studies.

Modeling Global Genomic Instability in Chronic Myeloid Leukemia (CML) Using Patient-Derived Induced Pluripotent Stem Cells (iPSCs).

METHODS: We used a patient-specific induced pluripotent stem cell (iPSC) line treated with the mutagenic agent N-ethyl-N-nitrosourea (ENU). Genomic instability was validated using γ-H2AX and micronuclei assays and CGH array for genomic events. RESULTS: An increased number of progenitors (x5-Fold), which proliferated in liquid cultures with a blast cell morphology, was observed in the mutagenized condition as compared to the unmutagenized one. CGH array performed for both conditions in two different time points reveals several cancer genes in the ENU-treated condition, some known to be altered in leukemia (BLM, IKZF1, NCOA2, ALK, EP300, ERG, MKL1, PHF6 and TET1). Transcriptome GEO-dataset GSE4170 allowed us to associate 125 of 249 of the aberrations that we detected in CML-iPSC with the CML progression genes already described during progression from chronic and AP to BC. Among these candidates, eleven of them have been described in CML and related to tyrosine kinase inhibitor resistance and genomic instability. CONCLUSIONS: These results demonstrated that we have generated, for the first time to our knowledge, an in vitro genetic instability model, reproducing genomic events described in patients with BC.