RSK2 is a new Pim2 target with pro-survival functions in FLT3-ITD-positive acute myeloid leukemia.

Acute myeloid leukemia (AML) with the FLT3 internal tandem duplication (FLT3-ITD AML) accounts for 20-30% of AML cases. This subtype usually responds poorly to conventional therapies, and might become resistant to FLT3 tyrosine kinase inhibitors (TKIs) due to molecular bypass mechanisms. New therapeutic strategies focusing on resistance mechanisms are therefore urgently needed. Pim kinases are FLT3-ITD oncogenic targets that have been implicated in FLT3 TKI resistance. However, their precise biological function downstream of FLT3-ITD requires further investigation. We performed high-throughput transcriptomic and proteomic analyses in Pim2-depleted FLT3-ITD AML cells and found that Pim2 predominantly controlled apoptosis through Bax expression and mitochondria disruption. We identified ribosomal protein S6 kinase A3 (RSK2), a 90 kDa serine/threonine kinase involved in the mitogen-activated protein kinase cascade encoded by the RPS6KA3 gene, as a novel Pim2 target. Ectopic expression of an RPS6KA3 allele rescued the viability of Pim2-depleted cells, supporting the involvement of RSK2 in AML cell survival downstream of Pim2. Finally, we showed that RPS6KA3 knockdown reduced the propagation of human AML cells in vivo in mice. Our results point to RSK2 as a novel Pim2 target with translational therapeutic potential in FLT3-ITD AML.

Oncogenic FLT3-ITD supports autophagy via ATF4 in acute myeloid leukemia.

In acute myeloid leukemia (AML), internal tandem duplication mutations in the FLT3 tyrosine kinase receptor (FLT3-ITD) account for up to 25% of cases and are associated with a poor outcome. In order to better target this AML subtype, a comprehensive view of how FLT3-ITD impacts AML cell biology is required. Here, we found that FLT3-ITD expression increased basal autophagy in AML cells, and that both pharmacological and genetic inhibition of the receptor reduced autophagy in primary AML samples and cell lines. Conditional expression of shRNAs against key autophagy proteins demonstrated that autophagy is required for AML cell proliferation in vitro and for leukemic cells survival in a mouse model of xenograft. Importantly, autophagy inhibition also overcame FLT3 inhibitor resistance both in vitro and in vivo. The transcription factor ATF4 was identified as an essential actor of FLT3-ITD-induced autophagy. Cellular levels of ATF4 were highly dependent on FLT3-ITD activity, and downregulation of ATF4 inhibited autophagy-dependent AML cell proliferation and improved overall mouse survival similarly to autophagy inhibition. These results suggest that targeting autophagy or ATF4 in patients expressing FLT3 mutations may represent a novel promising and innovative therapeutic strategy for AML.

High mTORC1 activity drives glycolysis addiction and sensitivity to G6PD inhibition in acute myeloid leukemia cells.

Alterations in metabolic activities are cancer hallmarks that offer a wide range of new therapeutic opportunities. Here we decipher the interplay between mTORC1 activity and glucose metabolism in acute myeloid leukemia (AML). We show that mTORC1 signaling that is constantly overactivated in AML cells promotes glycolysis and leads to glucose addiction. The level of mTORC1 activity determines the sensitivity of AML cells to glycolysis inhibition as switch-off mTORC1 activity leads to glucose-independent cell survival that is sustained by an increase in mitochondrial oxidative phosphorylation. Metabolic analysis identified the pentose phosphate pathway (PPP) as an important pro-survival pathway for glucose metabolism in AML cells with high mTORC1 activity and provided a clear rational for targeting glucose-6-phosphate dehydrogenase (G6PD) in AML. Indeed, our analysis of the cancer genome atlas AML database pinpointed G6PD as a new biomarker in AML, as its overexpression correlated with an adverse prognosis in this cohort. Targeting the PPP using the G6PD inhibitor 6-aminonicotinamide induces in vitro and in vivo cytotoxicity against AML cells and synergistically sensitizes leukemic cells to chemotherapy. Our results demonstrate that high mTORC1 activity creates a specific vulnerability to G6PD inhibition that may work as a new AML therapy.

A robust and rapid xenograft model to assess efficacy of chemotherapeutic agents for human acute myeloid leukemia.

Relevant preclinical mouse models are crucial to screen new therapeutic agents for acute myeloid leukemia (AML). Current in vivo models based on the use of patient samples are not easy to establish and manipulate in the laboratory. Our objective was to develop robust xenograft models of human AML using well-characterized cell lines as a more accessible and faster alternative to those incorporating the use of patient-derived AML cells. Five widely used AML cell lines representing various AML subtypes were transplanted and expanded into highly immunodeficient non-obese diabetic/LtSz-severe combined immunodeficiency IL2Rγc(null) mice (for example, cell line-derived xenografts). We show here that bone marrow sublethal conditioning with busulfan or irradiation has equal efficiency for the xenotransplantation of AML cell lines. Although higher number of injected AML cells did not change tumor engraftment in bone marrow and spleen, it significantly reduced the overall survival in mice for all tested AML cell lines. On the basis of AML cell characteristics, these models also exhibited a broad range of overall mouse survival, engraftment, tissue infiltration and aggressiveness. Thus, we have established a robust, rapid and straightforward in vivo model based on engraftment behavior of AML cell lines, all vital prerequisites for testing new therapeutic agents in preclinical studies.

Mitochondrial energetic and AKT status mediate metabolic effects and apoptosis of metformin in human leukemic cells.

Previous reports demonstrate that metformin, an anti-diabetic drug, can decrease the risk of cancer and inhibit cancer cell growth. However, its mechanism in cancer cells is still unknown. Metformin significantly blocks cell cycle and inhibits cell proliferation and colony formation of leukemic cells. However, the apoptotic response to metformin varies. Furthermore, daily treatment with metformin induces apoptosis and reduces tumor growth in vivo. While metformin induces early and transient activation of AMPK, inhibition of AMPKα1/2 does not abrogate anti-proliferative or pro-apoptotic effects of metformin. Metformin decreases electron transport chain complex I activity, oxygen consumption and mitochondrial ATP synthesis, while stimulating glycolysis for ATP and lactate production, pentose phosphate pathway for purine biosynthesis, fatty acid metabolism, as well as anaplerotic and mitochondrial gene expression. Importantly, leukemic cells with high basal AKT phosphorylation, glucose consumption or glycolysis exhibit a markedly reduced induction of the Pasteur effect in response to metformin and are resistant to metformin-induced apoptosis. Accordingly, glucose starvation or treatment with deoxyglucose or an AKT inhibitor induces sensitivity to metformin. Overall, metformin elicits reprogramming of intermediary metabolism leading to inhibition of cell proliferation in all leukemic cells and apoptosis only in leukemic cells responding to metformin with AKT phosphorylation and a strong Pasteur effect.

The short form of RON is expressed in acute myeloid leukemia and sensitizes leukemic cells to cMET inhibitors.

Several receptor tyrosine kinases (TKs) are involved in the pathogenesis of acute myeloid leukemia (AML). Here, we have assessed the expression of the Recepteur d'Origine Nantais (RON) in leukemic cell lines and samples from AML patients. In a series of 86 AML patients, we show that both the full length and/or the short form (sf) of RON are expressed in 51% and 43% of cases, respectively. Interestingly, sfRON is not expressed in normal CD34+ hematopoietic cells and induces part of its oncogenic signaling through interaction with the Src kinase Lyn. sfRON-mediated signaling in leukemic cells also involves mTORC1, the proapoptotic bcl2-family member, BAD, but not the phosphatidylinositol 3-kinase/Akt pathway. Furthermore, the expression of sfRON was specifically downregulated by 5-azacytidine (AZA). Conversely, AZA could induce the expression of sfRON in sfRON-negative leukemic cells suggesting that the activity of this drug in AML and myelodysplastic syndromes could involve modulation of TKs. cMET/RON inhibitors exhibited an antileukemic activity exclusively in AML samples and cell lines expressing sfRON. These results might support clinical trials evaluating cMET/RON inhibitors in AML patients expressing sfRON.

A robust xenotransplantation model for acute myeloid leukemia.

Xenotransplantation of human acute myeloid leukemia (AML) in immunocompromised animals has been critical for defining leukemic stem cells. However, existing immunodeficient strains of mice have short life spans and low levels of AML cell engraftment, hindering long-term evaluation of primary human AML biology. A recent study suggested that NOD/LtSz-scid IL2Rgammac null (NSG) mice have enhanced AML cell engraftment, but this relied on technically challenging neonatal injections. Here, we performed extensive analysis of AML engraftment in adult NSG mice using tail vein injection. Of the 35 AML samples analyzed, 66% showed bone marrow engraftment over 0.1%. Further, 37% showed high levels of engraftment (>10%), with some as high as 95%. A 2-44-fold expansion of AML cells was often seen. Secondary and tertiary recipients showed consistent engraftment, with most showing further AML cell expansion. Engraftment did not correlate with French-American-British subtype or cytogenetic abnormalities. However, samples with FLT3 mutations showed a higher probability of engraftment than FLT3 wild type. Importantly, animals developed organomegaly and a wasting illness consistent with advanced leukemia. We conclude that the NSG xenotransplantation model is a robust model for human AML cell engraftment, which will allow better characterization of AML biology and testing of new therapies.

Metabolomic, proteomic and biophysical analyses of Arabidopsis thaliana cells exposed to a caesium stress. Influence of potassium supply.

The incorporation and localisation of 133Cs in a plant cellular model and the metabolic response induced were analysed as a function of external K concentration using a multidisciplinary approach. Sucrose-fed photosynthetic Arabidopsis thaliana suspension cells, grown in a K-containing or K-depleted medium, were submitted to a 1 mM Cs stress. Cell growth, strongly diminished in absence of K, was not influenced by Cs. In contrast, the chlorophyll content, affected by a Cs stress superposed to K depletion, did not vary under the sole K depletion. The uptake of Cs was monitored in vivo using 133Cs NMR spectroscopy while the final K and Cs concentrations were determined using atomic absorption spectrometry. Cs absorption rate and final concentration increased in a K-depleted external medium; in vivo NMR revealed that intracellular Cs was distributed in two kinds of compartment. Synchrotron X-ray fluorescence microscopy indicated that one could be the chloroplasts. In parallel, the cellular response to the Cs stress was analysed using proteomic and metabolic profiling. Proteins up- and down-regulated in response to Cs, in presence of K+ or not, were analysed by 2D gel electrophoresis and identified by mass spectrometry. No salient feature was detected excepting the overexpression of antioxidant enzymes, a common response of Arabidopsis cells stressed whether by Cs or by K-depletion. 13C and 31P NMR analysis of acid extracts showed that the metabolome impact of the Cs stress was also a function of the K nutrition. These analyses suggested that sugar metabolism and glycolytic fluxes were affected in a way depending upon the medium content in K+. Metabolic flux measurements using 13C labelling would be an elegant way to pursue on this line. Using our experimental system, a progressively stronger Cs stress might point out other specific responses elicited by Cs.

The protective function of the xanthophyll cycle in photosynthesis.

The rapid conversion of the carotenoid violaxanthin to zeaxanthin via antheraxanthin (xanthophyll cycle) in potato leaves exposed at 23 degrees C to a strong white light of 2000 microE.m-2.s-1 was associated with a slight inhibition of photosynthetic electron transport (as estimated from chlorophyll fluorescence measurements) and a low lipid peroxidation (as estimated from ethane measurements). When the xanthophyll cycle was blocked by dithiothreitol (3 mM) or low temperature (3 degrees C), photoinhibition of electron transport was exacerbated and pronounced lipid peroxidation occurred concomitantly. Accumulation of zeaxanthin and antheraxanthin in potato leaves by a non-photoinhibitory light treatment at 23 degrees C (900 microE.m-2.s-1 for 1 h) considerably reduced the level of lipid peroxidation during subsequent light stress at 3 degrees C. The presented results indicate that one of the functions of the xanthophyll cycle could be the protection of thylakoid membranes against lipid peroxidation, suggesting that zeaxanthin and antheraxanthin synthesized in strong light are present as free pigments in the membrane lipid bilayer.