The Combination of Gefitinib With ATRA and ATO Induces Myeloid Differentiation in Acute Promyelocytic Leukemia Resistant Cells

In approximately 15% of patients with acute myeloid leukemia (AML), total and phosphorylated EGFR proteins have been reported to be increased compared to healthy CD34(+) samples. However, it is unclear if this subset of patients would benefit from EGFR signaling pharmacological inhibition. Pre-clinical studies on AML cells provided evidence on the pro-differentiation benefits of EGFR inhibitors when combined with ATRA or ATO in vitro. Despite the success of ATRA and ATO in the treatment of patients with acute promyelocytic leukemia (APL), therapy-associated resistance is observed in 5-10% of the cases, pointing to a clear need for new therapeutic strategies for those patients. In this context, the functional role of EGFR tyrosine-kinase inhibitors has never been evaluated in APL. Here, we investigated the EGFR pathway in primary samples along with functional in vitro and in vivo studies using several APL models. We observed that total and phosphorylated EGFR (Tyr992) was expressed in 28% and 19% of blast cells from APL patients, respectively, but not in healthy CD34(+) samples. Interestingly, the expression of the EGF was lower in APL plasma samples than in healthy controls. The EGFR ligand AREG was detected in 29% of APL patients at diagnosis, but not in control samples. In vitro, treatment with the EGFR inhibitor gefitinib (ZD1839) reduced cell proliferation and survival of NB4 (ATRA-sensitive) and NB4-R2 (ATRA-resistant) cells. Moreover, the combination of gefitinib with ATRA and ATO promoted myeloid cell differentiation in ATRA- and ATO-resistant APL cells. In vivo, the combination of gefitinib and ATRA prolonged survival compared to gefitinib- or vehicle-treated leukemic mice in a syngeneic transplantation model, while the gain in survival did not reach statistical difference compared to treatment with ATRA alone. Our results suggest that gefitinib is a potential adjuvant agent that can mitigate ATRA and ATO resistance in APL cells. Therefore, our data indicate that repurposing FDA-approved tyrosine-kinase inhibitors could provide new perspectives into combination therapy to overcome drug resistance in APL patients

PLZF-RARΑ ENHANCES METABOLIC PLASTICITY AND ROS SCAVENGING IN AML CELLS

BACKGROUND PLZF-RARα (ZBTB16-RARα) fusion protein binds retinoic acid response elements (RARE) to repress transcription of genes essential for myeloid maturation and differentiation, driving a rare acute promyelocytic leukemia (APL) variant (2% of APLs). Metabolic reprogramming is the hallmark of cancer: it confers increasing flexibility in nutrient uptake and supports cell growth and malignant progression. AIM We studied PLZF-RARα bearing cells’ metabolism in order to define possible targets for tailored therapeutic strategies. METHODS To define PLZF-RARα induced metabolism features via Seahorse Bioscience XFe96 analysis, we used an inducible cellular system: U937-B412 cells (B412) containing a ZnSO4 inducible construct, and control U937-MT (MT) cells. To carry out the metabolomic profiling of lysates and supernatants samples of B412 and MT cells, we used 1H-NMR spectroscopy. The NMR spectra were acquired in a 600 MHz spectrometer, and they were analyzed with the Chenomx NMR suite 9.0 software. The statistical analysis was performed using the MetaboAnalyst 5.0 platform (https://www.metaboanalyst.ca). In addition, we carried out the ROS MitoSOX-based assay and analyzed the ROS and glycolytic related NRF2, PFKP, and HK2 by Western blot and Immunofluorescence analysis. PLZF-RARα expression induces: (i) mitochondrial respiration enhancement [basal oxidative phosphorylation (OXPHOS) (MT: 95±6 B412: 135±14 (pmol/min)/6x104 cells), p<0.0001), respiratory reserve (MT: 93±41 B412: 155±55 (pmol/min)/6x104 cells), p=0.01) and ATP production (MT: 65±20 vs B412: 96±23 (pmol/min)/6x104 cells), p=0.008)] (Fig 1A).; (ii) glycolysis reduction (MT: 665±55 B412: 574±47 (pmol/min)/6x104 cells), p=0.01) (Fig 1B). These results were confirmed by enhanced mitochondrial ATP production and reduction of glycolytic ATP (XF Real-Time ATP Rate Assay); (iii) enhanced substrate flexibility (pyruvate, glutamine and fatty acids). RESULTS By NMR, we identified around 50 metabolites in the lysates and supernatants. The heat map highlights differences between the metabolic composition of lysates and supernatants (Fig 1C). The analysis of the metabolome data is in progress. However, preliminary results show an increased concentration of TCA intermediates in B412 vs MT lysates, which is in line with an enhanced mitochondrial respiration for B412. Regarding the supernatants, in B412, we detected a differential uptake of some amino acids for B412 vs MT. For instance, the increased consumption of Threonine and Asparagine, which could be converted to Acetyl-coA and Oxaloacetate, respectively, is in accordance with increased mitochondrial metabolism. Also, the higher consumption of Glycerol in B412s can be supporting lipid synthesis. 2-OH-Butyrate, a by-product of glutathione production which provides an indication of the antioxidant activity of glutathione, is more secreted into the medium in the presence of PLZF-RARα. Despite NRF2 whole cellular levels are decreased in the presence of PLZF-RARα, its nuclear fraction at six hours increases. Importantly, after ascorbic treatment (3 mM), PLZF-RARα expressing cells increased their ROS scavenger capacity, abating ROS quantity with respect to control cells (Fig 1D). These results are in line with the increased resistance to therapy showed by PLZF-RARa AML patients. CONCLUSIONS Our results show how PLZF-RARα influences cellular metabolism, explaining its capability to induce flexibility and resistance to therapy

Clinical significance of mitochondrial DNA content in acute promyelocytic leukaemia

Although a growing body of evidence demonstrates that altered mtDNA content (mtDNAc) has clinical implications in several types of solid tumours, its prognostic relevance in acute promyelocytic leukaemia (APL) patients remains largely unknown. Here, we show that patients with higher-than-normal mtDNAc had better outcomes regardless of tumour burden. These results were more evident in patients with low-risk of relapse. The multivariate Cox proportional hazard model demonstrated that high mtDNAc was independently associated with a decreased cumulative incidence of relapse. Altogether, our data highlights the possible role of mitochondrial metabolism in APL patients treated with ATRA