Ciblage thérapeutique d'AMPK dans les leucémies aiguës myéloïdes

Acute myeloid leukemia (AML) is a heterogeneous disease with poor prognosis despite intensive treatments. Virtually all recurrent molecular alterations in AML functionally converge to cause signal transduction pathway dysregulation that drives cellular proliferation and survival. The mammalian target of rapamycin complex 1 (mTORC1) is a rapamycin-sensitive signaling node defined by the interaction between mTOR and raptor. Constitutive mTORC1 activity is nearly universal in AML. However, pharmacologic inhibition with rapamycin or second-generation mTOR kinase inhibitors has shown limited anti-leukemic activity in both preclinical models as well as in clinical trials, suggesting that addiction to this oncogene is not a recurrent event in AML. Here we report that sustained mTORC1 activity is nonetheless essential for the cytotoxicity induced by pharmacologic activation of AMP-activated protein kinase (AMPK) in AML. Our studies employed a novel AMPK activator called GSK621. Using CRISPR/Cas9 and shRNA-mediated silencing of the AMPKa1 catalytic subunit, we showed that AMPK activity was necessary for the anti-leukemic response induced by this agent. GSK621-induced AMPK activation precipitated autophagy, and blocking autophagy via shRNA-mediated knockdown of ATG5 or ATG7 protected AML cells from cytotoxicity resulting from treatment with GSK621, suggesting that autophagy promotes cell death in the context of active AMPK. GSK621 cytotoxicity was consistently observed across twenty different AML cell lines, primary AML patient samples and AML xenografts in vivo. GSK621-induced AMPK activation also impaired the self-renewal capacity of MLL-ENL- and FLT3-ITD-induced murine leukemias as measured by serial methylcellulose replating assays. Strikingly, GSK621 did not induce cytotoxicity in normal CD34+ hematopoietic progenitor cells. We hypothesized that the differential sensitivity to GSK621 could be due to the difference in amplitude of mTORC1 activation between AML and normal CD34+ cells. In contrast to most reported cellular models in which AMPK inhibits mTORC1, sustained mTORC1 activity was seen following GSK621-induced AMPK activation in AML. Inhibition of mTORC1 either pharmacologically (using rapamycin) or genetically (using shRNAs targeting raptor and mTOR) abrogated AMPK-induced cytotoxicity in AML cells, including primary AML patient samples. The same synthetic lethality could be recapitulated in normal CD34+ progenitors by constitutive activation of mTORC1 using a lentivirally-transduced myrAKT construct. We further observed that the level of ATF4 protein is under a transcriptionnal control by mTORC1 and a translational control by AMPK (through eIF2A), and explains the synthetic lethal relationship between AMPK and mTORC1. Taken together, these data show that the magnitude of mTORC1 activity determines the degree of cytotoxicity triggered by AMPK activation. Our results therefore support AMPK activation as a promising therapeutic strategy in AML and other mTORC1-active malignancies which warrants further investigations in clinical trials.Les leucémies aiguës myéloïdes (LAM) représentent un groupe d’hémopathies malignes agressives, de pronostic sombre en dépit des traitements intensifs actuellement proposés. Malgré une grande hétérogénéité clinique et moléculaire, les cellules de LAM sont caractérisées par l’activation de voies de signalisation essentielles à leur prolifération et leur survie, comme par exemple celle du complexe mTORC1 (mammalian target of rapamycin complex 1). Cependant, l’utilisation clinique d’inhibiteurs tels que la rapamycine ou des inhibiteurs catalytiques s’est avérée décevante, ce qui suggère qu’il n’y a pas d’addiction oncogénique à mTORC1 dans les LAM. Au cours de ce travail, nous avons démontré que l’activation de mTORC1 est au contraire une condition nécessaire à l’induction de la mort cellulaire en réponse à l’activation d’AMPK (AMP-activated protein kinase), établissant une relation de létalité synthétique entre ces deux voies. Pour cela, nous avons utilisé un nouveau composé activateur spécifique d’AMPK, le GSK621. En invalidant la sous-unité catalytique AMPKα1 par ARN interférence ou par le système CRISPR/Cas9, nous avons démontré que les effets antileucémiques de ce composé sont bien dépendants de l’activation d’AMPK. Nous avons observé que ce composé favorise l’autophagie, et que ce processus est impliqué dans la mort des cellules leucémiques puisque l’inhibition des protéines ATG5 ou ATG7 a un effet protecteur sur les cellules leucémiques. Les effets antileucémiques du composé GSK621 ont été confirmés sur des cellules primaires, ainsi que sur un panel de vingt lignées de LAM, et dans un modèle murin de xénogreffe. De façon intéressante, l’activation d’AMPK pourrait également compromettre la survie des cellules souches leucémiques, comme en atteste l’atténuation du potentiel clonogénique en méthylcellulose de cellules murines transformées par MLL-ENL ou FLT3-ITD. Nous avons observé que le composé GSK 621 n’avait pas de toxicité envers les progéniteurs hématopoïétiques normaux, ouvrant ainsi une fenêtre thérapeutique intéressante. Comme l’activation d’AMPK conduit dans de nombreux modèles cellulaires à l’inhibition de mTORC1, et comme l’activation de mTORC1 est observée dans les cellules de LAM mais pas dans les progéniteurs hématopoïétiques normaux, nous avons proposé l’hypothèse que le niveau d’activation de mTORC1 déterminait les effets de l’activateur d’AMPK. Pour cela, nous avons inhibé mTORC1 dans les cellules leucémiques d’une part, et activé mTORC1 dans les progéniteurs normaux d’autre part. De façon inattendue, mTORC1 échappe au contrôle d’AMPK dans les LAM, et nous avons observé que l’activation de mTORC1 est une condition nécessaire et suffisante pour que le composé GSK621 entraîne la mort des cellules. Le substrat moléculaire de cette létalité synthétique est le facteur de transcription proapoptotique ATF4, dont la transcription est favorisée par mTORC1, et la traduction par AMPK via la phosphorylation d’eIF2A. Ces travaux proposent donc que malgré l’absence d’addiction oncogénique, l’activation de mTORC1 dans les LAM représente une opportunité thérapeutique originale via une relation de létalité synthétique avec l’activation d’AMPK. Ils constituent un rationnel au développement clinique d’activateurs d’AMPK dans les LAM, voire dans d’autres cancers ayant une activation constitutive de mTORC1

A gene-expression profiling score for prediction of outcome in patients with follicular lymphoma: a retrospective training and validation analysis in three international cohorts

Patients with follicular lymphoma (FL) have heterogeneous outcomes. Predictor models able to distinguish, at diagnosis, patients at high versus low risk of progression are still needed. A training set of fresh-frozen tumour biopsies was prospectively obtained from 160 untreated patients with high-tumour-burden follicular lymphoma enrolled in the phase 3 randomised PRIMA trial, in which rituximab maintenance was evaluated after rituximab plus chemotherapy induction (median follow-up 6·6 years [IQR 6·0-7·0]). RNA of sufficient quality was obtained for 149 of 160 cases, and Affymetrix U133 Plus 2.0 microarrays were used for gene-expression profiling. We did a multivariate Cox regression analysis to identify genes with expression levels associated with progression-free survival independently of maintenance treatment in a subgroup of 134 randomised patients. Expression levels from 95 curated genes were then determined by digital expression profiling (NanoString technology) in 53 formalin-fixed paraffin-embedded samples of the training set to compare the technical reproducibility of expression levels for each gene between technologies. Genes with high correlation (>0·75) were included in an L2-penalised Cox model adjusted on rituximab maintenance to build a predictive score for progression-free survival. The model was validated using NanoString technology to digitally quantify gene expression in 488 formalin-fixed, paraffin-embedded samples from three independent international patient cohorts from the PRIMA trial (n=178; distinct from the training cohort), the University of Iowa/Mayo Clinic Lymphoma SPORE project (n=201), and the Barcelona Hospital Clinic (n=109). All tissue samples consisted of pretreatment diagnostic biopsies and were confirmed as follicular lymphoma grade 1-3a. The patients were all treated with regimens containing rituximab and chemotherapy, possibly followed by either rituximab maintenance or ibritumomab-tiuxetan consolidation. We determined an optimum threshold on the score to predict patients at low risk and high risk of progression. The model, including the multigene score and the threshold, was initially evaluated in the three validation cohorts separately. The sensitivity and specificity of the score for the prediction of the risk of lymphoma progression at 2 years were assessed on the combined validation cohorts. FINDINGS: In the training cohort, the expression levels of 395 genes were associated with a risk of progression. 23 genes reflecting both B-cell biology and tumour microenvironment with correlation coefficients greater than 0·75 between the two technologies and sample types were retained to build a predictive model that identified a population at an increased risk of progression (p<0·0001). In a multivariate Cox model for progression-free survival adjusted on rituximab maintenance treatment and Follicular Lymphoma International Prognostic Index 1 (FLIPI-1) score, this predictor independently predicted progression (adjusted hazard ratio [aHR] of the high-risk group compared with the low-risk group 3·68, 95% CI 2·19-6·17 [p<0·0001]). The 5-year progression-free survival was 26% (95% CI 16-43) in the high-risk group and 73% (64-83) in the low-risk group. The predictor performances were confirmed in each of the individual validation cohorts (aHR comparing high-risk to low-risk groups 2·57 [95% CI 1·65-4·01] in cohort 1; 2·12 [1·32-3·39] in cohort 2; and 2·11 [1·01-4·41] in cohort 3). In the combined validation cohort, the median progression-free survival was 3·1 years (95% CI 2·4-4·8) in the high-risk group and 10·8 years (10·1-not reached) in the low-risk group (p<0·0001). The risk of lymphoma progression at 2 years was 38% (95% CI 29-46) in the high-risk group and 19% (15-24) in the low-risk group. In a multivariate analysis, the score predicted progression-free survival independently of anti-CD20 maintenance treatment and of the FLIPI score (aHR for the combined cohort 2·30, 95% CI 1·72-3·07). INTERPRETATION: We developed and validated a robust 23-gene expression-based predictor of progression-free survival that is applicable to routinely available formalin-fixed, paraffin-embedded tumour biopsies from patients with follicular lymphoma at time of diagnosis. Applying this score could allow individualised therapy for patients according to their risk category

Vitamin D Receptor Controls Cell Stemness in Acute Myeloid Leukemia and in Normal Bone Marrow.

Vitamin D (VD) is a known differentiating agent, but the role of VD receptor (VDR) is still incompletely described in acute myeloid leukemia (AML), whose treatment is based mostly on antimitotic chemotherapy. Here, we present an unexpected role of VDR in normal hematopoiesis and in leukemogenesis. Limited VDR expression is associated with impaired myeloid progenitor differentiation and is a new prognostic factor in AML. In mice, the lack of Vdr results in increased numbers of hematopoietic and leukemia stem cells and quiescent hematopoietic stem cells. In addition, malignant transformation of Vdr-/- cells results in myeloid differentiation block and increases self-renewal. Vdr promoter is methylated in AML as in CD34+ cells, and demethylating agents induce VDR expression. Association of VDR agonists with hypomethylating agents promotes leukemia stem cell exhaustion and decreases tumor burden in AML mouse models. Thus, Vdr functions as a regulator of stem cell homeostasis and leukemic propagation

The Genetic Basis of Hepatosplenic T-cell Lymphoma

Hepatosplenic T cell lymphoma (HSTL) is a rare and lethal lymphoma; the genetic drivers of this disease are unknown. Through whole exome sequencing of 68 HSTLs, we define recurrently mutated driver genes and copy number alterations in the disease. Chromatin modifying genes including SETD2, INO80 and ARID1B were commonly mutated in HSTL, affecting 62% of cases. HSTLs manifest frequent mutations in STAT5B (31%), STAT3 (9%), and PIK3CD (9%) for which there currently exist potential targeted therapies. In addition, we noted less frequent events in EZH2, KRAS and TP53. SETD2 was the most frequently silenced gene in HSTL. We experimentally demonstrated that SETD2 acts as a tumor suppressor gene. In addition, we found that mutations in STAT5B and PIK3CD activate critical signaling pathways important to cell survival in HSTL. Our work thus defines the genetic landscape of HSTL and implicates novel gene mutations linked to HSTL pathogenesis and potential treatment targets

A French multicentric prospective prognostic cohort with epidemiological, clinical, biological and treatment information to improve knowledge on lymphoma patients: study protocol of the "REal world dAta in LYmphoma and survival in adults" (REALYSA) cohort.

BACKGROUND: Age-adjusted lymphoma incidence rates continue to rise in France since the early 80's, although rates have slowed since 2010 and vary across subtypes. Recent improvements in patient survival in major lymphoma subtypes at population level raise new questions about patient outcomes (i.e. quality of life, long-term sequelae). Epidemiological studies have investigated factors related to lymphoma risk, but few have addressed the extent to which socioeconomic status, social institutional context (i.e. healthcare system), social relationships, environmental context (exposures), individual behaviours (lifestyle) or genetic determinants influence lymphoma outcomes, especially in the general population. Moreover, the knowledge of the disease behaviour mainly obtained from clinical trials data is partly biased because of patient selection. METHODS: The REALYSA ("REal world dAta in LYmphoma and Survival in Adults") study is a real-life multicentric cohort set up in French areas covered by population-based cancer registries to study the prognostic value of epidemiological, clinical and biological factors with a prospective 9-year follow-up. We aim to include 6000 patients over 4 to 5 years. Adult patients without lymphoma history and newly diagnosed with one of the following 7 lymphoma subtypes (diffuse large B-cell, follicular, marginal zone, mantle cell, Burkitt, Hodgkin, mature T-cell) are invited to participate during a medical consultation with their hematologist. Exclusion criteria are: having already received anti-lymphoma treatment (except pre-phase) and having a documented HIV infection. Patients are treated according to the standard practice in their center. Clinical data, including treatment received, are extracted from patients' medical records. Patients' risk factors exposures and other epidemiological data are obtained at baseline by filling out a questionnaire during an interview led by a clinical research assistant. Biological samples are collected at baseline and during treatment. A virtual tumor biobank is constituted for baseline tumor samples. Follow-up data, both clinical and epidemiological, are collected every 6 months in the first 3 years and every year thereafter. DISCUSSION: This cohort constitutes an innovative platform for clinical, biological, epidemiological and socio-economic research projects and provides an opportunity to improve knowledge on factors associated to outcome of lymphoma patients in real life. TRIAL REGISTRATION: 2018-A01332-53, ClinicalTrials.gov identifier: NCT03869619

AMPK is a therapeutic target in acute meloid leukemias

Les leucémies aiguës myéloïdes (LAM) représentent un groupe d’hémopathies malignes agressives, de pronostic sombre en dépit des traitements intensifs actuellement proposés. Malgré une grande hétérogénéité clinique et moléculaire, les cellules de LAM sont caractérisées par l’activation de voies de signalisation essentielles à leur prolifération et leur survie, comme par exemple celle du complexe mTORC1 (mammalian target of rapamycin complex 1). Cependant, l’utilisation clinique d’inhibiteurs tels que la rapamycine ou des inhibiteurs catalytiques s’est avérée décevante, ce qui suggère qu’il n’y a pas d’addiction oncogénique à mTORC1 dans les LAM. Au cours de ce travail, nous avons démontré que l’activation de mTORC1 est au contraire une condition nécessaire à l’induction de la mort cellulaire en réponse à l’activation d’AMPK (AMP-activated protein kinase), établissant une relation de létalité synthétique entre ces deux voies. Pour cela, nous avons utilisé un nouveau composé activateur spécifique d’AMPK, le GSK621. En invalidant la sous-unité catalytique AMPKα1 par ARN interférence ou par le système CRISPR/Cas9, nous avons démontré que les effets antileucémiques de ce composé sont bien dépendants de l’activation d’AMPK. Nous avons observé que ce composé favorise l’autophagie, et que ce processus est impliqué dans la mort des cellules leucémiques puisque l’inhibition des protéines ATG5 ou ATG7 a un effet protecteur sur les cellules leucémiques. Les effets antileucémiques du composé GSK621 ont été confirmés sur des cellules primaires, ainsi que sur un panel de vingt lignées de LAM, et dans un modèle murin de xénogreffe. De façon intéressante, l’activation d’AMPK pourrait également compromettre la survie des cellules souches leucémiques, comme en atteste l’atténuation du potentiel clonogénique en méthylcellulose de cellules murines transformées par MLL-ENL ou FLT3-ITD. Nous avons observé que le composé GSK 621 n’avait pas de toxicité envers les progéniteurs hématopoïétiques normaux, ouvrant ainsi une fenêtre thérapeutique intéressante. Comme l’activation d’AMPK conduit dans de nombreux modèles cellulaires à l’inhibition de mTORC1, et comme l’activation de mTORC1 est observée dans les cellules de LAM mais pas dans les progéniteurs hématopoïétiques normaux, nous avons proposé l’hypothèse que le niveau d’activation de mTORC1 déterminait les effets de l’activateur d’AMPK. Pour cela, nous avons inhibé mTORC1 dans les cellules leucémiques d’une part, et activé mTORC1 dans les progéniteurs normaux d’autre part. De façon inattendue, mTORC1 échappe au contrôle d’AMPK dans les LAM, et nous avons observé que l’activation de mTORC1 est une condition nécessaire et suffisante pour que le composé GSK621 entraîne la mort des cellules. Le substrat moléculaire de cette létalité synthétique est le facteur de transcription proapoptotique ATF4, dont la transcription est favorisée par mTORC1, et la traduction par AMPK via la phosphorylation d’eIF2A. Ces travaux proposent donc que malgré l’absence d’addiction oncogénique, l’activation de mTORC1 dans les LAM représente une opportunité thérapeutique originale via une relation de létalité synthétique avec l’activation d’AMPK. Ils constituent un rationnel au développement clinique d’activateurs d’AMPK dans les LAM, voire dans d’autres cancers ayant une activation constitutive de mTORC1.Acute myeloid leukemia (AML) is a heterogeneous disease with poor prognosis despite intensive treatments. Virtually all recurrent molecular alterations in AML functionally converge to cause signal transduction pathway dysregulation that drives cellular proliferation and survival. The mammalian target of rapamycin complex 1 (mTORC1) is a rapamycin-sensitive signaling node defined by the interaction between mTOR and raptor. Constitutive mTORC1 activity is nearly universal in AML. However, pharmacologic inhibition with rapamycin or second-generation mTOR kinase inhibitors has shown limited anti-leukemic activity in both preclinical models as well as in clinical trials, suggesting that addiction to this oncogene is not a recurrent event in AML. Here we report that sustained mTORC1 activity is nonetheless essential for the cytotoxicity induced by pharmacologic activation of AMP-activated protein kinase (AMPK) in AML. Our studies employed a novel AMPK activator called GSK621. Using CRISPR/Cas9 and shRNA-mediated silencing of the AMPKa1 catalytic subunit, we showed that AMPK activity was necessary for the anti-leukemic response induced by this agent. GSK621-induced AMPK activation precipitated autophagy, and blocking autophagy via shRNA-mediated knockdown of ATG5 or ATG7 protected AML cells from cytotoxicity resulting from treatment with GSK621, suggesting that autophagy promotes cell death in the context of active AMPK. GSK621 cytotoxicity was consistently observed across twenty different AML cell lines, primary AML patient samples and AML xenografts in vivo. GSK621-induced AMPK activation also impaired the self-renewal capacity of MLL-ENL- and FLT3-ITD-induced murine leukemias as measured by serial methylcellulose replating assays. Strikingly, GSK621 did not induce cytotoxicity in normal CD34+ hematopoietic progenitor cells. We hypothesized that the differential sensitivity to GSK621 could be due to the difference in amplitude of mTORC1 activation between AML and normal CD34+ cells. In contrast to most reported cellular models in which AMPK inhibits mTORC1, sustained mTORC1 activity was seen following GSK621-induced AMPK activation in AML. Inhibition of mTORC1 either pharmacologically (using rapamycin) or genetically (using shRNAs targeting raptor and mTOR) abrogated AMPK-induced cytotoxicity in AML cells, including primary AML patient samples. The same synthetic lethality could be recapitulated in normal CD34+ progenitors by constitutive activation of mTORC1 using a lentivirally-transduced myrAKT construct. We further observed that the level of ATF4 protein is under a transcriptionnal control by mTORC1 and a translational control by AMPK (through eIF2A), and explains the synthetic lethal relationship between AMPK and mTORC1. Taken together, these data show that the magnitude of mTORC1 activity determines the degree of cytotoxicity triggered by AMPK activation. Our results therefore support AMPK activation as a promising therapeutic strategy in AML and other mTORC1-active malignancies which warrants further investigations in clinical trials