Littérature scientifique sur le sujet « PML-RARA, LAMP, APL »

Abstract The Hippo signaling pathway, also known as the Salvador-Warts-Hippo pathway, is a well-known oncogenic signaling pathway associated with organ size regulation, cell proliferation, and apoptosis. In this study, we analyzed the association of Hippo signaling pathway genes in patients with acute myeloid leukemia (AML) using whole-exome sequencing (WES) and whole-transcriptome sequencing (WTS) data. We used Seoul National University Hospital (SNUH) AML patient data (n=16) collected from December 2015 to January 2019 for analysis. Additionally, public datasets of AML patients, TCGA-LAML (n=179) and LeuceGene (n=110) were used. we performed hierarchical clustering using Hippo pathway gene expression from AML patient samples. As a result, we discovered a gene expression signature in which the expressions of tumor suppressor genes among the Hippo pathway genes were significantly decreased. We divided AML patients into the case group and the control group according to the presence or absence of the corresponding gene expression signature and attempted to identify differences in biological features between the two groups. First, in the process of comparing differences in genetic alteration, it was confirmed that the frequency of PML-RARA fusion was significantly higher in the case group. We further analyzed the data of the AML patients in the case group without PML-RARA fusion to discover other biological features. To discover the correlation between the Hippo pathway and other oncogenic signaling pathways on gene expression level, we performed a correlation analysis. As a result, it was confirmed that the expression of the Hippo pathway gene showed a high correlation with the gene expression of the Notch signaling pathway and the Myc signaling pathway in the patients of the case group. In addition, we performed DEGs analysis to find new therapeutic target candidate genes from the case group. As a result, we identified a significantly upregulated gene, LAMP5.In conclusion, we identified the gene expression signatures of the Hippo signaling pathway in AML patients. Also, we discovered a correlation between the Hippo pathway and other signaling pathways and suggested potential therapeutic candidate genes using gene expression signatures of the Hippo signaling pathway genes. And we will perform further analysis about validating therapeutic candidate genes related to the Hippo signaling pathway gene expression signature. Citation Format: Heejun Jang, Sungyoung Lee, Hongseok Yun, Yongil Koh, Sung-Soo Yoon. Association of Hippo signaling pathway in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3359.

Abstract Abstract 1447 Background: There is a growing realization that outcome of acute myeloid leukemia (AML) is strongly correlated to whether minimal residual disease (MRD) - as quantified by either multicolor flow cytometry (MFC) or real-time quantitative polymerase chain reaction (qPCR) - is demonstrable at crucial clinical decision time points. While FCM is applicable in up to 95% of all cases through the detection of leukemia-associated immunophenotypes (LAIPs), its major drawback is the lack of sensitivity in most cases and antigen shift, which is seen in up to 20% of cases. In contrast, while its sensitivity is generally considered to be higher than that of FCM, qPCR can only be applied in less than 80% of the patients. We have previously reported human Myeloid Inhibitory C-type Lectin (hMICL) and the interleukin-3 receptor alpha (CD123) to be potent and widely applicable markers in MRD detection by MFC, but the sensitivity of this approach compared to qPCR techniques remains to be elucidated (Clinical Cytometry, Part B, in press). Problem formulation: No previous studies have to our knowledge established the sensitivity levels of markers within these methodologies. Data from such experimentation would allow for better decision making as to what marker to apply in the given patient. Hypothesis: We hypothesized that the direct comparison between MRD technologies might yield hard data as to optimal choice of methods in the pre-clinical setting, and ultimately also for design of future FCM based MRD protocols. Results: Initially, we determined the validity of hMICL/CD123 FCM assay by performing two-fold dilution series of cryopreserved AML cells in highly purified T-cells, the latter being hMICL-/CD123-/CD34-/CD117-. Dilutions at 1: 1 to 1: 214 displayed linearity in MRD drop and resulted in a coefficient of correlation of 0.99 (P = <0.01) for all experiments. The sensitivity was calculated from the plateau-phase at of the titration curve at 1: 216 and reached a maximal sensitivity of 1: 30.000 in this setting. In order to evaluate the sensitivity of the hMICL/CD123 technique compared to standard qPCR methods in experiments, we admixed leukemic blasts from 5 patients with either known fusion transcript positive and WT-1 + disease in normal bone marrow. Sensitivities were calculated from the plateau phase at 216. The most sensitive LAIP was defined in each patient from internal standard references generated from normal donors and regenerating BM from acute lymphoblastic leukemia patients. The detection limits of MFC ranged from 10−3 to below 10−4 for the most sensitive hMICL/CD123 combinations. While the fusion transcript qPCR assays were found to be the most sensitive, the hMICL/CD123 FCM one stood up surprisingly well in 3 of 5 cases of fusion transcript positive disease and approached the qPCR assay. In all 5 cases the FCM did better than the WT1-assay (an example of titration curves and relative MRD values for a PML-RARA + patient is given in the Figure). In the clinical setting, we analyzed a total of 69 unselected AML patients consecutively diagnosed at our department. A total of 10 different hMICL/CD123 LAIPs were analyzed closely, of which one or more were present in 90% of the patients. By calculating log differences between diagnostic AML samples and normal and regenerating BM we found the frequency of LAIP+ cells to be more than 4 logs higher in leukemia than in normal BM for the most sensitive combinations of LAIPs. Conclusion: These data are the first to directly compare MRD sensitivity of FCM and qPCR in spiking experiments and in a larger patient material. They reveal that a new assay involving hMICL and CD123, which are stable markers throughout the disease in these patients, has a surprisingly high sensitivity, though not quite at the level of standard qPCR assays. These data can be used for the design of future studies of cytoreduction in AML, where individualized therapy is warranted. Disclosures: No relevant conflicts of interest to declare.

Quantification of minimal residual disease (MRD) is becoming increasingly important to guide therapy in patients with acute myeloid leukemia (AML). While MFC can be applied to more patients with AML than QPCR, the latter has the advantage of a higher sensitivity in many cases. We compared data obtained by both methods in parallel in bone marrow samples in 160 patients at diagnosis and at 469 follow-up checkpoints. MFC was applied at diagnosis with a comprehensive panel of antibodies to identify leukemia-associated aberrant immunophenotypes (LAIP) useful for MRD monitoring. QPCR targeted on the leukemia-specific fusion transcripts AML1-ETO, AML1-EVI1, CBFB-MYH11, MLL-AF10, MLL-AF6, MLL-AF9, MLL-ELL, MLL-ENL, and PML-RARA as well as overexpression of EVI1, length mutations of FLT3, and partial tandem duplications of MLL. In order to adjust for differences in the percentages of bone marrow cells covered by the respective LAIP by MFC at diagnosis and for the heterogeneity of transcript levels detected by QPCR at diagnosis, the logarithmic difference (LD) was calculated for each follow-up sample in comparison to the diagnostic sample. There was a significant correlation between MFC and QPCR with regard to the LD from diagnosis to follow-up checkpoint (r=0.645, p=0.000001). Concordant results with regard to negativity between QPCR (no signal) and MFC (<0.01% positive cells) was found in 301/469 (64.2%) samples (both methods positive, 270 (57.6%); both methods negative, 31 (6.6%)). In 44 samples (9.4%) QPCR detected positivity and MFC negativity while in 124 samples (26.4%) MFC detected positivity and QPCR negativity (sensitivity of QPCR was lower than 1:100,000 in some cases). In 133 patients clinical follow-up data was available allowing the analysis of the prognostic impact of MRD levels. Cytogenetics were favorable, intermediate, and unfavorable in 86, 30, and 17 cases, respectively. Median age was 46 years (range, 17–83). Median event-free survival (EFS) was 22.1 months, overall survival (OS) at three years was 77%. The median LDs for MFC and QPCR at the checkpoint 1 (up to day 21), 2 (day 22–60), 3 (day 61–120), 4 (day 121–365), and 5 (after day 365) were 2.40 and 0.62, 2.05 and 1.55, 2.51 and 3.34, 2.71 and 3.70, and 2.60 and 3.45, respectively. Separating patients according to these median LDs resulted in a better EFS and OS for cases with higher LDs at all five checkpoints for each method. Significant differences in EFS were observed at checkpoints 2 (MFC, 22.1 vs. 12.6 months, p=0.0379; QPCR, median not reached vs. 9.9, p=0.0081), 3 (QPCR, 30.9 vs. 14.1 months, p=0.0011), 4 (MFC, median not reached vs. 16.9 months, p=0.0007; QPCR, median not reached vs. 15.1 months, p=0.0102), and 5 (QPCR, median not reached vs. 17.2, p=0.0008). Cox regression analysis taking into consideration cytogenetics, age, WBC count, and bone marrow blast count at diagnosis identified the LD at checkpoint 4 determined by MFC and the LD at checkpoints 2 and 5 determined by QPCR as independent prognostic factors. The results of our analyses confirm that both MFC and QPCR are highly sensitive methods capable of quantifying MRD in AML. While data are concordant for both methods in many cases, either of the two has advantages in distinct cases depending on the individual MRD marker. Clinical trials should consider MRD monitoring by both methods in order to prove their respective roles in risk prediction and treatment stratification.

Abstract Nuclear Hormone Receptors (NHRs) are a large superfamily of ligand dependent transcription factors regulating a plethora of genes involved in metabolism, growth and differentiation. Recent studies identified the overwhelming role played by NHR in determining hematopoiesis as well as HSC function and fate, suggesting that defective proliferation and maturation of progenitors in hematopoietic malignancies could be attributed to altered expression of these transcriptional regulators. NHRs are attractive but relatively unexplored drug targets and there is limited data on their expression profile in hematological malignancies. Our preliminary screening of AML cell lines and primary cells revealed differential expression of PPARg and RXRα among AML cell lines and decreased expression of these targets in resistant cell lines vs. sensitive ones (Fig.1a). We extended this study aiming to identify the expression pattern of all NHRs in myeloid malignancies, compare with ex-vivo chemoresistance and prognostic markers. Myeloid leukemia cell lines AML(n=12) carrying AML-ETO, Inv-16, PML-RAR fusion transcript, NPM1 or FLT3 mutation and CML(n=8), pooled primary samples harboring only FLT3 ITD mutation and only NPM1 mutation, CD34+ cells and total cellular RNA samples from normal donors were included in the study. Bone marrow samples from AML patients and peripheral blood samples from healthy volunteers were collected after informed consent. PBSC from normal donors (n=7) were enriched for CD34+ cells by magnetic enrichment. RNA extraction followed by purification and cDNA synthesis were done as per manufacturer’s recommendation. The expression of 42 NHRs and 42 co-regulators was profiled by qRT PCR using RT2 PCR Profiler array (SABiosciences, Germany). Expression of each gene was normalized to the average of 5 housekeeping genes (GAPDH, b-actin, b2-microglobulin, HGPRT & RPLP0) and differential expression was calculated by normalizing this with normal total RNA samples to determine fold change. Data normalization and differential gene expression were computed using SABiosciences web-based analysis software. Ex-vivo cytotoxicity to cytarabine and daunorubicin was analyzed using MTT assay and IC50 was calculated using Adapt software. Based on Ara-C and Dnr IC50, AML samples were categorized as sensitive or resistant (IC50<6.25µM and >6.25µM for Ara-C;<0.5µM and >0.5µM for Dnr). FLT3-ITD and NPM1 mutations were screened using PCR followed by GeneScan methods. Upon analysis we observed steroid receptors except PPARG, NR5A1, RXRG, NR1D2, glucocorticoid receptors, androgen receptors and their co-regulatory molecules were expressed across all cell lines. In contrast, expression of certain NHRs such as NR2E3, NR1I2, NR1H4, NR0B1, NR0B2, ESRRG, ESRRB, NR1D1 and the co-regulator HDAC7 were less or at undetectable levels, while estrogen receptors and its co-regulatory molecules except PPARGC1B, steroid receptors HNF4A, NR2F1, NR2F2, RARG and NR3C2 were moderately expressed across the cell lines (Fig.1b). Analysis also revealed significantly reduced expression of ESR2, ESRRG, NR2F2, NR2F6 and THRB and increased PPARG in AML compared to CML cell lines. Comparison between CD34+ progenitor population and well differentiated hematopoietic cells demonstrated >6 fold change in NR4A1 expression. Likewise, expression of several differentiation markers NR1H3, NR1D2, RORA, RORB, RARA and VDR were elevated in cells with increased degree of maturation. The receptor inducing terminal differentiation of erythrocytes THRA, had lesser expression (<60 Fold) in erythroleukemic cell lines K562, HEL and LAMA-84 compared to normal (Fig.1c). In Ara-C resistant cell lines, expression of NR1I3, AHR, and HNF4A were up-regulated while PPARG and RXRA were down-regulated. NPM1 positive cell lines and patient samples had increased expression of PPARG, PPARGC1A, NOTCH2, NR1H3 and NR2F2 than the FLT3 mutated group (Fig.1d) and similar expression of PPARG and RXRA was validated in NPM mutated primary AML blasts (Fig.1e). Our comprehensive analysis of NHRs and its co regulatory molecules provides us with insights on NHR expression on differentiation and drug resistance across myeloid leukemia cell lines with various genetic and molecular characteristics. This could be further explored to identify potential novel drug targets to be used as combination therapy in myeloid leukemias to overcome drug resistance. Disclosures: No relevant conflicts of interest to declare.

We have developed a deep-scale proteome and phosphoproteome database from 44 representative Acute Myeloid Leukemia (AML) patients from the LAML TCGA dataset, and 6 healthy bone marrow derived controls. After confirming data quality, we orthogonally validated several previously undescribed features of AML revealed by the proteomic data. We identified examples of post-transcriptionally regulated proteins both globally (i.e. in all AML samples), and also, in patients with recurrent AML driver mutations. For example, samples with IDH1/2 mutations displayed elevated levels of the 2‑oxoglutarate-dependent histone demethylases KDM4A/B/C, despite no changes in mRNA levels for these genes; we confirmed this finding in vitro. In samples with NPMc mutations, we identified several nuclear importins with post‑transcriptionally increased protein abundance, and showed that they interact with NPMc, but not wildtype NPM1. We identified two cell surface proteins (CD180 and MRC1/CD206) expressed on AML blasts of many patients (but not healthy CD34+ stem/progenitor cells) that could represent novel targets for immunologic therapies, and confirmed these targets via flow cytometry. Finally, we detected nearly 30,000 phosphosites in these samples; globally, AML samples were associated with the abnormal phosphorylation of specific residues in PTPN11, STAT3, AKT1 and PRKCD. FLT3‑TKD samples were associated with increased phosphorylation of activating tyrosines on the cytoplasmic Src-family tyrosine kinases FGR and HCK, and related signaling proteins. PML-RARA-initiated AML samples displayed a unique phosphorylation signature, and TP53-mutant samples showed abundant phosphorylation of serine-183 on TP53 itself. This publicly available database will serve as a foundation for further investigations of protein dysregulation in AML pathogenesis.

Acute promyelocytic leukemia (APL) accounts for 10% of cases of acute myeloid leukemia and is characterized by a particularly aggressive disease progression. Its peculiarity is the presence at diagnosis, 80% of cases, of a severe hemorrhagic syndrome. This dysfunction is often manifested in the form of hemorrhage at brain level that, before the advent of the revolutionary therapy with ATRA (All-Trans Retinoic Acid) caused the early death of almost 20% of patients. At the genetic level, the disease is caused in 99% of cases by a balanced translocation between chromosomes 15 and 17, leading to the formation of the chimeric protein PML-RAR. In RARA gene, the breakpoint constantly occurs in intron 2, while is variable in PML gene, resulting in three different isoforms (bcr1, bcr2 and bcr3). Due to the fast and dramatic progression of the disease and to exploit the availability of life-saving drug it is necessary to have a very quick method of diagnosis, therefore the object of this work is the development of a molecular assay for the ultra-rapid diagnosis of APL, based on the molecular technology LAMP (loop mediated isothermal amplification), a non-PCR method for the amplification of nucleic acids, very rapid and highly specific. LAMP is a quite recent technology, developed in Japan, originally based on the detection of the amplification signal with turbidimetry. The detection method has been greatly improved at DiaSorin, exploiting the use of fluorescent probes. This innovation allowed the detection of different targets in real-time and in multiplex format, thanks to the use of probes emitting at different wavelenghts. A further point of improvement has been the introduction of an engineered polymerase can perform the reverse transcription of RNA in a single step and in a single tube. Two assays have been developed, based on RT-Q-LAMP technology, ultra-rapid and simple to perform, which, starting directly from RNA, are able to identify and discriminate in 15 minutes, the three transcripts bcr1, bcr2 and bcr3, together with an internal control for the validation of negative results. Sensitivity and specificity of the assays have been evaluated on plasmids and validated on positive (n = 88) and negative (n = 94) clinical samples. The results obtained with RT-Q-LAMP assays have been compared with the reference method (PCR or sequencing), giving 100% agreement on the final result. The assays have proven to be highly specific on RNAs negative for the PML-RARA translocations (n = 282) and on water samples (NTC, n = 627). Moreover, both assays demonstrated to be very robust, even when used on suboptimal RNA, with low concentration or poor purity and quality. Compared to current methods, such as PCR, expensive in terms of time, reagents, and which require specialized staff, these new RT-Q-LAMP assays, thanks to the characteristics mentioned above, prove to be extremely competitive and suitable for actual clinical needs.