An In Vivo CRISPR Screening Platform for Prioritizing Therapeutic Targets in AML

CRISPR-Cas9-based genetic screens have successfully identified cell type-dependent liabilities in cancer, including acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because most of these screens have been performed in vitro using established cell lines, evaluating the physiologic relevance of these targets is critical. We have established a CRISPR screening approach using orthotopic xenograft models to validate and prioritize AML-enriched dependencies in vivo, including in CRISPR-competent AML patient-derived xenograft (PDX) models tractable for genome editing. Our integrated pipeline has revealed several targets with translational value, including SLC5A3 as a metabolic vulnerability for AML addicted to exogenous myo-inositol and MARCH5 as a critical guardian to prevent apoptosis in AML. MARCH5 repression enhanced the efficacy of BCL2 inhibitors such as venetoclax, further highlighting the clinical potential of targeting MARCH5 in AML. Our study provides a valuable strategy for discovery and prioritization of new candidate AML therapeutic targets. SIGNIFICANCE: There is an unmet need to improve the clinical outcome of AML. We developed an integrated in vivo screening approach to prioritize and validate AML dependencies with high translational potential. We identified SLC5A3 as a metabolic vulnerability and MARCH5 as a critical apoptosis regulator in AML, both of which represent novel therapeutic opportunities.This article is highlighted in the In This Issue feature, p. 275

Epitope-engineered human hematopoietic stem cells are shielded from CD123-targeted immunotherapy

Targeted eradication of transformed or otherwise dysregulated cells using monoclonal antibodies (mAb), antibody-drug conjugates (ADC), T cell engagers (TCE), or chimeric antigen receptor (CAR) cells is very effective for hematologic diseases. Unlike the breakthrough progress achieved for B cell malignancies, there is a pressing need to find suitable antigens for myeloid malignancies. CD123, the interleukin-3 (IL-3) receptor alpha-chain, is highly expressed in various hematological malignancies, including acute myeloid leukemia (AML). However, shared CD123 expression on healthy hematopoietic stem and progenitor cells (HSPCs) bears the risk for myelotoxicity. We demonstrate that epitope-engineered HSPCs were shielded from CD123-targeted immunotherapy but remained functional, while CD123-deficient HSPCs displayed a competitive disadvantage. Transplantation of genome-edited HSPCs could enable tumor-selective targeted immunotherapy while rebuilding a fully functional hematopoietic system. We envision that this approach is broadly applicable to other targets and cells, could render hitherto undruggable targets accessible to immunotherapy, and will allow continued posttransplant therapy, for instance, to treat minimal residual disease (MRD)

Régulation de l'expression protéique des récepteurs à activité tyrosine kinase FLT3 et KIT dans les leucémies aigües myéloïdes

Les mutations FLT3-ITD et KITD816V sont fréquemment retrouvées dans les leucémies aiguës myéloïdes où elles sont associées à un pronostic défavorable. Ces deux récepteurs à activité tyrosine kinase (RTK) mutés sont des acteurs clés de la leucémogenèse régulant la prolifération, la survie et la différenciation cellulaire. L'objectif de ce travail de thèse a été d'étudier la régulation de l'expression protéique de FLT3 en réponse aux inhibiteurs du protéasome, le rôle de l'autophagie dans les LAM KITD816V et l'impact du 2-deoxy-D-glucose sur la localisation intracellulaire des récepteurs. Les travaux réalisés ont démontré trois manières originales de cibler des cellules portant les oncogènes FLT3-ITD ou KIT en jouant sur leur dégradation, leur localisation intracellulaire et l'autophagie.FLT3-ITD and KITD816V mutations are recurrently found in acute myeloid leukemia, where they are associated with a poor prognosis. These two Tyrosine Kinase Receptors (TKR) are involved in leukemogenesis, regulating proliferation, survival and cell differentiation. The aim of this thesis was to study the regulation of FLT3 protein expression in response to proteasome inhibitors, the role of autophagy in KITD816V-driven AML and the impact of 2-deoxy-D-glucose (2-DG) on the intracellular localization of TKRs. Our studies investigated three original ways to target cells bearing FLT3-ITD or oncogenic KIT mutations playing on their degradation, intracellular localization and autophagy

Inhibition of the proteasome and proteaphagy enhances apoptosis in FLT3‐ITD‐driven acute myeloid leukemia

International audienceAcute myeloid leukaemia (AML) is a clonal disorder that affects hematopoietic stem cells or myeloid progenitors. One of the most common mutations that results in AML occurs in the gene encoding fms‐like tyrosine kinase 3 (FLT3). Previous studies demonstrated that AML cells expressing FLT3‐ITD are more sensitive to the proteasome inhibitor (PI) Bortezomib (Bz), than FLT3 wild‐type cells, and this cytotoxicity is mediated by autophagy. Here we show that proteasome inhibition with Bz results in modest but consistent proteaphagy in MOLM‐14 leukemic cells expressing the FLT3‐ITD mutation, but not in OCI‐AML3 leukemic cells with wild type FLT3. Chemical inhibition of autophagy with Bafilomycin A (BafA) simultaneously blocked proteaphagy and resulted in accumulation of the p62 autophagy receptor in Bz‐treated MOLM‐14 cells. The use of ubiquitin traps (TUBEs) revealed that ubiquitin plays an important role in proteasome‐autophagy crosstalk. The p62 inhibitor Verteporfin (VT) blocked proteaphagy and, importantly, resulted in accumulation of high molecular weight forms of p62 and FLT3‐ITD in Bz‐treated MOLM‐14 cells. Both autophagy inhibitors enhanced Bz‐induced apoptosis in FLT3‐ITD‐driven leukemic cells, underlining the therapeutic potential of these treatments

III-V-semiconductor vertically-coupled whispering-gallery mode resonators made by selective lateral oxidation

International audienceIntegrated whispering-gallery mode resonators are attractive devices which have found applications as selective filters, low-threshold lasers, high-speed modulators, high-sensitivity sensors and even as nonlinear converters. Their performance is governed by the level of detrimental (scattering, bulk, bending) loss incurred and the usable loss represented by the coupling rate between the resonator and its access waveguide. Practically, the latter parameter can be more accurately controlled when the resonator lies above the access waveguide, in other words, when the device uses a vertical integration scheme. So far, when using such an integration technique, the process involved a rather technically challenging step being either a planarization or a substrate transfer step. In this presentation, we propose and demonstrate an alternative method to fabricate vertically-coupled whispering-gallery mode resonators on III-V semiconductor epitaxial structures which has the benefit of being planarization-free and performed as single-side top-down process. The approach relies on a selective lateral thermal oxidation of aluminum-rich AlGaAs layers to define the buried access waveguide and enhance the vertical confinement of the whispering-gallery mode into the resonator. As a first experimental proof-of-principle of this approach, 75 µm-diameter micro-disk devices exhibiting quality factor reaching ~4500 have been successfully made

AMP-Activated Protein Kinase Contributes to Apoptosis Induced by the Bcl-2 Inhibitor Venetoclax in Acute Myeloid Leukemia

The treatment of acute myeloid leukemia (AML) remains a challenge especially among the elderly. The Bcl-2 inhibitor venetoclax recently showed significant survival benefits in AML patients when combined to low-dose cytarabine or azacitidine. Bcl-2 inhibition initiate mitochondrial apoptosis, but also respiration and cellular ATP production in AML. AMP-Activated Protein Kinase (AMPK) is a central energy sensor activated by increased AMP:ATP ratio to restore the cellular energy balance. Unexpectedly, we observed that venetoclax inhibited AMPK activity through caspase-dependent degradation of AMPK subunits in AML cells. On the other hand, genetic models of AMPK invalidation and re-expression suggested that AMPK participated to the early stages of apoptotic response through a negative regulation of multi-domain anti-apoptotic effectors such as Mcl-1 or Bcl-xL. Together our results suggested a new link between AMPK and Bcl-2-dependent mitochondrial apoptosis that participated to the anti-leukemic activity of venetoclax in AML.</p

Coupled-mode analysis of vertically-coupled AlGaAs/AlOx microdisk resonators

International audienceThis article reports the experimental and theoretical assessments of the optical characteristics of recently-introduced vertically-coupled microdisk resonators made by selective oxidation of AlGaAs multilayer structures. Experimental measurements show that the Q-factors are in the 10 3 to 10 4 range for diameters ranging from 75 to 300 µm. To establish the origins of this limited performance a coupled-mode-theory-based model of the single-access-waveguide-coupled resonator system was developed. It includes features which are specific to oxide-based vertically-coupled resonators, namely losses towards the slab waveguide lying under the resonator and a coupling region with an asymmetric and multilayer structure. Setting this simulation tool required the proposal and validation of a general criterion to select an appropriate set of decomposition permittivity profiles to be able to accurately model the characteristics of these more complex couplers using the coupled-mode-theory approach. This theoretical development is generic and can be now deployed to simulate any device which includes multi-waveguide couplers with arbitrary piece-wise-constant profile of the dielectric permittivity. Exploiting this particular development and experimental measurements of the disk sidewall roughness and of the coupling lengths, the calculated and experimental Q-factors are found to be in good agreement and allow establishing that the current performance is limited by the scattering losses and the slab-leakage losses for small-and large-diameter devices respectively

Integrating rare genetic variants into DPYD pharmacogenetic testing may help preventing fluoropyrimidine-induced toxicity

International audienceVariability in genes involved in drug pharmacokinetics or drug response can be responsible for suboptimal treatment efficacy or predispose to adverse drug reactions. In addition to common genetic variations, large-scale sequencing studies have uncovered multiple rare genetic variants predicted to cause functional alterations in genes encoding proteins implicated in drug metabolism, transport and response. To understand the functional importance of rare genetic variants in DPYD , a pharmacogene whose alterations can cause severe toxicity in patients exposed to fluoropyrimidine-based regimens, massively parallel sequencing of the exonic regions and flanking splice junctions of the DPYD gene was performed in a series of nearly 3000 patients categorized according to pre-emptive DPD enzyme activity using the dihydrouracil/uracil ([UH 2 ]/[U]) plasma ratio as a surrogate marker of DPD activity. Our results underscore the importance of integrating next-generation sequencing-based pharmacogenomic interpretation into clinical decision making to minimize fluoropyrimidine-based chemotherapy toxicity without altering treatment efficacy

The FibromiR miR-214-3p Is Upregulated in Duchenne Muscular Dystrophy and Promotes Differentiation of Human Fibro-Adipogenic Muscle Progenitors

International audienceFibrosis is a deleterious invasion of tissues associated with many pathological conditions, such as Duchenne muscular dystrophy (DMD) for which no cure is at present available for its prevention or its treatment. Fibro-adipogenic progenitors (FAPs) are resident cells in the human skeletal muscle and can differentiate into myofibroblasts, which represent the key cell population responsible for fibrosis. In this study, we delineated the pool of microRNAs (miRNAs) that are specifically modulated by TGFβ1 in FAPs versus myogenic progenitors (MPs) by a global miRNome analysis. A subset of candidates, including several “FibromiRs”, was found differentially expressed between FAPs and MPs and was also deregulated in DMD versus healthy biopsies. Among them, the expression of the TGFβ1-induced miR-199a~214 cluster was strongly correlated with the fibrotic score in DMD biopsies. Loss-of-function experiments in FAPs indicated that a miR-214-3p inhibitor efficiently blocked expression of fibrogenic markers in both basal conditions and following TGFβ1 stimulation. We found that FGFR1 is a functional target of miR-214-3p, preventing the signaling of the anti-fibrotic FGF2 pathway during FAP fibrogenesis. Overall, our work demonstrates that the « FibromiR » miR-214-3p is a key activator of FAP fibrogenesis by modulating the FGF2/FGFR1/TGFβ axis, opening new avenues for the treatment of DMD

Dendrogenin A Synergizes with Cytarabine to Kill Acute Myeloid Leukemia Cells In Vitro and In Vivo

International audienceDendrogenin A (DDA) is a mammalian cholesterol metabolite that displays potent antitumor properties on acute myeloid leukemia (AML). DDA triggers lethal autophagy in cancer cells through a biased activation of the oxysterol receptor LXRβ, and the inhibition of a sterol isomerase. We hypothesize that DDA could potentiate the activity of an anticancer drug acting through a different molecular mechanism, and conducted in vitro and in vivo combination tests on AML cell lines and patient primary tumors. We report here results from tests combining DDA with antimetabolite cytarabine (Ara-C), one of the main drugs used for AML treatment worldwide. We demonstrated that DDA potentiated and sensitized AML cells, including primary patient samples, to Ara-C in vitro and in vivo. Mechanistic studies revealed that this sensitization was LXRβ-dependent and was due to the activation of lethal autophagy. This study demonstrates a positive in vitro and in vivo interaction between DDA and Ara-C, and supports the clinical evaluation of DDA in combination with Ara-C for the treatment of AML