Pre-Clinical Evaluation of the Proteasome Inhibitor Ixazomib against Bortezomib-Resistant Leukemia Cells and Primary Acute Leukemia Cells.

At present, 20-30% of children with acute leukemia still relapse from current chemotherapy protocols, underscoring the unmet need for new treatment options, such as proteasome inhibition. Ixazomib (IXA) is an orally available proteasome inhibitor, with an improved safety profile compared to Bortezomib (BTZ). The mechanism of action (proteasome subunit inhibition, apoptosis induction) and growth inhibitory potential of IXA vs. BTZ were tested in vitro in human (BTZ-resistant) leukemia cell lines. Ex vivo activity of IXA vs. BTZ was analyzed in 15 acute lymphoblastic leukemia (ALL) and 9 acute myeloid leukemia (AML) primary pediatric patient samples. BTZ demonstrated more potent inhibitory effects on constitutive ß5 and immunoproteasome ß5i proteasome subunit activity; however, IXA more potently inhibited ß1i subunit than BTZ (70% vs. 29% at 2.5 nM). In ALL/AML cell lines, IXA conveyed 50% growth inhibition at low nanomolar concentrations, but was ~10-fold less potent than BTZ. BTZ-resistant cells (150-160 fold) displayed similar (100-fold) cross-resistance to IXA. Finally, IXA and BTZ exhibited anti-leukemic effects for primary ex vivo ALL and AML cells; mean LC50 (nM) for IXA: 24 ± 11 and 30 ± 8, respectively, and mean LC50 for BTZ: 4.5 ± 1 and 11 ± 4, respectively. IXA has overlapping mechanisms of action with BTZ and showed anti-leukemic activity in primary leukemic cells, encouraging further pre-clinical in vivo evaluation.

Harnessing Gene Expression Profiles for the Identification of Ex Vivo Drug Response Genes in Pediatric Acute Myeloid Leukemia.

Novel treatment strategies are of paramount importance to improve clinical outcomes in pediatric AML. Since chemotherapy is likely to remain the cornerstone of curative treatment of AML, insights in the molecular mechanisms that determine its cytotoxic effects could aid further treatment optimization. To assess which genes and pathways are implicated in tumor drug resistance, we correlated ex vivo drug response data to genome-wide gene expression profiles of 73 primary pediatric AML samples obtained at initial diagnosis. Ex vivo response of primary AML blasts towards cytarabine (Ara C), daunorubicin (DNR), etoposide (VP16), and cladribine (2-CdA) was associated with the expression of 101, 345, 206, and 599 genes, respectively (p < 0.001, FDR 0.004-0.416). Microarray based expression of multiple genes was technically validated using qRT-PCR for a selection of genes. Moreover, expression levels of BRE, HIF1A, and CLEC7A were confirmed to be significantly (p < 0.05) associated with ex vivo drug response in an independent set of 48 primary pediatric AML patients. We present unique data that addresses transcriptomic analyses of the mechanisms underlying ex vivo drug response of primary tumor samples. Our data suggest that distinct gene expression profiles are associated with ex vivo drug response, and may confer a priori drug resistance in leukemic cells. The described associations represent a fundament for the development of interventions to overcome drug resistance in AML, and maximize the benefits of current chemotherapy for sensitive patients.

Gene expression profiles associated with pediatric relapsed AML.

Development of relapse remains a problem for further improvements in the survival of pediatric AML patients. While virtually all patients show a good response to initial treatment, more patients respond poorly when treated at relapse. The cellular characteristics of leukemic blast cells that allow survival of initial treatment, relapse development and subsequent resistance to salvage treatment remain largely elusive. Therefore, we studied if leukemic blasts at relapse biologically resemble their initial diagnosis counterparts. We performed microarray gene expression profiling on paired initial and relapse samples of 23 pediatric AML patients. In 11 out of 23 patients, gene expression profiles of initial and corresponding relapse samples end up in different clusters in unsupervised analysis, indicating altered gene expression profiles. In addition, shifts in type I/II mutational status were found in 5 of these 11 patients, while shifts were found in 3 of the remaining 12 patients. Although differentially expressed genes varied between patients, they were commonly related to hematopoietic differentiation, encompassed genes involved in chromatin remodeling and showed associations with similar transcription factors. The top five were CEBPA, GFI1, SATB1, KLF2 and TBP. In conclusion, the leukemic blasts at relapse are biologically different from their diagnosis counterparts. These differences may be exploited for further development of novel treatment strategies.

Clinical relevance of molecular aberrations in paediatric acute myeloid leukaemia at first relapse.

Outcome for relapsed paediatric acute myeloid leukaemia (AML) remains poor. Strong prognostic factors at first relapse are lacking, which hampers optimization of therapy. We assessed the frequency of molecular aberrations (FLT3, NRAS, KRAS, KIT, WT1 and NPM1 genes) at first relapse in a large set (n = 198) of relapsed non-French-American-British M3, non-Down syndrome AML patients that received similar relapse treatment. We correlated molecular aberrations with clinical and biological factors and studied their prognostic relevance. Hotspot mutations in the analysed genes were detected in 92 out of 198 patients (46·5%). In 72 of these 92 patients (78%), molecular aberrations were mutually exclusive for the currently analysed genes. FLT3-internal tandem repeat (ITD) (18% of total group) mutations were most frequent, followed by NRAS (10·2%), KRAS (8%), WT1 (8%), KIT (8%), NPM1 (5%) and FLT3-tyrosine kinase domain (3%) mutations. Presence of a WT1 aberration was an independent risk factor for second relapse (Hazard Ratio [HR] = 2·74, P = 0·013). In patients who achieved second complete remission (70·2%), WT1 and FLT3-ITD aberrations were independent risk factors for poor overall survival (HR = 2·32, P = 0·038 and HR = 1·89, P = 0·045 respectively). These data show that molecular aberrations at first relapse are of prognostic relevance and potentially useful for risk group stratification of paediatric relapsed AML and for identification of patients eligible for personalized treatment.

High-frequency type I/II mutational shifts between diagnosis and relapse are associated with outcome in pediatric AML: implications for personalized medicine.

Although virtually all pediatric patients with acute myeloid leukemia (AML) achieve a complete remission after initial induction therapy, 30%-40% of patients will encounter a relapse and have a dismal prognosis. To prevent relapses, personalized treatment strategies are currently being developed, which target specific molecular aberrations. To determine relevance of established AML type I/II mutations that may serve as therapeutic targets, we assessed frequencies of these mutations and their persistence during disease progression in a large group (n = 69) of paired diagnosis and relapse pediatric AML specimens. In 26 of 42 patients (61%) harboring mutations at either stage of the disease, mutation status changed between diagnosis and relapse, particularly in FLT3, WT1, and RAS genes. Presence or gain of type I/II mutations at relapse was associated with a shorter time to relapse (TTR), whereas absence or loss correlated with longer TTR. Moreover, an adverse outcome was found for patients with activating mutations at relapse, which was statistically significant for FLT3/ITD and WT1 mutations. These findings suggest that mutational shifts affect disease progression. We hence propose that risk stratification, malignant cell detection, and selection of personalized treatment should be based on status of type I/II mutations both at initial diagnosis and during follow-up.

Strategies for the analysis of in vitro radiation sensitivity and prediction of interaction with potential radiation modifying agents.

PURPOSE: To better predict radiation-drug interactions in in vitro model systems, thorough assessment of the effects of in vitro exposure is required. The aim of this article is to show that both clonogenic capacity and cellular proliferation, which represent important different elements of tumour conduct, can be considered when assessing in vitro radio sensitisation. METHODS: A model was designed that can predict radiation-drug interactions based on changes in clonogenic capacity and cell proliferation by radiation modifying agents. RESULTS: Using this mechanistical model, the effect of combined exposure to radiation and potential drugs can be tested on both established cell lines and primary cells. In addition, we could obtain more information about the mechanisms underlying the radiation-drug interaction by assessing the results of in vitro exposure on tumour cell proliferation and clonogenic capacity according to our model. CONCLUSIONS: The significance of our model is not to replace the clonogenic gold standard but to give additional information about the radiation-drug combination by determining cell proliferation. Moreover, the advantage is that the interaction can also be predicted in cases where a clonogenic assay is not possible. Additional research into the biological effect of potential radio-sensitisers is warranted for future (pre)clinical studies.