In-vitro modeling of TKI resistance in the high-risk B-cell acute lymphoblastic leukemia fusion gene RANBP2-ABL1 - implications for targeted therapy.

Acute lymphoblastic leukemia remains a leading cause of cancer-related death in children. Furthermore, subtypes such as Ph-like ALL remain at high-risk of relapse, and treatment resistance remains a significant clinical issue. The patient-derived Ph-like ALL RANBP2-ABL1 fusion gene was transduced into Ba/F3 cells and allowed to become resistant to the tyrosine kinase inhibitors (TKIs) imatinib or dasatinib, followed by secondary resistance to ponatinib. RANBP2-ABL1 Ba/F3 cells developed the clinically relevant ABL1 p.T315I mutation and upon secondary resistance to ponatinib, developed compound mutations, including a novel ABL1 p.L302H mutation. Significantly, compound mutations were targetable with a combination of asciminib and ponatinib. In-vitro modeling of Ph-like ALL RANBP2-ABL1 has identified kinase domain mutations in response to TKI treatment, that may have important clinical ramifications. Early detection of mutations is paramount to guide treatment strategies and improve survival in this high-risk group of patients.

Gene expression signature that predicts early molecular response failure in chronic-phase CML patients on frontline imatinib.

In chronic-phase chronic myeloid leukemia (CP-CML) patients treated with frontline imatinib, failure to achieve early molecular response (EMR; EMR failure: BCR-ABL1 >10% on the international scale at 3 months) is predictive of inferior outcomes. Identifying patients at high-risk of EMR failure at diagnosis provides an opportunity to intensify frontline therapy and potentially avoid EMR failure. We studied blood samples from 96 CP-CML patients at diagnosis and identified 365 genes that were aberrantly expressed in 13 patients who subsequently failed to achieve EMR, with a gene signature significantly enriched for stem cell phenotype (eg, Myc, ß-catenin, Hoxa9/Meis1), cell cycle, and reduced immune response pathways. We selected a 17-gene panel to predict EMR failure and validated this signature on an independent patient cohort. Patients classified as high risk with our gene expression signature (HR-GES) exhibited significantly higher rates of EMR failure compared with low-risk (LR-GES) patients (78% vs 5%; P < .0001), with an overall accuracy of 93%. Furthermore, HR-GES patients who received frontline nilotinib had a relatively low rate of EMR failure (10%). However, HR-GES patients still had inferior deep molecular response achievement rate by 24 months compared with LR-GES patients. This novel multigene signature may be useful for selecting patients at high risk of EMR failure on standard therapy who may benefit from trials of more potent kinase inhibitors or other experimental approaches.

Increased peroxisome proliferator-activated receptor γ activity reduces imatinib uptake and efficacy in chronic myeloid leukemia mononuclear cells.

Imatinib is actively transported by organic cation transporter-1 (OCT-1) influx transporter, and low OCT-1 activity in diagnostic chronic myeloid leukemia blood mononuclear cells is significantly associated with poor molecular response to imatinib. Herein we report that, in diagnostic chronic myeloid leukemia mononuclear cells and BCR-ABL1+ cell lines, peroxisome proliferator-activated receptor γ agonists (GW1929, rosiglitazone, pioglitazone) significantly decrease OCT-1 activity; conversely, peroxisome proliferator-activated receptor γ antagonists (GW9662, T0070907) increase OCT-1 activity. Importantly, these effects can lead to corresponding changes in sensitivity to BCR-ABL kinase inhibition. Results were confirmed in peroxisome proliferator-activated receptor γ-transduced K562 cells. Furthermore, we identified a strong negative correlation between OCT-1 activity and peroxisome proliferator-activated receptor γ transcriptional activity in diagnostic chronic myeloid leukemia patients (n=84; P<0.0001), suggesting that peroxisome proliferator-activated receptor γ activation has a negative impact on the intracellular uptake of imatinib and consequent BCR-ABL kinase inhibition. The inter-patient variability of peroxisome proliferator-activated receptor γ activation likely accounts for the heterogeneity observed in patient OCT-1 activity at diagnosis. Recently, the peroxisome proliferator-activated receptor γ agonist pioglitazone was reported to act synergistically with imatinib, targeting the residual chronic myeloid leukemia stem cell pool. Our findings suggest that peroxisome proliferator-activated receptor γ ligands have differential effects on circulating mononuclear cells compared to stem cells. Since the effect of peroxisome proliferator-activated receptor γ activation on imatinib uptake in mononuclear cells may counteract the clinical benefit of this activation in stem cells, caution should be applied when combining these therapies, especially in patients with high peroxisome proliferator-activated receptor γ transcriptional activity.

Modelling Predictors of Molecular Response to Frontline Imatinib for Patients with Chronic Myeloid Leukaemia.

BACKGROUND: Treatment of patients with chronic myeloid leukaemia (CML) has become increasingly difficult in recent years due to the variety of treatment options available and challenge deciding on the most appropriate treatment strategy for an individual patient. To facilitate the treatment strategy decision, disease assessment should involve molecular response to initial treatment for an individual patient. Patients predicted not to achieve major molecular response (MMR) at 24 months to frontline imatinib may be better treated with alternative frontline therapies, such as nilotinib or dasatinib. The aims of this study were to i) understand the clinical prediction 'rules' for predicting MMR at 24 months for CML patients treated with imatinib using clinical, molecular, and cell count observations (predictive factors collected at diagnosis and categorised based on available knowledge) and ii) develop a predictive model for CML treatment management. This predictive model was developed, based on CML patients undergoing imatinib therapy enrolled in the TIDEL II clinical trial with an experimentally identified achieving MMR group and non-achieving MMR group, by addressing the challenge as a machine learning problem. The recommended model was validated externally using an independent data set from King Faisal Specialist Hospital and Research Centre, Saudi Arabia. PRINCIPLE FINDINGS: The common prognostic scores yielded similar sensitivity performance in testing and validation datasets and are therefore good predictors of the positive group. The G-mean and F-score values in our models outperformed the common prognostic scores in testing and validation datasets and are therefore good predictors for both the positive and negative groups. Furthermore, a high PPV above 65% indicated that our models are appropriate for making decisions at diagnosis and pre-therapy. Study limitations include that prior knowledge may change based on varying expert opinions; hence, representing the category boundaries of each predictive factor could dramatically change performance of the models.

An oncogenic role for sphingosine kinase 2.

While both human sphingosine kinases (SK1 and SK2) catalyze the generation of the pleiotropic signaling lipid sphingosine 1-phosphate, these enzymes appear to be functionally distinct. SK1 has well described roles in promoting cell survival, proliferation and neoplastic transformation. The roles of SK2, and its contribution to cancer, however, are much less clear. Some studies have suggested an anti-proliferative/pro-apoptotic function for SK2, while others indicate it has a pro-survival role and its inhibition can have anti-cancer effects. Our analysis of gene expression data revealed that SK2 is upregulated in many human cancers, but only to a small extent (up to 2.5-fold over normal tissue). Based on these findings, we examined the effect of different levels of cellular SK2 and showed that high-level overexpression reduced cell proliferation and survival, and increased cellular ceramide levels. In contrast, however, low-level SK2 overexpression promoted cell survival and proliferation, and induced neoplastic transformation in vivo. These findings coincided with decreased nuclear localization and increased plasma membrane localization of SK2, as well as increases in extracellular S1P formation. Hence, we have shown for the first time that SK2 can have a direct role in promoting oncogenesis, supporting the use of SK2-specific inhibitors as anti-cancer agents.

Inhibition kinetics and regulation of sphingosine kinase 1 expression in prostate cancer cells: functional differences between sphingosine kinase 1a and 1b.

Sphingosine kinase 1 catalyses the formation of the bioactive lipid, sphingosine 1-phosphate and is a target for anti-cancer agents. We demonstrate here that 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole (SKi, also referred to as SKI-II), FTY720 (Fingolimod), and (S)-FTY720 vinylphosphonate inhibit sphingosine kinase 1 activity with distinct kinetics, indicating that these compounds exhibit different binding modalities with sphingosine kinase 1. Thus, SKi is a mixed inhibitor of sphingosine and ATP binding, whereas FTY720 is competitive with sphingosine and uncompetitive with ATP, and (S)-FTY720 vinylphosphonate is uncompetitive with sphingosine and is a mixed inhibitor with respect to ATP. A novel 'see-saw' model is proposed for the binding of inhibitor to catalytic and allosteric sites, the latter dependent on substrate binding, that provides an explanation for the different inhibitor kinetics. In addition, we demonstrate that the expression level and properties unique to an N-terminal 86 amino-acid isoform variant of sphingosine kinase 1 (SK1b) in prostate cancer cells reduce its sensitivity to SKi-induced proteasomal degradation in comparison to SK1a, i.e. these two N-terminal variants of sphingosine kinase 1 (SK1a and SK1b) have different properties. The reduced sensitivity of SK1b to proteasomal degradation in response to SKi is translated into specific changes in ceramide and S1P levels that leads to apoptosis of androgen-sensitive but not androgen-independent LNCaP prostate cancer cells. Therefore, our proposed 'see-saw' model might be usefully employed in the design of sphingosine kinase inhibitors to promote apoptosis of chemotherapeutic resistant cancer cells.

The sphingosine kinase 1 inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole induces proteasomal degradation of sphingosine kinase 1 in mammalian cells.

Sphingosine kinase 1 (SK1) is an enzyme that catalyzes the phosphorylation of sphingosine to produce the bioactive lipid sphingosine 1-phosphate (S1P). We demonstrate here that the SK1 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) induces the proteasomal degradation of SK1 in human pulmonary artery smooth muscle cells, androgen-sensitive LNCaP prostate cancer cells, MCF-7 and MCF-7 HER2 breast cancer cells and that this is likely mediated by ceramide as a consequence of catalytic inhibition of SK1 by SKi. Moreover, SK1 is polyubiquitinated under basal conditions, and SKi appears to increase the degradation of SK1 by activating the proteasome. In addition, the proteasomal degradation of SK1a and SK1b in androgen-sensitive LNCaP cells is associated with the induction of apoptosis. However, SK1b in LNCaP-AI cells (androgen-independent) is less sensitive to SKi-induced proteasomal degradation and these cells are resistant to SKi-induced apoptosis, thereby implicating the ubiquitin-proteasomal degradation of SK1 as an important mechanism controlling cell survival.

Eukaryotic elongation factor 1A interacts with sphingosine kinase and directly enhances its catalytic activity.

Sphingosine 1-phosphate (S1P) has many important roles in mammalian cells, including contributing to the control of cell survival and proliferation. S1P is generated by sphingosine kinases (SKs), of which two mammalian isoforms have been identified (SK1 and SK2). To gain a better understanding of SK regulation, we have used a yeast two-hybrid screen to identify SK1-interacting proteins and established elongation factor 1A (eEF1A) as one such protein that associates with both SK1 and SK2. We show the direct interaction of eEF1A with the SKs in vitro, whereas the physiological relevance of this association was demonstrated by co-immunoprecipitation of the endogenous proteins from cell lysates. Although the canonical role of eEF1A resides in protein synthesis, it has also been implicated in other roles, including regulating the activity of some signaling enzymes. Thus, we examined the potential role of eEF1A in regulation of the SKs and show that eEF1A is able to directly increase the activity of SK1 and SK2 approximately 3-fold in vitro. Substrate kinetics demonstrated that eEF1A increased the catalytic rate of both SKs, while having no observable effect on substrate affinities of these enzymes for either ATP or sphingosine. Overexpression of eEF1A in quiescent Chinese hamster ovary cells increased cellular SK activity, whereas a small interfering RNA-mediated decrease in eEF1A levels in MCF7 cells substantially reduced cellular SK activity and S1P levels, supporting the in vivo physiological relevance of this interaction. Thus, this study has established a novel mechanism of regulation of both SK1 and SK2 that is mediated by their interaction with eEF1A.

Cellular signalling by sphingosine kinase and sphingosine 1-phosphate.

Sphingosine kinases, through the formation of the bioactive phospholipid sphingosine 1-phosphate, have been implicated in a diverse range of cellular processes, including cell proliferation, apoptosis, calcium homeostasis, angiogenesis and vascular maturation. The last few years have seen a number of significant advances in understanding of the mechanisms of action, activation, cellular localisation and biological roles of these enzymes. Here we review the current understanding of the regulation of and cellular signalling by sphingosine kinase and sphingosine 1-phosphate and discuss recent findings implicating sphingosine kinase as a potential therapeutic target for the control of cancer, inflammation and a number of other diseases. We suggest that, since the activation and subcellular localization of these enzymes appear to play critical roles in their biological functions, targeting these processes may provide more specific therapeutic options than direct catalytic inhibitors.

Phosphorylation-dependent translocation of sphingosine kinase to the plasma membrane drives its oncogenic signalling.

Sphingosine kinase (SK) 1 catalyzes the formation of the bioactive lipid sphingosine 1-phosphate, and has been implicated in several biological processes in mammalian cells, including enhanced proliferation, inhibition of apoptosis, and oncogenesis. Human SK (hSK) 1 possesses high instrinsic catalytic activity which can be further increased by a diverse array of cellular agonists. We have shown previously that this activation occurs as a direct consequence of extracellular signal-regulated kinase 1/2-mediated phosphorylation at Ser225, which not only increases catalytic activity, but is also necessary for agonist-induced translocation of hSK1 to the plasma membrane. In this study, we report that the oncogenic effects of overexpressed hSK1 are blocked by mutation of the phosphorylation site despite the phosphorylation-deficient form of the enzyme retaining full instrinsic catalytic activity. This indicates that oncogenic signaling by hSK1 relies on a phosphorylation-dependent function beyond increasing enzyme activity. We demonstrate, through constitutive localization of the phosphorylation-deficient form of hSK1 to the plasma membrane, that hSK1 translocation is the key effect of phosphorylation in oncogenic signaling by this enzyme. Thus, phosphorylation of hSK1 is essential for oncogenic signaling, and is brought about through phosphorylation-induced translocation of hSK1 to the plasma membrane, rather than from enhanced catalytic activity of this enzyme.