A Nine-Gene Expression Signature Distinguished a Patient with Chronic Lymphocytic Leukemia Who Underwent Prolonged Periodic Fasting

Background and Objectives: This study aimed to investigate the causes of continuous deep fluctuations in the absolute lymphocyte count (ALC) in an untreated patient with Chronic Lymphocytic Leukemia (CLL), who has had a favorable prognosis since the time of diagnosis. Up until now, the patient has voluntarily chosen to adopt a predominantly vegetarian and fruitarian diet, along with prolonged periods of total fasting (ranging from 4 to 39 days) each year. Materials and Methods: For this purpose, we decided to analyze the whole transcriptome profiling of peripheral blood (PB) CD19+ cells from the patient (#1) at different time-points vs. the same cells of five other untreated CLL patients who followed a varied diet. Consequently, the CLL patients were categorized as follows: the 1st group comprised patient #1 at 20 different time-points (16 time-points during nutrition and 4 time-points during fasting), whereas the 2nd group included only one time point for each of the patients (#2, #3, #4, #5, and #6) as they followed a varied diet. We performed microarray experiments using a powerful tool, the Affymetrix Human Clariom™ D Pico Assay, to generate high-fidelity biomarker signatures. Statistical analysis was employed to identify differentially expressed genes and to perform sample clustering. Results: The lymphocytosis trend in patient #1 showed recurring fluctuations since the time of diagnosis. Interestingly, we observed that approximately 4–6 weeks after the conclusion of fasting periods, the absolute lymphocyte count was reduced by about half. The gene expression profiling analysis revealed that nine genes were statistically differently expressed between the 1st group and the 2nd group. Specifically, IGLC3, RPS26, CHPT1, and PCDH9 were under expressed in the 1st group compared to the 2nd group of CLL patients. Conversely, IGHV3-43, IGKV3D-20, PLEKHA1, CYBB, and GABRB2 were over-expressed in the 1st group when compared to the 2nd group of CLL patients. Furthermore, clustering analysis validated that all the samples from patient #1 clustered together, showing clear separation from the samples of the other CLL patients. Conclusions: This study unveiled a small gene expression signature consisting of nine genes that distinguished an untreated CLL patient who followed prolonged periods of total fasting, maintaining a gradual growth trend of lymphocytosis, compared to five untreated CLL patients with a varied diet. Future investigations focusing on patient #1 could potentially shed light on the role of prolonged periodic fasting and the implication of this specific gene signature in sustaining the lymphocytosis trend and the favorable course of the disease

Identification of a Candidate Gene Set Signature for the Risk of Progression in IgM MGUS to Smoldering/Symptomatic Waldenström Macroglobulinemia (WM) by a Comparative Transcriptome Analysis of B Cells and Plasma Cells

Waldenström Macroglobulinemia (WM) is a B-cell lymphoma characterized by the precursor condition IgM monoclonal gammopathies of undetermined significance (IgM MGUS). We performed a gene expression profiling study to compare the transcriptome signatures of bone marrow (BM) B-cells and plasma cells of 36 WM patients, 13 IgM MGUS cases, and 7 healthy subjects used as controls (CTRLs) by Affymetrix microarray. We determined 2038 differentially expressed genes (DEGs) in CD19+ cells and 29 DEGs genes in CD138+ cells, respectively. The DEGs identified in B-cells were associated with KEGG pathways, mainly involved in hematopoietic cell lineage antigens, cell adhesion/focal adhesion/transmembrane proteins, adherens junctions, Wnt-signaling pathway, BCR-signaling pathway, calcium signaling pathway, complement/coagulation cascade, platelet activation, cytokine-cytokine receptor interactions, and signaling pathways responsible for cell cycle, apoptosis, proliferation and survival. In conclusion, we showed the deregulation of groups of genes belonging to KEGG pathways in the comparison among WM vs. IgM MGUS vs. CTRLs in B-cells. Interestingly, a small set of genes in B-cells displayed a common transcriptome expression profile between WM and IgM MGUS compared to CTRLs, suggesting its possible role in the risk of transformation of IgM MGUS to WM

FZD6 triggers Wnt-signalling driven by WNT10BIVS1 expression and highlights new targets in T cell acute lymphoblastic leukemia

Wnt/Fzd signaling has been implicated in hematopoietic stem cell maintenance and in acute leukemia establishment. In our previous work we described a recurrent rearrangement involving the WNT10B locus (WNT10BR ), characterized by the expression of WNT10BIVS1 transcript variant, in acute myeloid leukemia. To determine the occurrence of WNT10BR in T-cell acute lymphoblastic leukemia (T-ALL), we retrospectively analysed an Italian cohort of patients (n=20) and detected a high incidence (13/20) of WNT10BIVS1 expression. To address genes involved in WNT10B molecular response, we have designed a Wnt targeted RNA sequencing panel. Identifying Wnt agonists and antagonists, it results that the expression of FZD6, LRP5, and PROM1 genes stands out in WNT10BIVS1 positive patients compared to negative ones. Using MOLT4 and MUTZ-2 as leukemic cell models, which are characterized by the expression of WNT10BIVS1 , we have observed that WNT10B drives major Wnt activation to the FZD6 receptor complex through receipt of ligand. Additionally, short hairpin RNAs (shRNAs)-mediated gene silencing and small molecules-mediated inhibition of WNTs secretion, have been observed to interfere with the WNT10B/FZD6 interaction. We have therefore identified that WNT10BIVS1 knockdown, or pharmacological interference by the LGK974 porcupine (PORCN) inhibitor, reduces WNT10B/FZD6 protein complex formation and significantly impairs intracellular effectors and leukemic expansion. These results describe the molecular circuit induced by WNT10B and suggest WNT10B/FZD6 as a new target in the T-ALL treatment strategy. This article is protected by copyright. All rights reserved

Nilotinib interferes with cell cycle, ABC transporters and JAK-STAT signaling pathway in CD34+/lin- cells of patients with chronic phase chronic myeloid leukemia after 12 months of treatment

Chronic myeloid leukemia (CML) is characterized by the constitutive tyrosine kinase activity of the oncoprotein BCR-ABL1 in myeloid progenitor cells that activates multiple signal transduction pathways leading to the leukemic phenotype. The tyrosine-kinase inhibitor (TKI) nilotinib inhibits the tyrosine kinase activity of BCR-ABL1 in CML patients. Despite the success of nilotinib treatment in patients with chronic-phase (CP) CML, a population of Philadelphia-positive (Ph+) quiescent stem cells escapes the drug activity and can lead to drug resistance. The molecular mechanism by which these quiescent cells remain insensitive is poorly understood. The aim of this study was to compare the gene expression profiling (GEP) of bone marrow (BM) CD34+/lin- cells from CP-CML patients at diagnosis and after 12 months of nilotinib treatment by microarray, in order to identify gene expression changes and the dysregulation of pathways due to nilotinib action. We selected BM CD34+/lin- cells from 78 CP-CML patients at diagnosis and after 12 months of first-line nilotinib therapy and microarray analysis was performed. GEP bioinformatic analyses identified 2,959 differently expressed probes and functional clustering determined some significantly enriched pathways between diagnosis and 12 months of nilotinib treatment. Among these pathways, we observed the under expression of 26 genes encoding proteins belonging to the cell cycle after 12 months of nilotinib treatment which led to the up-regulation of chromosome replication, cell proliferation, DNA replication, and DNA damage checkpoint at diagnosis. We demonstrated the under expression of the ATP-binding cassette (ABC) transporters ABCC4, ABCC5, and ABCD3 encoding proteins which pumped drugs out of the cells after 12 months of nilotinib. Moreover, GEP data demonstrated the deregulation of genes involved in the JAK-STAT signaling pathway. The down-regulation of JAK2, IL7, STAM, PIK3CA, PTPN11, RAF1, and SOS1 key genes after 12 months of nilotinib could demonstrate the up-regulation of cell cycle, proliferation and differentiation via MAPK and PI3K-AKT signaling pathways at diagnosis

Nilotinib-induced bone marrow CD34+/lin-Ph+ cells early clearance in newly diagnosed CP-Chronic Myeloid Leukemia: Final report of the PhilosoPhi34 study

none21Chronic Myeloid Leukemia is a clonal disorder characterized by the presence of the Ph-chromosome and the BCR-ABL tyrosine-kinase (TK). Target-therapy with Imatinib has greatly improved its outcome. Deeper and faster responses are reported with the second-generation TKI Nilotinib. Sustained responses may enable TKI discontinuation. However, even in a complete molecular response, some patients experience disease recurrence possibly due to persistence of quiescent leukemic CD34+/lin-Ph+ stem cells (LSCs). Degree and mechanisms of LSCs clearance during TKI treatment are not clearly established. The PhilosoPhi34 study was designed to verify the in-vivo activity and timecourse of first-line Nilotinib therapy on BM CD34+/lin-Ph+ cells clearance. Eighty-seven CP-CML patients were enrolled. BM cells were collected and tested for Ph+ residual cells, at diagnosis, 3, 6 and 12 months of treatment. FISH analysis of unstimulated CD34+/lin- cells in CCyR patients were positive in 8/65 (12.3%), 5/71 (7%), 0/69 (0%) evaluable tests, respectively. Per-Protocol analysis response rates were as follows: CCyR 95% at 12 months, MR4.5 31% and 46% at 12 and 36 months, respectively. An exploratory Gene Expression Profiling (GEP) study of CD34+/lin- cells was performed on 30 patients at diagnosis and after, on 79 patients at diagnosis vs 12 months of nilotinib treatment vs 10 healthy subjects. Data demonstrated some genes significantly different expressed: NFKBIA, many cell cycle genes, ABC transporters, JAK-STAT signaling pathway (JAK2). In addition, a correlation between different expression of some genes (JAK2, OLFM4, ICAM1, NFKBIA) among patients at diagnosis and their achievement of an early and deeper MR was observed.nonePungolino, Ester; D'adda, Mariella; De Canal, Gabriella; Trojani, Alessandra; Perego, Alessandra; Elena, Chiara; Lunghi, Francesca; Turrini, Mauro; Borin, Lorenza; Iurlo, Alessandra; Latargia, Maria Luisa; Carraro, Maria Cristina; Spina, Francesco; Artale, Salvatore; Anghilieri, Michela; Molteni, Alfredo; Caramella, Marianna; Baruzzo, Giacomo; Nichelatti, Michele; Di Camillo, Barbara; Cairoli, RobertoPungolino, Ester; D'Adda, Mariella; De Canal, Gabriella; Trojani, Alessandra; Perego, Alessandra; Elena, Chiara; Lunghi, Francesca; Turrini, Mauro; Borin, Lorenza; Iurlo, Alessandra; Latargia, Maria Luisa; Carraro, Maria Cristina; Spina, Francesco; Artale, Salvatore; Anghilieri, Michela; Molteni, Alfredo; Caramella, Marianna; Baruzzo, Giacomo; Nichelatti, Michele; Di Camillo, Barbara; Cairoli, Robert

Wide-transcriptome analysis and cellularity of bone marrow CD34+/lin- cells of patients with chronic-phase chronic myeloid leukemia at diagnosis vs. 12 months of first-line nilotinib treatment

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder with heterogeneous biological and clinical features. The biomolecular mechanisms of CML response to tyrosine-kinase inhibitors are not fully defined

Occurrence of L1M Elements in Chromosomal Rearrangements Associated to Chronic Myeloid Leukemia (CML): Insights from Patient-Specific Breakpoints Characterization

Chronic myeloid leukemia (CML) is a rare myeloproliferative disorder caused by the reciprocal translocation t(9;22)(q34;q11) in hematopoietic stem cells (HSCs). This chromosomal translocation results in the formation of an extra-short chromosome 22, called a Philadelphia chromosome (Ph), containing the BCR-ABL1 fusion gene responsible for the expression of a constitutively active tyrosine kinase that causes uncontrolled growth and replication of leukemic cells. Mechanisms behind the formation of this chromosomal rearrangement are not well known, even if, as observed in tumors, repetitive DNA may be involved as core elements in chromosomal rearrangements. We have participated in the explorative investigations of the PhilosoPhi34 study to evaluate residual Ph+ cells in patients with negative FISH analysis on CD34+/lin- cells with gDNA qPCR. Using targeted next-generation deep sequencing strategies, we analyzed the genomic region around the t(9;22) translocations of 82 CML patients and one CML cell line and assessed the relevance of interspersed repeat elements at breakpoints (BP). We found a statistically higher presence of LINE elements, in particular belonging to the subfamily L1M, in BP cluster regions of both chromosome 22 and 9 compared to the whole human genome. These data suggest that L1M elements could be potential drivers of t(9;22) translocation leading to the generation of the BCR-ABL1 chimeric gene and the expression of the active BCR-ABL1-controlled tyrosine kinase chimeric protein responsible for CML