Harnessing Gene Expression Profiles for the Identification of Ex Vivo Drug Response Genes in Pediatric Acute Myeloid Leukemia.
ABSTRACT: 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.
Project description:BACKGROUND:Variation in terms of outcome and toxic side effects of treatment exists among acute myeloid leukemia (AML) patients on chemotherapy with cytarabine (Ara-C) and daunorubicin (Dnr). Candidate Ara-C metabolizing gene expression in primary AML cells is proposed to account for this variation. METHODS:Ex vivo Ara-C sensitivity was determined in primary AML samples using MTT assay. mRNA expression of candidate Ara-C metabolizing genes were evaluated by RQPCR analysis. Global gene expression profiling was carried out for identifying differentially expressed genes between exvivo Ara-C sensitive and resistant samples. RESULTS:Wide interindividual variations in ex vivo Ara-C cytotoxicity were observed among samples from patients with AML and were stratified into sensitive, intermediately sensitive and resistant, based on IC50 values obtained by MTT assay. RNA expression of deoxycytidine kinase (DCK), human equilibrative nucleoside transporter-1 (ENT1) and ribonucleotide reductase M1 (RRM1) were significantly higher and cytidine deaminase (CDA) was significantly lower in ex vivo Ara-C sensitive samples. Higher DCK and RRM1 expression in AML patient's blast correlated with better DFS. Ara-C resistance index (RI), a mathematically derived quotient was proposed based on candidate gene expression pattern. Ara-C ex vivo sensitive samples were found to have significantly lower RI compared with resistant as well as samples from patients presenting with relapse. Patients with low RI supposedly highly sensitive to Ara-C were found to have higher incidence of induction death (p = 0.002; RR: 4.35 [95% CI: 1.69-11.22]). Global gene expression profiling undertaken to find out additional contributors of Ara-C resistance identified many apoptosis as well as metabolic pathway genes to be differentially expressed between Ara-C resistant and sensitive samples. CONCLUSION:This study highlights the importance of evaluating expression of candidate Ara-C metabolizing genes in predicting ex vivo drug response as well as treatment outcome. RI could be a predictor of ex vivo Ara-C response irrespective of cytogenetic and molecular risk groups and a potential biomarker for AML treatment outcome and toxicity. Original submitted 22 December 2014; Revision submitted 9 April 2015.
Project description:ABCB1 is a member of the ATP binding cassette transporter family and high ABCB1 activity is considered as a poor prognostic factor in acute myeloid leukemia (AML) treated with intensive chemotherapy, its direct relation with drug resistance remains unclear. We evaluated ABCB1 activity in relation with clinical parameters and treatment response to standard chemotherapy in 321 patients with de novo AML. We assessed multiple clinical relationships of ABCB1 activity-ex vivo drug resistance, gene expression, and the ABCB1 inhibitor quinine were evaluated. ABCB1 activity was observed in 58% of AML and was linked to low white blood cell count, high expression of CD34, absence of FLT3-ITD, and absence of mutant NPM1. Moreover, ABCB1 activity was associated with worse overall- and event-free survival. However, ABCB1 activity did not directly lead to ex vivo drug resistance to anthracyclines. We found that ABCB1 was highly correlated with gene expressions of BAALC, CD34, CD200, and CD7, indicating that ABCB1 expression maybe a passenger characteristic of high-risk AML. Furthermore, ABCB1 was inversely correlated to HOX cluster genes and CD33. Thus, low ABCB1 AML patients benefited specifically from anti-CD33 treatment by gemtuzumab ozogamicin in addition to standard chemotherapy. We showed prognostic importance of ABCB1 gene expression, protein expression, and activity. Furthermore, ABCB1 was not directly linked to drug resistance, ABCB1 inhibition did not improve outcome of high ABCB1 AML patients and thus high ABCB1 may represent a passenger characteristic of high-risk AML.
Project description:Cytarabine (Ara-C) and Daunorubicin (Dnr) forms the backbone of acute myeloid leukemia (AML) therapy. Drug resistance and toxic side effects pose a major threat to treatment success and hence alternate less toxic therapies are warranted. NF-E2 related factor-2 (Nrf2), a master regulator of antioxidant response is implicated in chemoresistance in solid tumors. However, little is known about the role of Nrf2 in AML chemoresistance and the effect of pharmacological inhibitor brusatol in modulating this resistance. Primary AML samples with high ex-vivo IC50 to Ara-C, ATO, Dnr had significantly high NRF2 RNA expression. Gene-specific knockdown of NRF2 improved sensitivity to these drugs in resistant AML cell lines by decreasing the expression of downstream antioxidant targets of Nrf2 by compromising the cell's ability to scavenge the ROS. Treatment with brusatol, a pharmacological inhibitor of Nrf2, improved sensitivity to Ara-C, ATO, and Dnr and reduced colony formation capacity. AML cell lines stably overexpressing NRF2 showed increased resistance to ATO, Dnr and Ara-C and increased expression of downstream targets. This study demonstrates that Nrf2 could be an ideal druggable target in AML, more so to the drugs that function through ROS, suggesting the possibility of using Nrf2 inhibitors in combination with chemotherapeutic agents to modulate drug resistance in AML.
Project description:Despite initial remission, ?60-70% of adult and 30% of pediatric patients experience relapse or refractory AML. Studies so far have identified base line gene expression profiles of pathogenic and prognostic significance in AML; however, the extent of change in gene expression post-initiation of treatment has not been investigated. Exposure of leukemic cells to chemotherapeutic agents such as cytarabine, a mainstay of AML chemotherapy, can trigger adaptive response by influencing leukemic cell transcriptome and, hence, development of resistance or refractory disease. It is, however, challenging to perform such a study due to lack of availability of specimens post-drug treatment. The primary objective of this study was to identify in vivo cytarabine-induced changes in leukemia cell transcriptome and to evaluate their impact on clinical outcome. The results highlight genes relevant to cytarabine resistance and support the concept of targeting cytarabine-induced genes as a means of improving response.
Project description:Acute myeloid leukemia (AML) is a heterogeneous malignancy with the most common genomic alterations in NPM1, DNMT3A, and FLT3. Midostaurin was the first FLT3 inhibitor FDA approved for AML and is standard of care for FLT3 mutant patients undergoing induction chemotherapy [1, 2]. As there is a spectrum of response, we hypothesized that biological factors beyond FLT3 could play a role in drug sensitivity and that select FLT3-ITD negative samples may also demonstrate sensitivity. Thus, we aimed to identify features that would predict response to midostaurin in FLT3 mutant and wild-type samples. We performed an ex vivo drug sensitivity screen on primary and relapsed AML samples with corresponding targeted sequencing and RNA sequencing. We observed a correlation between FLT3-ITD mutations and midostaurin sensitivity as expected and observed KRAS and TP53 mutations correlating with midostaurin resistance in FLT3-ITD negative samples. Further, we identified genes differentially expressed in sensitive vs. resistant samples independent of FLT3-ITD status. Within FLT3-ITD mutant samples, over-expression of RGL4, oncogene and regulator of the Ras-Raf-MEK-ERK cascade, distinguished resistant from sensitive samples. Overall, this study highlights the complexity underlying midostaurin response. And, our results suggest that therapies that target both FLT3 and MAPK/ERK signaling may help circumvent some cases of resistance.
Project description:This study reports the establishment of a bone marrow mononuclear cell (BMMC) 3D culture model and the application of this model to define sensitivity and resistance biomarkers of acute myeloid leukaemia (AML) patient bone marrow samples in response to Cytarabine (Ara-C) treatment. By mimicking physiological bone marrow microenvironment, the growth conditions were optimized by using frozen BMMCs derived from healthy donors. Healthy BMMCs are capable of differentiating into major hematopoietic lineages and various types of stromal cells in this platform. Cryopreserved BMMC samples from 49 AML patients were characterized for ex vivo growth and sensitivity to Ara-C. RNA sequencing was performed for 3D and 2D cultures to determine differential gene expression patterns. Specific genetic mutations and/or gene expression signatures associated with the ability of the ex vivo expansion and response to Ara-C were elucidated by whole-exome and RNA sequencing. Data analysis identified unique gene expression signatures and novel genetic mutations associated with sensitivity to Ara-C treatment of proliferating AML specimens and can be used as predictive therapeutic biomarkers to determine the optimal treatment regimens. Furthermore, these data demonstrate the translational value of this ex vivo platform which should be widely applicable to evaluate other therapies in AML.
Project description:Here we report the case of a 30-year-old woman with relapsed acute myeloid leukemia (AML) who was treated with all-trans retinoic acid (ATRA) as part of investigational therapy (NCT02273102). The patient died from rapid disease progression following eight days of continuous treatment with ATRA. Karyotype analysis and RNA-Seq revealed the presence of a novel t(4;15)(q31;q22) reciprocal translocation involving the TMEM154 and RASGRF1 genes. Analysis of primary cells from the patient revealed the expression of TMEM154-RASGRF1 mRNA and the resulting fusion protein, but no expression of the reciprocal RASGRF1-TMEM154 fusion. Consistent with the response of the patient to ATRA therapy, we observed a rapid proliferation of t(4;15) primary cells following ATRA treatment ex vivo. Preliminary characterization of the retinoid response of t(4;15) AML revealed that in stark contrast to non-t(4;15) AML, these cells proliferate in response to specific agonists of RARα and RARγ. Furthermore, we observed an increase in the levels of nuclear RARγ upon ATRA treatment. In summary, the identification of the novel t(4;15)(q31;q22) reciprocal translocation opens new avenues in the study of retinoid resistance and provides potential for a new biomarker for therapy of AML.
Project description:<h4>Background</h4>Although current chemotherapy regimens have remarkably improved the cure rate of pediatric acute promyelocytic leukemia (APL) over the past decade, more than 20% of patients still die of the disease, and the 5-year cumulative incidence of relapse is 17%. The precise gene pathways that exert critical control over the determination of cell lineage fate during the development of pediatric APL remain unclear.<h4>Methods</h4>In this study, we analyzed miR-125b expression in 169 pediatric acute myelogenous leukemia (AML) samples including 76 APL samples before therapy and 38 APL samples after therapy. The effects of enforced expression of miR-125b were evaluated in leukemic cell and drug-resistant cell lines.<h4>Results</h4>miR-125b is highly expressed in pediatric APL compared with other subtypes of AML and is correlated with treatment response, as well as relapse of pediatric APL. Our results further demonstrated that miR-125b could promote leukemic cell proliferation and inhibit cell apoptosis by regulating the expression of tumor suppressor BCL2-antagonist/killer 1 (Bak1). Remarkably, miR-125b was also found to be up-regulated in leukemic drug-resistant cells, and transfection of a miR-125b duplex into AML cells can increase their resistance to therapeutic drugs,<h4>Conclusions</h4>These findings strongly indicate that miR-125b plays an important role in the development of pediatric APL at least partially mediated by repressing BAK1 protein expression and could be a potential therapeutic target for treating pediatric APL failure.
Project description:OBJECTIVE:This study aimed to investigate the function and mechanism of neddylation of HDAC1 underlying drug resistance of AML cells. METHODS:Evaluation experiments of effects of HDAC1 on drug resistance of AML cells were performed with AML cell transfected with constructs overexpressing HDAC1 or multi-drug resistance AML cells transfected with siRNA for HDAC1 through observing cell viability, percentage of apoptotic cell, doxorubicin-releasing index and multidrug resistance associated protein 1 (MRP1) expression. Neddylation or ubiquitination of HDAC1 was determined by immunoprecipitation or Ni2+ pull down assay followed by western blot. The role of HDAC1 was in vivo confirmed by xenograft in mice. RESULTS:HDAC1 was significantly upregulated in refractory AML patients, and in drug-resistant AML cells (HL-60/ADM and K562/A02). Intracellular HDAC1 expression promoted doxorubicin resistance of HL-60, K562, and primary bone marrow cells (BMCs) of remission AML patients as shown by increasing cell viability and doxorubicin-releasing index, inhibiting cell apoptosis. Moreover, HDAC1 protein level in AML cells was regulated by the Nedd8-mediated neddylation and ubiquitination, which further promoted HDAC1 degradation. In vivo, HDAC1 overexpression significantly increased doxorubicin resistance; while HDACs inhibitor Panobinostat markedly improved the inhibitory effect of doxorubicin on tumor growth. Furthermore, HDAC1 silencing by Panobinostat and/or lentivirus mediated RNA interference against HDAC1 effectively reduced doxorubicin resistance, resulting in the inhibition of tumor growth in AML bearing mice. CONCLUSION:Our findings suggested that HDAC1 contributed to the multidrug resistance of AML and its function turnover was regulated, at least in part, by post-translational modifications, including neddylation and ubiquitination.
Project description:BACKGROUND:While conventional chemotherapy is effective at eliminating the bulk of leukemic cells, chemotherapy resistance in acute myeloid leukemia (AML) is a prevalent problem that hinders conventional therapies and contributes to disease relapse, and ultimately patient death. We have recently shown that allogeneic double negative T cells (DNTs) are able to target the majority of primary AML blasts in vitro and in patient-derived xenograft models. However, some primary AML blast samples are resistant to DNT cell therapy. Given the differences in the modes of action of DNTs and chemotherapy, we hypothesize that DNT therapy can be used in combination with conventional chemotherapy to further improve their anti-leukemic effects and to target chemotherapy-resistant disease. METHODS:Drug titration assays and flow-based cytotoxicity assays using ex vivo expanded allogeneic DNTs were performed on multiple AML cell lines to identify therapy-resistance. Primary AML samples were also tested to validate our in vitro findings. Further, a xenograft model was employed to demonstrate the feasibility of combining conventional chemotherapy and adoptive DNT therapy to target therapy-resistant AML. Lastly, blocking assays with neutralizing antibodies were employed to determine the mechanism by which chemotherapy increases the susceptibility of AML to DNT-mediated cytotoxicity. RESULTS:Here, we demonstrate that KG1a, a stem-like AML cell line that is resistant to DNTs and chemotherapy, and chemotherapy-resistant primary AML samples both became more susceptible to DNT-mediated cytotoxicity in vitro following pre-treatment with daunorubicin. Moreover, chemotherapy treatment followed by adoptive DNT cell therapy significantly decreased bone marrow engraftment of KG1a in a xenograft model. Mechanistically, daunorubicin increased the expression of NKG2D and DNAM-1 ligands on KG1a; blocking of these pathways attenuated DNT-mediated cytotoxicity. CONCLUSIONS:Our results demonstrate the feasibility and benefit of using DNTs as an immunotherapy after the administration of conventional chemotherapy.