Mitochondrial fusion is a therapeutic vulnerability of acute myeloid leukemia

Mitochondrial metabolism recently emerged as a critical dependency in acute myeloid leukemia (AML). The shape of mitochondria is tightly regulated by dynamin GTPase proteins, which drive opposing fusion and fission forces to consistently adapt bioenergetics to the cellular context. Here, we showed that targeting mitochondrial fusion was a new vulnerability of AML cells, when assayed in patient-derived xenograft (PDX) models. Genetic depletion of mitofusin 2 (MFN2) or optic atrophy 1 (OPA1) or pharmacological inhibition of OPA1 (MYLS22) blocked mitochondrial fusion and had significant anti-leukemic activity, while having limited impact on normal hematopoietic cells ex vivo and in vivo. Mechanistically, inhibition of mitochondrial fusion disrupted mitochondrial respiration and reactive oxygen species production, leading to cell cycle arrest at the G$_{0}$/G$_{1}$ transition. These results nominate the inhibition of mitochondrial fusion as a promising therapeutic approach for AML

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

The NADPH oxidase NOX5 protects against apoptosis in ALK-positive anaplastic large-cell lymphoma cell lines

Reactive oxygen species (ROS) are key modulators of apoptosis and carcinogenesis. One of the important sources of ROS is NADPH oxidases (NOXs). The isoform NOX5 is highly expressed in lymphoid tissues, but it has not been detected in any common Hodgkin or non-Hodgkin lymphoma cell lines. In diverse, nonlymphoid malignant cells NOX5 exerts an antiapoptotic effect. Apoptosis suppression is the hallmark feature of a rare type of lymphoma, termed anaplastic lymphoma kinase-positive (ALK(+)) anaplastic large-cell lymphoma (ALCL), and a major factor in the therapy resistance and relapse of ALK(+) ALCL tumors. We applied RT-PCR and Western blot analysis to detect NOX5 expression in three ALK(+) ALCL cell lines (Karpas-299, SR-786, SUP-M2). We investigated the role of NOX5 in apoptosis by small-interfering RNA (siRNA)-mediated gene silencing and chemical inhibition of NOX5 using FACS analysis and examining caspase 3 cleavage in Karpas-299 cells. We used immunohistochemistry to detect NOX5 in ALK(+) ALCL pediatric tumors. NOX5 mRNA was uniquely detected in ALK(+) ALCL cells, whereas cell lines of other lymphoma classes were devoid of NOX5. Transfection of NOX5-specific siRNA and chemical inhibition of NOX5 abrogated calcium-induced superoxide production and increased caspase 3-mediated apoptosis in Karpas-299 cells. Immunohistochemistry revealed focal NOX5 reactivity in pediatric ALK(+) ALCL tumor cells. These results indicate that NOX5-derived ROS contribute to apoptosis blockage in ALK(+) ALCL cell lines and suggest NOX5 as a potential pharmaceutical target to enhance apoptosis and thus to suppress tumor progression and prevent relapse in pediatric ALK(+) ALCL patients that resist classical therapeutic approaches

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

Mechanisms of HIV Receptor and Co-Receptor Down-Regulation by Prostratin: Role of Conventional and Novel PKC Isoforms

Prostratin is an unusual non-tumour promoting phorbol ester with potential as an inductive adjuvant therapy for highly active antiretroviral therapy (HAART) due to its ability to up-regulate viral expression from latent provirus. In addition, prostratin is also able to inhibit de novo HIV infection most probably because it induces down-regulation of HIV receptors from the surface of target cells. In this study, we investigate the mechanisms by which prostratin down-regulates HIV receptor and co-receptor surface expression in lymphocytic and monocytic cell lines. Our results indicate that prostratin induces down-regulation of surface expression of CD4 and CXCR4, but not CCR5, in various cell lines. Down-regulation of CD4 and CXCR4 by prostratin is achieved by internalization through receptor-mediated endocytosis and/or macropinocytosis, which is then followed by degradation of these molecules. Because prostratin is a protein kinase C (PKC) activator, we next examined the potential contribution of distinct PKC isoforms to down-regulate CD4 and CXCR4 in response to prostratin stimulation. Although exposure of cells to prostratin or phorbol-myristate-acetate (PMA) induces the translocation of several PKC isoforms to the plasma membrane, the use of specific PKC inhibitors revealed that novel PKCs are the main mediators of the prostratin-induced CD4 down-regulation, whereas both conventional and novel PKCs contribute to CXCR4 down-regulation. Altogether these results showed that prostratin, through the activation of conventional and/or novel PKC isoforms, rapidly reduces cell surface expression of CD4 and CXCR4, but not CCR5, by inducing their internalization and degradation

Reduced expression of the NADPH oxidase NOX4 is a hallmark of adipocyte differentiation

Adipocyte differentiation is a complex process regulated among other factors by insulin and the production of reactive oxygen species (ROS). NOX4 is a ROS generating NADPH oxidase enzyme mediating insulin's action in 3T3L1 adipocytes. In the present paper we show that NOX4 is expressed at high levels both in white and brown preadipocytes and that differentiation into adipocytes results in a decrease in their NOX4 mRNA content. These in vitro results were confirmed in vivo by demonstrating that in intact adipose tissue the majority of NOX4 expressing cells are localized within the preadipocyte containing stromal/vascular fraction, rather than in the portion consisting of mature adipocytes. In line with these observations, quantification of NOX4 mRNA in fat derived from different rodent models of insulin resistance indicated that alteration in NOX4 expression reflects changes in the ratio of adipocyte/interstitial fractions. In conclusion, we reveal that decreased NOX4 mRNA content is a hallmark of adipocyte differentiation and that NOX4 expression measured in whole adipose tissue is not an unequivocal indicator of intact or impaired insulin action

Deficiency in the NADPH oxidase 4 predisposes towards diet-induced obesity

NADPH oxidase 4 (NOX4) is a reactive oxygen species (ROS) producing NADPH oxidase that regulates redox homeostasis in diverse insulin-sensitive cell types. In particular, NOX4-derived ROS is a key modulator of adipocyte differentiation and mediates insulin receptor signaling in mature adipocytes in vitro. Our study was aimed at investigating the role of NOX4 in adipose tissue differentiation, whole body metabolic homeostasis and insulin sensitivity in vivo

Beta-cell-specific expression of NOX5 aggravates high fat diet-induced impairment of islet insulin secretion in mice

NADPH oxidases (NOX-es) produce reactive oxygen species and modulate β-cell insulin secretion. Islets of Type2 diabetic subjects present elevated expression of NOX5. Here we sought to characterize regulation of NOX5 expression in human islets in vitro and to uncover the relevance of NOX5 in islet function in vivo using a novel mouse model expressing NOX5 in doxycycline-inducible, β-cell-specific manner (RIP/rtTA/NOX5 mice). In situ hybridization and immunohistochemistry employed on pancreatic sections demonstrated NOX5 mRNA and protein expressions in human islets. In cultures of dispersed islets NOX5 protein was observed in somatostatin-positive (δ) cells in basal (2.8mM glucose) conditions. siRNA-mediated knock-down of NOX5 in human islets cultured in basal glucose concentrations resulted in diminished glucose-induced insulin secretion in vitro. However, when islets were preincubated in high (16.7mM) glucose media for 12 hours, NOX5 appeared also in insulin-positive (β) cells. In vivo, mice with β-cell NOX5 expression developed aggravated impairment of glucose-induced insulin secretion compared to control mice when challenged with 14 weeks of high-fat diet. Similarly, in vitro palmitate pre-incubation resulted in more severe reduction of insulin release in islets of RIP/rtTA/NOX5 mice compared to their control littermates. Decreased insulin secretion was most distinct in response to theophylline stimulation suggesting impaired cAMP-mediated signaling due to increased phophodiesterase activation. Our data provide the first insight into the complex regulation and function of NOX5 in islets implying an important role for NOX5 in δ-cell mediated intra-islet crosstalk in physiological circumstances but also identifying it as an aggravating factor in β-cell failure in diabetic conditions

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