Metabolic priming by multiple enzyme systems supports glycolysis, HIF1α stabilisation, and human cancer cell survival in early hypoxia.

Adaptation to chronic hypoxia occurs through changes in protein expression, which are controlled by hypoxia-inducible factor 1α (HIF1α) and are necessary for cancer cell survival. However, the mechanisms that enable cancer cells to adapt in early hypoxia, before the HIF1α-mediated transcription programme is fully established, remain poorly understood. Here we show in human breast cancer cells, that within 3 h of hypoxia exposure, glycolytic flux increases in a HIF1α-independent manner but is limited by NAD+ availability. Glycolytic ATP maintenance and cell survival in early hypoxia rely on reserve lactate dehydrogenase A capacity as well as the activity of glutamate-oxoglutarate transaminase 1 (GOT1), an enzyme that fuels malate dehydrogenase 1 (MDH1)-derived NAD+. In addition, GOT1 maintains low α-ketoglutarate levels, thereby limiting prolyl hydroxylase activity to promote HIF1α stabilisation in early hypoxia and enable robust HIF1α target gene expression in later hypoxia. Our findings reveal that, in normoxia, multiple enzyme systems maintain cells in a primed state ready to support increased glycolysis and HIF1α stabilisation upon oxygen limitation, until other adaptive processes that require more time are fully established.

Improved efficacy of quizartinib in combination therapy with PI3K inhibition in primary FLT3-ITD AML cells.

Acute myeloid leukemia is a heterogeneous hematopoietic malignancy, characterized by uncontrolled clonal proliferation of abnormal myeloid progenitor cells, with poor outcomes. The internal tandem duplication (ITD) mutation of the Fms-like receptor tyrosine kinase 3 (FLT3) (FLT3-ITD) represents the most common genetic alteration in AML, detected in approximately 30% of AML patients, and is associated with high leukemic burden and poor prognosis. Therefore, this kinase has been regarded as an attractive druggable target for the treatment of FLT3-ITD AML, and selective small molecule inhibitors, such as quizartinib, have been identified and trialled. However, clinical outcomes have been disappointing so far due to poor remission rates, also because of acquired resistance. A strategy to overcome resistance is to combine FLT3 inhibitors with other targeted therapies. In this study, we investigated the preclinical efficacy of the combination of quizartinib with the pan PI3K inhibitor BAY-806946 in FLT3-ITD cell lines and primary cells from AML patients. We show here that BAY-806946 enhanced quizartinib cytotoxicity and, most importantly, that this combination increases the ability of quizartinib to kill CD34+ CD38-leukemia stem cells, whilst sparing normal hematopoietic stem cells. Because constitutively active FLT3 receptor tyrosine kinase is known to boost aberrant PI3K signaling, the increased sensitivity of primary cells to the above combination can be the mechanistic results of the disruption of signaling by vertical inhibition.

Mapping the cellular response to electron transport chain inhibitors reveals selective signaling networks triggered by mitochondrial perturbation.

Mitochondrial perturbation is a key event in chemical-induced organ toxicities that is incompletely understood. Here, we studied how electron transport chain (ETC) complex I, II, or III (CI, CII and CIII) inhibitors affect mitochondrial functionality, stress response activation, and cell viability using a combination of high-content imaging and TempO-Seq in HepG2 hepatocyte cells. CI and CIII inhibitors perturbed mitochondrial membrane potential (MMP) and mitochondrial and cellular ATP levels in a concentration- and time-dependent fashion and, under conditions preventing a switch to glycolysis attenuated cell viability, whereas CII inhibitors had no effect. TempO-Seq analysis of changes in mRNA expression pointed to a shared cellular response to CI and CIII inhibition. First, to define specific ETC inhibition responses, a gene set responsive toward ETC inhibition (and not to genotoxic, oxidative, or endoplasmic reticulum stress) was identified using targeted TempO-Seq in HepG2. Silencing of one of these genes, NOS3, exacerbated the impact of CI and CIII inhibitors on cell viability, indicating its functional implication in cellular responses to mitochondrial stress. Then by monitoring dynamic responses to ETC inhibition using a HepG2 GFP reporter panel for different classes of stress response pathways and applying pathway and gene network analysis to TempO-Seq data, we looked for downstream cellular events of ETC inhibition and identified the amino acid response (AAR) as being triggered in HepG2 by ETC inhibition. Through in silico approaches we provide evidence indicating that a similar AAR is associated with exposure to mitochondrial toxicants in primary human hepatocytes. Altogether, we (i) unravel quantitative, time- and concentration-resolved cellular responses to mitochondrial perturbation, (ii) identify a gene set associated with adaptation to exposure to active ETC inhibitors, and (iii) show that ER stress and an AAR accompany ETC inhibition in HepG2 and primary hepatocytes.

Synergistic cytotoxicity of dual PI3K/mTOR and FLT3 inhibition in FLT3-ITD AML cells.

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy, characterized by a heterogeneous genetic landscape and complex clonal evolution, with poor outcomes. Mutation at the internal tandem duplication of FLT3 (FLT3-ITD) is one of the most common somatic alterations in AML, associated with high relapse rates and poor survival due to the constitutive activation of the FLT3 receptor tyrosine kinase and its downstream effectors, such as PI3K signaling. Thus, aberrantly activated FLT3-kinase is regarded as an attractive target for therapy for this AML subtype, and a number of small molecule inhibitors of this kinase have been identified, some of which are approved for clinical practice. Nevertheless, acquired resistance to these molecules is often observed, leading to severe clinical outcomes. Therapeutic strategies to tackle resistance include combining FLT3 inhibitors with other antileukemic agents. Here, we report on the preclinical activity of the combination of the FLT3 inhibitor quizartinib with the dual PI3K/mTOR inhibitor PF-04691502 in FLT3-ITD cells. Briefly, we show that the association of these two molecules displays synergistic cytotoxicity in vitro in FLT3-ITD AML cells, triggering 90% cell death at nanomolar concentrations after 48 h.

Targeting PI3K/Akt/mTOR in AML: Rationale and Clinical Evidence.

Acute myeloid leukemia (AML) is a highly heterogeneous hematopoietic malignancy characterized by excessive proliferation and accumulation of immature myeloid blasts in the bone marrow. AML has a very poor 5-year survival rate of just 16% in the UK; hence, more efficacious, tolerable, and targeted therapy is required. Persistent leukemia stem cell (LSC) populations underlie patient relapse and development of resistance to therapy. Identification of critical oncogenic signaling pathways in AML LSC may provide new avenues for novel therapeutic strategies. The phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) signaling pathway, is often hyperactivated in AML, required to sustain the oncogenic potential of LSCs. Growing evidence suggests that targeting key components of this pathway may represent an effective treatment to kill AML LSCs. Despite this, accruing significant body of scientific knowledge, PI3K/Akt/mTOR inhibitors have not translated into clinical practice. In this article, we review the laboratory-based evidence of the critical role of PI3K/Akt/mTOR pathway in AML, and outcomes from current clinical studies using PI3K/Akt/mTOR inhibitors. Based on these results, we discuss the putative mechanisms of resistance to PI3K/Akt/mTOR inhibition, offering rationale for potential candidate combination therapies incorporating PI3K/Akt/mTOR inhibitors for precision medicine in AML.

Multiparametric assessment of mitochondrial respiratory inhibition in HepG2 and RPTEC/TERT1 cells using a panel of mitochondrial targeting agrochemicals.

Evidence is mounting for the central role of mitochondrial dysfunction in several pathologies including metabolic diseases, accelerated ageing, neurodegenerative diseases and in certain xenobiotic-induced organ toxicity. Assessing mitochondrial perturbations is not trivial and the outcomes of such investigations are dependent on the cell types used and assays employed. Here we systematically investigated the effect of electron transport chain (ETC) inhibitors on multiple mitochondrial-related parameters in two human cell types, HepG2 and RPTEC/TERT1. Cells were exposed to a broad range of concentrations of 20 ETC-inhibiting agrochemicals and capsaicin, consisting of inhibitors of NADH dehydrogenase (Complex I, CI), succinate dehydrogenase (Complex II, CII) and cytochrome bc1 complex (Complex III, CIII). A battery of tests was utilised, including viability assays, lactate production, mitochondrial membrane potential (MMP) and the Seahorse bioanalyser, which simultaneously measures extracellular acidification rate [ECAR] and oxygen consumption rate [OCR]. CI inhibitors caused a potent decrease in OCR, decreased mitochondrial membrane potential, increased ECAR and increased lactate production in both cell types. Twenty-fourhour exposure to CI inhibitors decreased viability of RPTEC/TERT1 cells and 3D spheroid-cultured HepG2 cells in the presence of glucose. CI inhibitors decreased 2D HepG2 viability only in the absence of glucose. CII inhibitors had no notable effects in intact cells up to 10 µM. CIII inhibitors had similar effects to the CI inhibitors. Antimycin A was the most potent CIII inhibitor, with activity in the nanomolar range. The proposed CIII inhibitor cyazofamid demonstrated a mitochondrial uncoupling signal in both cell types. The study presents a comprehensive example of a mitochondrial assessment workflow and establishes measurable key events of ETC inhibition.

Correction to: Multiparametric assessment of mitochondrial respiratory inhibition in HepG2 and RPTEC/TERT1 cells using a panel of mitochondrial targeting agrochemicals.

In the original publication of the article.