Leukemogenic Process Research Articles

RAPSYN-mediated neddylation of BCR-ABL alternatively determines the fate of Philadelphia chromosome-positive leukemia

Philadelphia chromosome-positive (Ph+) leukemia is a fatal hematological malignancy. Although standard treatments with tyrosine kinase inhibitors (TKIs) have achieved remarkable success in prolonging patient survival, intolerance, relapse, and TKI resistance remain serious issues for patients with Ph+ leukemia. Here, we report a new leukemogenic process in which RAPSYN and BCR-ABL co-occur in Ph+ leukemia, and RAPSYN mediates the neddylation of BCR-ABL. Consequently, neddylated BCR-ABL enhances the stability by competing its c-CBL-mediated degradation. Furthermore, SRC phosphorylates RAPSYN to activate its NEDD8 E3 ligase activity, promoting BCR-ABL stabilization and disease progression. Moreover, in contrast to in vivo ineffectiveness of PROTAC-based degraders, depletion of RAPSYN expression, or its ligase activity decreased BCR-ABL stability and, in turn, inhibited tumor formation and growth. Collectively, these findings represent an alternative to tyrosine kinase activity for the oncoprotein and leukemogenic cells and generate a rationale of targeting RAPSYN-mediated BCR-ABL neddylation for the treatment of Ph+ leukemia.

RAPSYN-mediated neddylation of BCR-ABL alternatively determines the fate of Philadelphia chromosome-positive leukemia.

Philadelphia chromosome-positive (Ph+) leukemia is a fatal hematological malignancy. Although standard treatments with tyrosine kinase inhibitors (TKIs) have achieved remarkable success in prolonging patient survival, intolerance, relapse, and TKI resistance remain serious issues for patients with Ph+ leukemia. Here, we report a new leukemogenic process in which RAPSYN and BCR-ABL co-occur in Ph+ leukemia, and RAPSYN mediates the neddylation of BCR-ABL. Consequently, neddylated BCR-ABL enhances the stability by competing its c-CBL-mediated degradation. Furthermore, SRC phosphorylates RAPSYN to activate its NEDD8 E3 ligase activity, promoting BCR-ABL stabilization and disease progression. Moreover, in contrast to in vivo ineffectiveness of PROTAC-based degraders, depletion of RAPSYN expression, or its ligase activity decreased BCR-ABL stability and, in turn, inhibited tumor formation and growth. Collectively, these findings represent an alternative to tyrosine kinase activity for the oncoprotein and leukemogenic cells and generate a rationale of targeting RAPSYN-mediated BCR-ABL neddylation for the treatment of Ph+ leukemia.

Therapeutic applications of CRISPR/Cas9 mediated targeted gene editing in acute lymphoblastic leukemia: current perspectives, future challenges, and clinical implications.

Acute Lymphoblastic Leukemia (ALL) is the predominant hematological malignancy in pediatric populations, originating from B- or T-cell precursors within the bone marrow. The disease exhibits a high degree of heterogeneity, both at the molecular level and in terms of clinical presentation. A complex interplay between inherited and acquired genetic alterations contributes to disease pathogenesis, often resulting in the disruption of cellular functions integral to the leukemogenic process. The advent of CRISPR/Cas9 as a gene editing tool has revolutionized biological research, underscoring its potential to modify specific genomic loci implicated in cancer. Enhanced understanding of molecular alterations in ALL has facilitated significant advancements in therapeutic strategies. In this review, we scrutinize the application of CRISPR/Cas9 as a tool for identifying genetic targets to improve therapy, circumvent drug resistance, and facilitate CAR-T cell-based immunotherapy. Additionally, we discuss the challenges and future prospects of CRISPR/Cas9 applications in ALL.

The antitumor effects of WNT5A against hematological malignancies.

The bone marrow (BM) microenvironment (niche) is abnormally altered in acute myeloid leukemia (AML), leading to deficient secretion of proteins, soluble factors, and cytokines by mesenchymal stromal cells (MSC) that modifies the crosstalk between MSC and hematopoietic cells. We focused on a WNT gene/protein family member, WNT5A, which is downregulated in leukemia and correlated with disease progression and poor prognosis. We demonstrated that WNT5A protein upregulated the WNT non-canonical pathway only in leukemic cells, without modulating normal cell behavior. We also introduced a novel WNT5A-mimicking compound, Foxy-5. Our results showed reduction of crucial biological functions that are upregulated in leukemia cells, including ROS generation, cell proliferation, and autophagy, as well as G0/G1 cell cycle arrest. Additionally, Foxy-5 induced early-stage macrophage cell differentiation, a crucial process during leukemia development. At a molecular level, Foxy-5 led to the downregulation of two overexpressed leukemia pathways, PI3K and MAPK, which resulted in a disarrangement of actin polymerization with consequent impairment of CXCL12-induced chemotaxis. Notably, in a novel tri-dimensional bone marrow-mimicking model, Foxy-5 led to reduced leukemia cell growth and similar results were observed in a xenograft in vivo model. Overall, our findings highlight the pivotal role of WNT5A in leukemia and demonstrate that Foxy-5 acts as a specific antineoplastic agent in leukemia, counterbalancing several leukemic oncogenic processes related to the crosstalk in the bone marrow niche, and represents a promising therapeutic option for AML. WNT5A, a WNT gene/protein family member, is naturally secreted by mesenchymal stromal cells and contributes to the maintenance of the bone marrow microenvironment. WNT5A downregulation is correlated with disease progression and poor prognosis. The treatment with Foxy-5, a WNT5A mimetizing compound, counterbalanced several leukemogenic processes that are upregulated in leukemia cells, including ROS generation, cell proliferation, and autophagy and disruption of PI3K and MAPK signaling pathways.

AML-131 ERG: From a Fortuitous Discovery to Potential Treatment for AML

The ETS transcription factor ERG is essential for the maintenance of hematopoietic stem cells. However, it has also been implicated as an oncogene in the development of acute leukemia. Our studies and those of others have demonstrated that ERG directly contributes to the initiation and maintenance of lymphoid and myeloid acute leukemia subtypes. Nevertheless, ERG co-factors critically involved in leukemogenesis remain largely uncharacterized. Here we report a critical role for the conserved amino-acid proline at position 199, at the 3' end of the PNT domain, for ERG's leukemogenic activity. We specifically demonstrate in functional and gene expression assays that P199 is required for ERG-induced myeloid differentiation restriction and for self-renewal and maintenance of murine hematopoietic stem and progenitor cells (HSPC). To identify key protein interactions associated with leukemia progression induced by ERG, we used proximity ligation-mass spectrometry. The proximity maps of wildtype (WT)-ERG and mutated form of ERG at position 199 (P199L-ERG) were compared in HEK293 cells. Most significantly, the mutation severely impaired ERG's interaction with components of the NCoR-HDAC3 complex, resulting in a 40% reduction in the number of spectral counts in comparison with WT-ERG. ChIP-sequencing for histone markers conducted on ER-Hoxb8 cells (murine GMPs) demonstrated a decrease in H3K27ac signature at over 1500 unique sites in cells transduced with WT-ERG compared to those transduced with the mutant. Interestingly, these sites were predominantly associated with enhancers of genes expressed in mature myeloid cells. Furthermore, in gene expression assays conducted on ER-Hoxb8 cells stably expressing human ERG and treated with an HDAC3 inhibitor, myeloid differentiation genes and pathways associated with acute leukemia were most affected. Finally, we examined the significance of the ERG/NCoR-HDAC3 interaction in human leukemia models. Targeting HDAC3 using pharmacological and genetic tools led to a significant antileukemic effect in NSG mice transplanted with ERG-dependent human leukemia lines. Our findings indicate that the aberrant overexpression of ERG maintains HSPCs in an undifferentiated state and promotes AML development. We suggest that the interaction between ERG and the NCoR-HDAC3 complex has an important role in the leukemogenic process, and that HDAC3 inhibition could be beneficial in AML characterized by high ERG expression.

P417: A SINGLE AMINO ACID AT THE PNT DOMAIN OF ERG MEDIATES ITS LEUKEMOGENIC ACTIVITY THROUGH INTERACTION WITH THE NCOR-HDAC3 CO-REPRESSOR COMPLEX.

Background: The ETS transcription factor ERG is essential for the maintenance of hematopoietic stem cells. However, it has also been implicated as an oncogene in the development of acute leukemia. Our studies and those of others have demonstrated that ERG directly contributes to the initiation and maintenance of lymphoid and myeloid acute leukemia subtypes. Nevertheless, ERG co-factors critically involved in leukemogenesis remain largely uncharacterized. Aims: Here we report a critical role for the conserved amino-acid proline at position 199, at the 3’ end of the PNT domain, for ERG’s leukemogenic activity. Methods: A genomic and proteomic analysis has been used to study the role of P199 in the leukemogenic pathway of ERG. Results: We specifically demonstrate in functional and gene expression assays that P199 is required for ERG-induced myeloid differentiation restriction and for self-renewal and maintenance of murine hematopoietic stem and progenitor cells (HSPC). As P199 is located within the PNT domain (protein interaction domain), we attempted to identify key protein interactions associated with leukemia progression induced by ERG. To this end, we used proximity ligation-mass spectrometry (BioID). In HEK293 cells, proximity maps of WT-ERG and a mutated form of ERG at position 199 (P199L-ERG) were compared. A total of 240 putative protein interactors with a significance analysis of interactome were identified. The hits were highly enriched with chromatin modifiers. Most significantly, the mutation severely impaired ERG’s interaction with components of the NCoR-HDAC3 complex, resulting in a 40% reduction in the number of spectral counts in comparison with wild-type ERG. ChIP sequencing for histone markers conducted on ER-Hoxb8 cells (transformed murine GMPs) demonstrated a decrease in H3K27ac signature at over 1500 unique sites in cells transduced with WT-ERG compared to those transduced with the mutant. Interestingly, these sites were predominantly associated with enhancers of genes expressed in mature myeloid cells. Next, we addressed the role of HDAC3 in ERG’s transcriptional activity. RNA sequencing of human ER-Hoxb8 cells treated with BRD3308 (an HDAC3 inhibitor) for 48 hours was performed. Interestingly, leukemia-associated pathways (Hoxa9-Meis1, CBFA2T3 and repression of myeloid differentiation genes) were ranked at the leading edge of gene sets that were significantly perturbed after HDAC3 inhibition. Moreover, we tested the significance of the ERG/NCoR-HDAC3 interaction in models of human leukemia. We demonstrated that ERG-dependent human AML cells as SKNO1 (AML1-ETO) and TF1 (CBFA2T3-ABDH12) were more sensitive to HDAC3 inhibition (RGFP966) as compared to ERG independent cells. Furthermore, RGFP966 exhibited a significant in-vivo antileukemic effect as measured by the burden of disease in the bone marrow of NSG mice transplanted with SKNO1 cells. By using the CRISPR-dCAS9 system we were able to show that leukemia development is abrogated in mice transplanted with SKNO1 cells expressing a reduced level of HDAC3. Remarkably, this effect was translated into a significant survival advantage. Summary/Conclusion: Taken together, our findings indicate that the aberrant overexpression of ERG maintains HSPCs in an undifferentiated state and promotes AML development. We suggest that the interaction between ERG and the NCoR-HDAC3 complex has an important role in the leukemogenic process, and that HDAC3 inhibition could be beneficial in AML characterized by high ERG expression.

Generation of a H9 Clonal Cell Line With Inducible Expression of NUP98-KDM5A Fusion Gene in the AAVS1 Safe Harbor Locus.

Pediatric acute myeloid leukemia (AML) is a rare and heterogeneous disease that remains the major cause of mortality in children with leukemia. To improve the outcome of pediatric AML we need to gain knowledge on the biological bases of this disease. NUP98-KDM5A (NK5A) fusion protein is present in a particular subgroup of young pediatric patients with poor outcome. We report the generation and characterization of human Embryonic Stem Cell (hESC) clonal lines with inducible expression of NK5A. Temporal control of NK5A expression during hematopoietic differentiation from hESC will be critical for elucidating its participation during the leukemogenic process.

Oxidative Stress and ROS-Mediated Signaling in Leukemia: Novel Promising Perspectives to Eradicate Chemoresistant Cells in Myeloid Leukemia.

Myeloid leukemic cells are intrinsically under oxidative stress due to impaired reactive oxygen species (ROS) homeostasis, a common signature of several hematological malignancies. The present review focuses on the molecular mechanisms of aberrant ROS production in myeloid leukemia cells as well as on the redox-dependent signaling pathways involved in the leukemogenic process. Finally, the relevance of new chemotherapy options that specifically exert their pharmacological activity by altering the cellular redox imbalance will be discussed as an effective strategy to eradicate chemoresistant cells.

Leukemogenic pathway of infant leukemia with MLL fusion

Acute lymphoblastic leukemia (ALL) in infants remains an intractable and difficult-to-treat leukemia as compared to other pediatric ALLs, for which considerable progress has been achieved in terms of treatment outcomes in recent years. The leukemic cells in infants with ALL frequently carry chromosome translocations involving 11q23, resulting in the rearrangement and fusion of the MLL (KMT2A) gene. Among many MLL fusion genes, MLL-AF4 (KMT2A-AFF1) fusion is characteristically observed in infants with ALL, representing a hallmark of poor prognosis. In MLL-AF4-positive infants with ALL, first leukemic cells with MLL-AF4 were generated in utero. Analysis of several murine and human leukemia models revealed that the target cells for tumorigenesis by MLL-AF4 were not the hematopoietic progenitor cells of the bone marrow, but the early hematopoietic progenitor cells present in the fetal liver during the embryonic period and possibly the undifferentiated cells prior to the commitment to hematopoietic cells in the fetus. Elucidation of the leukemogenic process of infant ALL with MLL-AF4 may lead to early, pre-symptomatic diagnosis of leukemia, resulting in the improvement of prognosis and prevention of the onset of ALL in infants.

Investigation of Leukemogenic Mechanisms using Metabolomic Approaches

In the bone marrow, the rapid distribution of white blood cells, which agglomerate and ultimately associate with normal blood cells, characterizes leukemogenic processes. Leukemogenic system AML is a complex neoplastic disorder characterized by naive myeloid cell upsurge and bone marrow incompetence. AML is the most common form of leukemia that affects the elderly. One of the prognostic characteristics of this response is the predominance of triggering the FMS-like tyrosine kinase 3 mutations (FLT3), in particular the probability of internal tandem duplication (FLT3-ITD). AML has a poor prognosis of the FLT3-ITD mutation. Metabolic profiling is the ability to understand metabolite modality. In this study, we attempted to understand AML with FLT3 from a metabolomic perspective. It has identified all the metabolites involved in Acute Myeloid Leukemia (AML) and their pathways. AML is a hematologic disease arising from the proliferation and intensification of malignant myeloid cells. In the past, fewer metabolites, such as inborn metabolism defects, were used to diagnose complex metabolic diseases and monogenic disorders. The results of this study have helped to create metabolic pathways for patients FLT3 / ITD, and we have further investigated how these metabolites are relevant from a network biology perspective. With Cytoscape and its plug-ins, such as Metscape, we studied molecular networks. Metabolisms of arachidonic acid and purine in cell metabolites during glycolysis and gluconeogenesis in plasma metabolites are pathways with the greatest impact of AML FLT3 / ITD, urea cycle, and metabolism of arginine, proline, glutamate, aspartate, and asparagine metabolism.

HDAC3 functions as a positive regulator in Notch signal transduction.

Aberrant Notch signaling plays a pivotal role in T-cell acute lymphoblastic leukemia (T-ALL) and chronic lymphocytic leukemia (CLL). Amplitude and duration of the Notch response is controlled by ubiquitin-dependent proteasomal degradation of the Notch1 intracellular domain (NICD1), a hallmark of the leukemogenic process. Here, we show that HDAC3 controls NICD1 acetylation levels directly affecting NICD1 protein stability. Either genetic loss-of-function of HDAC3 or nanomolar concentrations of HDAC inhibitor apicidin lead to downregulation of Notch target genes accompanied by a local reduction of histone acetylation. Importantly, an HDAC3-insensitive NICD1 mutant is more stable but biologically less active. Collectively, these data show a new HDAC3- and acetylation-dependent mechanism that may be exploited to treat Notch1-dependent leukemias.

Pathogenesis of chronic lymphocytic leukemia and the development of novel therapeutic strategies.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries and is characterized by the clonal expansion of mature CD5+ B cells. There have been substantial advances in the field of CLL research in the last decade, including the identification of recurrent mutations, and clarification of clonal architectures, signaling molecules, and the multistep leukemogenic process, providing a comprehensive understanding of CLL pathogenesis. Furthermore, the development of therapeutic approaches, especially that of molecular target therapies against CLL, has markedly improved the standard of care for CLL. This review focuses on the recent insights made in CLL leukemogenesis and the development of novel therapeutic strategies.

Pathophysiology of chronic lymphocytic leukemia and human B1 cell development.

Chronic lymphocytic leukemia (CLL), the most frequent type of leukemia in adults, is a lymphoproliferative disease characterized by the clonal expansion of mature CD5+ B cells in peripheral blood, bone marrow, and secondary lymphoid tissues. Over the past decade, substantial advances have been made in understanding the pathogenesis of CLL, including the identification of recurrent mutations, and clarification of clonal architectures, transcriptome analyses, and the multistep leukemogenic process. The biology of CLL is now better understood. The present review focuses on recent insights into CLL leukemogenesis, emphasizing the role of genetic lesions, and the multistep process initiating from very immature hematopoietic stem cells. Finally, we also review progress in the study of human B1 B cells, the putative normal counterparts of CLL cells.

Identification of Two DNMT3A Mutations Compromising Protein Stability and Methylation Capacity in Acute Myeloid Leukemia.

Somatic mutations of DNMT3A occur in about 20% of acute myeloid leukemia (AML) patients. They mostly consist in heterozygous missense mutations targeting a hotspot site at R882 codon, which exhibit a dominant negative effect and are associated with high myeloblast count, advanced age, and poor prognosis. Other types of mutations such as truncations, insertions, or single-nucleotide deletion also affect the DNMT3A gene, though with lower frequency. The present study aimed to characterize two DNMT3A gene mutations identified by next-generation sequencing (NGS), through analysis of protein stability and DNA methylation status at CpG islands. The first mutation was a single-nucleotide variant of DNMT3A at exon 20 causing a premature STOP codon (c.2385G > A; p.Trp795∗; NM_022552.4). The DNMT3A mutation load increased from 4.5% to 38.2% during guadecitabine treatment, with a dominant negative effect on CpG methylation and on protein expression. The second mutation was a novel insertion of 35 nucleotides in exon 22 of DNMT3A (NM_022552.4) that introduced a STOP codon too, after the amino acid Glu863 caused by a frameshift insertion (c.2586_2587insTCATGAATGAGAAAGAGGACATCTTATGGTGCACT; p. Thr862_Glu863fsins). The mutation, which was associated with reduced DNMT3A expression and CpG methylation, persisted at relapse with minor changes in the methylation profile and at protein level. Our data highlight the need to better understand the consequences of DNMT3A mutations other than R882 substitutions in the leukemogenic process in order to tailor patient treatments, thus avoiding therapeutic resistance and disease relapse.

Benzoquinone, a leukemogenic metabolite of benzene, catalytically inhibits the protein tyrosine phosphatase PTPN2 and alters STAT1 signaling

Protein tyrosine phosphatase, nonreceptor type 2 (PTPN2) is mainly expressed in hematopoietic cells, where it negatively regulates growth factor and cytokine signaling. PTPN2 is an important regulator of hematopoiesis and immune/inflammatory responses, as evidenced by loss-of-function mutations of PTPN2 in leukemia and lymphoma and knockout mice studies. Benzene is an environmental chemical that causes hematological malignancies, and its hematotoxicity arises from its bioactivation in the bone marrow to electrophilic metabolites, notably 1,4-benzoquinone, a major hematotoxic benzene metabolite. Although the molecular bases for benzene-induced leukemia are not well-understood, it has been suggested that benzene metabolites alter topoisomerases II function and thereby significantly contribute to leukemogenesis. However, several studies indicate that benzene and its hematotoxic metabolites may also promote the leukemogenic process by reacting with other targets and pathways. Interestingly, alterations of cell-signaling pathways, such as Janus kinase (JAK)/signal transducer and activator of transcription (STAT), have been proposed to contribute to benzene-induced malignant blood diseases. We show here that 1,4-benzoquinone directly impairs PTPN2 activity. Mechanistic and kinetic experiments with purified human PTPN2 indicated that this impairment results from the irreversible formation (kinact = 645 m-1·s-1) of a covalent 1,4-benzoquinone adduct at the catalytic cysteine residue of the enzyme. Accordingly, cell experiments revealed that 1,4-benzoquinone exposure irreversibly inhibits cellular PTPN2 and concomitantly increases tyrosine phosphorylation of STAT1 and expression of STAT1-regulated genes. Our results provide molecular and cellular evidence that 1,4-benzoquinone covalently modifies key signaling enzymes, implicating it in benzene-induced malignant blood diseases.

PS1013 IS QUERCETIN ABLE TO REVERT THE APOPTOTIC RESISTANCE TRIGGERED BY THE SHORT ISOFORM OF GATA‐1 IN MYELOID LEUKEMIA CELLS?

Background:Dysregulated trans‐acting factors are key elements in the leukemogenic process. Aberrant expression of GATA‐1, a master regulator of several hematopoietic genes has been reported in myeloid leukemias. Maintenance of a balanced expression of the two isoforms of the transcription factor GATA‐1, the full‐length protein (GATA‐1FL) and a shorter isoform (GATA‐1S), contributes to control hematopoiesis, whereas their dysregulation can alter the differentiation/proliferation potential of hematopoietic precursors (WY Lee et al. 2018; JD Crispino et al. 2017). Elevated GATA‐1S levels have been recognized as a poor prognostic factor thus further emphasizing the pro‐leukemic role of this isoform in hematological malignancies.Aims:We evaluated the oxidative stress associated with different GATA‐1 isoforms over‐expressed in the myeloid leukemia cell line K562 and their resistance to pro‐apoptotic stimuli.Methods:K562 cells were transiently transfected with expression vectors for GATA‐1FL and GATA‐1S isoforms. Cytoplasmatic ROS levels were evaluated by flow citometry with the CellROX Deep Red reagent (Thermo Fisher Scientific). Antioxidant capacity was evaluated by measurements of Cu/ZnSOD and MnSOD protein levels and of total and oxidized glutathione (GSH/GSSG‐Glo Assay kit, Promega). Cell viability and apoptosis rate were determined with MTT (Sigma Aldrich) and Annexin V/Pi (BD Biosciences) assays on cells over‐expressing GATA‐1 isoforms and treated with cis‐platin alone or co‐treated with quercetin and cis‐platin.Results:Cytoplasmatic ROS resulted to be significantly increased in cells over‐expressing GATA‐1FL (GATA‐1FL cells) compared to GATA‐1S cells. Total GSH levels were found reduced in GATA‐1FL cells and, conversely, increased in GATA‐1S cells in which we also observed an higher GSH/GSSG ratio. According to the different cytoplasmatic ROS levels detected in these cells, these findings further reinforce the evidence of an enhanced antioxidant capacity in GATA1S cells and of an higher oxidative stress status in GATA‐1FL cells. Accordingly, we also found a significant decrease in the MnSOD protein levels in GATA1S cells (Fig. 1A‐B). Furthermore, we were able to demonstrate a correlation between these data and the lower cell viability and higher apoptotic rate detected in GATA‐1FL cells exposed to a 10 microM dose of the pro‐apoptotic drug cis‐platin, in contrast with the strong resistance to apoptotis shown by GATA‐1S cells. The relationship between changes in proliferation and apoptosis with cell redox states was also demonstrated by analyzing the antioxidant effects of quercetin, a natural flavonoid compound, on transfected cells. According to cell viability data, after 24 hours of 50 and 150 microM quercetin treatment, GATA‐1S cells showed an enhanced apoptotic rate with respect to the GATA‐1FL counterpart. To confirm the changes in the apoptotic susceptibility induced by quercetin in GATA‐1FL and GATA‐1S cells, apoptosis was also evaluated after co‐treatment with 10 microM cis‐platin. Results clearly demonstrated the enhancer activity of quercetin on the apoptosis susceptibility in both cell types and, more interestingly, its ability to revert the resistance to apoptosis shown by GATA‐1S cells when treated with cis‐platin alone (Fig. 1C‐D).Summary/Conclusion:Our study highlights a mechanism to possibly circumvent the apoptotic resistance triggered by aberrant expression of GATA‐1 isoforms in myeloid leukemia cells. Expectedly, a better understanding of this mechanism can eventually lead to design more effective ROS‐based therapies in myeloid leukemias.image

GATA-1 isoforms differently contribute to the production and compartmentation of reactive oxygen species in the myeloid leukemia cell line K562.

Maintenance of a balanced expression of the two isoforms of the transcription factor GATA‐1, the full‐length protein (GATA‐1FL) and a shorter isoform (GATA‐1 S), contributes to control hematopoiesis, whereas their dysregulation can alter the differentiation/proliferation potential of hematopoietic precursors thereby eventually leading to a variety of hematopoietic disorders. Although it is well established that these isoforms play opposite roles in these remarkable processes, most of the molecular pathways involved remain unknown.Here, we demonstrate that GATA‐1FL and GATA‐1S are able to differently influence intracellular redox states and reactive oxygen species (ROS) compartmentation in the erythroleukemic K562 cell line, thus shedding novel mechanistic insights into the processes of cell proliferation and apoptosis resistance in myeloid precursors. Furthermore, given the role played by ROS signaling as a strategy to escape apoptosis and evade cell‐mediated immunity in myeloid cells, this study highlights a mechanism through which aberrant expression of GATA‐1 isoforms could play a role in the leukemogenic process.

Overexpression of TEL-MN1 Fusion Enhances Resistance of HL-60 Cells to Idarubicin

Background: The translocation t(12; 22) (p13;q12) is a recurrent but infrequent chromosome abnormality in human myeloid malignancies. To date, the role of TEL-MN1 fusion in leukemogenic process and drug resistance is still largely unknown. Methods: In the present study, the TEL-MN1 fusion was transfected into HL-60 cells to upregulate TEL-MN1 expression via a retroviral vector. MTT assay was employed to examine cell viability and flow cytometry was performed to evaluate cell apoptosis. Idarubicin was used to treat HL-60 cells for estimating the effect of TEL-MN1 fusion on the chemotherapy resistance. Results: The results showed that overexpression of TEL-MN1 in HL-60 cells could promote cell proliferation, suggesting that TEL-MN1 may be involved in the leukemogenesis process. HL-60 cells treated with idarubicin showed a weakened cell viability, whereas TEL-MN1 overexpression attenuated the idarubicin-induced inhibition of cell viability and acceleration of cell apoptosis of HL-60 cells. Conclusion: Taken together, our results indicated that TEL-MN1 fusion is an oncogene involved in the leukemogenesis process and TEL-MN1 overexpression enhanced resistance of HL-60 cells to idarubicin, which may provide a useful tool for studying the mechanism of leukemogenesis and drug resistance.

Fibroblast growth factor receptor signaling in pediatric B-cell precursor acute lymphoblastic leukemia

The FGF receptor signaling pathway is recurrently involved in the leukemogenic processes. Oncogenic fusions of FGFR1 with various fusion partners were described in myeloid proliferative neoplasms, and overexpression and mutations of FGFR3 are common in multiple myeloma. In addition, fibroblast growth factors are abundant in the bone marrow, and they were shown to enhance the survival of acute myeloid leukemia cells. Here we investigate the effect of FGFR stimulation on pediatric BCP-ALL cells in vitro, and search for mutations with deep targeted next-generation sequencing of mutational hotspots in FGFR1, FGFR2, and FGFR3. In 481 primary BCP-ALL cases, 28 samples from 19 unique relapsed BCP-ALL cases, and twelve BCP-ALL cell lines we found that mutations are rare (4/481 = 0.8%, 0/28 and 0/12) and do not affect codons which are frequently mutated in other malignancies. However, recombinant ligand FGF2 reduced the response to prednisolone in several BCP-ALL cell lines in vitro. We therefore conclude that FGFR signaling can contribute to prednisolone resistance in BCP-ALL cells, but that activating mutations in this receptor tyrosine kinase family are very rare.

Single Cell RNA-Sequencing to Uncover Cell Type-Specific Gene Expression Changes during Inv(16) Leukemia Initiation

The transcription factor RUNX1 and its binding partner CBFβ form a heterodimer whose activity is essential for normal blood cell development. A subset of acute myeloid leukemias (AMLs) are marked by chromosomal abnormalities such as inversions and translocations affecting RUNX1 or CBFβ, leading to malignant hematopoiesis. For example, an inversion of chromosome 16 is seen in patients with the M4Eo subtype of AML and leads to the expression of the fusion protein CBFβ-SMMHC. This fusion protein acts as a driver of leukemogenesis but there are still many unanswered mechanistic questions about the leukemogenic process as well as the molecular activity of the fusion protein. The conditional knock-in mouse model (Kuo et. al, 2006; PMID: 16413472) allows us to study the earliest molecular events in leukemogenesis, since it takes 3-4 months to develop leukemia after the expression of CBFβ-SMMHC is induced in the adult mice. Previous work indicated that CBFβ-SMMHC expression leads to the formation of pre-leukemic hematopoietic cell populations in the bone marrow. In this study, we have utilized single cell RNA-sequencing to profile dynamic changes of hematopoietic cell populations based on gene expression analysis in individual cells, in order to understand the function of the CBFβ-SMMHC fusion protein in hematopoietic cells and leukemogenesis.Flow cytometry was used to enrich for Cd117+ (c-Kit+) stem and progenitor cells from the total bone marrow of a) wild-type mice (n=2) b) pre-leukemic CBFβ-SMMHC expressing mice (n=2), and c) leukemic CBFβ-SMMHC expressing mice (n=2). The 10X Genomics Chromium platform was utilized to capture individual cells in droplets, with each cell and its transcripts uniquely tagged at the time of isolation. Prepared cDNA libraries were sequenced on an Illumina NextSeq 500. After post-sequence alignment and QC, expression matrices were imported into Seurat (Butler et. al, 2018; PMID: 29608179) for further analysis. In total, cDNAs from more than 14,000 cells were sequenced, each expressing between 1500-4000 genes. This high dimensional data was reduced using t-distributed stochastic neighbor embedding (t-SNE), a process that “clusters” the cells that are the most related to one another based on their transcriptional profile (See figure). Cluster identities can then be assigned based on canonical markers expressed within each cluster.In wild-type mice, tSNE clustering revealed at least 14 unique cell identities among the c-Kit+ cells comprising stem cells, multipotent progenitors, and cells of myeloid, erythroid, and lymphoid lineages. Similar heterogeneity was observed for the c-Kit+ cells from CBFβ-SMMHC expressing mice that were pre-leukemic. The c-Kit+ cells from leukemic CBFβ-SMMHC expressing mice were more homogeneous, indicating a dominant leukemia clone. In the two pre-leukemic mice examined, a small cluster of c-Kit+ cells express some markers of megakaryocyte-erythroid progenitors (MEP), but express numerous other genes that classify them as “abnormal”, a leukemogenic cell type that has been reported previously as abnormal myeloid progenitors (AMPs, Kuo et. al, 2006; PMID: 16413472). We have identified novel markers of this population, which expands as leukemia progresses. Interestingly, the top canonical pathway identified from markers in this AMP population is heme biosynthesis. In humans, upregulated heme biosynthesis has recently been reported in aggressive pediatric AMLs which overexpress MYCN (Fukuda et. al, 2017; PMID: 28768907). In the sequencing data from our mouse model, however, we see no alteration in Mycn, potentially suggesting an alternative driver of heme biosynthesis in inv[16] leukemogenesis.Lastly, we noted numerous other alterations when comparing the data from wild-type to pre-leukemic c-Kit+ cells, both in the number of cells occupying a cluster (cell type) as well as gene expression differences within a cell type. These data truly underscore the large impact CBFβ-SMMHC has on the hematopoietic landscape and, because these data were obtained on the single cell level, we will be able to examine the gene expression alterations the fusion protein drives in specific cell subtypes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.