differentiation block
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Blood ◽  
2021 ◽  
Author(s):  
Swagata Goswami ◽  
Rajeswaran Mani ◽  
Jessica Nunes ◽  
Chi-ling Chiang ◽  
Kevan Zapolnik ◽  
...  

Dysregulated cellular differentiation is a hallmark of acute leukemogenesis. Phosphatases are widely suppressed in cancers but have not been traditionally associated with differentiation. Herein, we identified that the silencing of Protein Phosphatase 2A (PP2A) directly contributes to differentiation block in acute myeloid leukemia (AML). Gene expression and mass cytometric profiling reveal that PP2A activation modulates cell cycle and transcriptional regulators that program terminal myeloid differentiation. Using a novel pharmacological agent OSU-2S in parallel with genetic approaches, we discovered that PP2A enforces c-Myc and p21 dependent terminal differentiation, proliferation arrest and apoptosis in AML. Finally, we demonstrate that PP2A activation decreases leukemia initiating stem cells, increases leukemic blast maturation, and improves overall survival in murine Tet2-/-Flt3ITD/WT and human AML models in-vivo. Our findings identify the PP2A/c-Myc/p21 axis as a critical regulator of the differentiation/proliferation switch in AML that can be therapeutically targeted in malignancies with dysregulated maturation fate.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1146-1146
Author(s):  
Raveen Stephen Stallon Illangeswaran ◽  
Sreeja Karathedath ◽  
Abhirup Bagchi ◽  
Bharathi M Rajamani ◽  
Balaji Balakrishnan ◽  
...  

Abstract The success of differentiation therapy is limited to acute promyelocytic leukemia (APL), and approaches to overcome the differentiation block in non-M3 AML have been unsuccessful. Nuclear hormone receptors (NHR) belong to ligand-inducible transcription factors that govern many cellular functions like differentiation, metabolism, and development. Retinoic Acid Receptor Alpha (RXRA) is a class of NHR that, when activated by all-trans retinoic acid (ATRA), successfully alleviates differentiation block in APL. To identify the NHRs/cofactors that could mediate or prevent differentiation in AML, we examined the differentially expressed NHRs and cofactors between ATRA sensitive (ATs) (NB4 and HL60) vs. ATRA resistant (ATr) AML cell lines (KG1a, Hel, K562, MV4-11, and OCI-AML3). Nuclear Receptor Interacting Protein 1 (NRIP1), a corepressor known to prevent transactivation of ligand-activated NHRs preferentially, was one of the top upregulated targets in the ATr cell lines (3.5 fold increase in RNA expression, figure 1a ). Immunoblot analysis also showed a significant increase in NRIP1 protein expression in the ATr than ATs cell lines (Figure 1b). Further, probing for NRIP1 expression in the publicly available TCGA and MILE AML study cohorts showed decreased NRIP1 expression in the APL cohort compared to other AML subtypes. Methylation profile from CCLE database of the NRIP1 promoter in AML cell lines showed ATs cell lines to be highly methylated compared to the ATr cell lines, suggesting the involvement of NRIP1 in mediating differentiation block in non-M3 AML (Figure 1c). To further dissect the role of NRIP1 in mediating this differentiation block, we carried out experiments in the AML cell line KG1a (having primitive blast features, high expression of NRIP1, and unresponsive to ATRA). Using CRISPR-cas9, we developed an NRIP1 knock-out (KO) cell line (Figure 1d). NRIP1 KO cell line showed a significant reduction in proliferation rate (Doubling time 26.2 vs. 36.5Hrs p<0.05). Further, cell cycle analysis revealed that NRIP1 KO leads to increased accumulation of cells in the G0 phase than in the S-phase (Figure 1e & f). We next assessed the sensitivity of the NRIP1 WT/KO cells to retinoic acids ATRA and bexarotene. Cells were treated with 1µM ATRA / bexarotene or in combination for 72 hours and evaluated for differentiation using CD11b marker by flow cytometry. NRIP1 KO alone leads to a marginal increase in basal CD11b expression compared to the WT cells (Mean CD11b expression 2.03% Vs 0.91%). ATRA treatment further increased the CD11b expression to 3.8% in KO cells compared to 1.6% in the WT cells. A similar increase in CD11b expression was observed in bexarotene-treated cells (3.7% Vs 1.24%). Combination of ATRA with bexarotene showed a 3-fold increase in CD11b expression in the KO cells compared to the WT (23.9% Vs 7.2%, Figure 1g). NRIP1 KO diminishes its repressive action on ligand-activated RARA (ATRA activated) and RXRA (Bexarotene-activated), thereby allowing synergistic differentiation induction by retinoic acids in AML cells. This study suggests a potential mechanism of differentiation inhibition mediated by corepressor NRIP1 in AML cells unresponsive to retinoic acids. Further in-depth analyses of molecular pathways governed by NRIP1 during ligand activation of NHRs are warranted to design differentiation therapies for AML. Figure 1 Figure 1. Disclosures Mathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor.


2021 ◽  
Author(s):  
Emanuele Gioacchino ◽  
Cansu Koyunlar ◽  
Joke Zink ◽  
Hans de Looper ◽  
Kirsten J Gussinklo ◽  
...  

The transcription factor GATA2 has pivotal roles in hematopoiesis. Germline GATA2 mutations result in GATA2 haploinsufficiency characterized by immunodeficiency, bone marrow failure and predispositions to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Clinical symptoms in GATA2 patients are diverse and mechanisms driving GATA2 related phenotypes are largely unknown. To explore the impact of GATA2 haploinsufficiency on hematopoiesis, we generated a zebrafish model carrying a heterozygous mutation in gata2b, an orthologue of GATA2. Morphological analysis revealed progression of myeloid and erythroid dysplasia in gata2b+/- kidney marrow (KM). Single cell RNA sequencing on KM cells showed that the erythroid dysplasia in gata2b+/- zebrafish was preceded by a differentiation block in erythroid progenitors, hallmarked by downregulation of cytoskeletal transcripts, aberrant proliferative signatures and ribosome biogenesis. Additionally, transcriptional and functional analysis of Gata2b haploinsufficient hematopoietic stem cells (HSCs) indicated that proliferative stress within the HSC compartment possibly contributes to the development of myeloid and erythroid dysplasia in gata2b+/- zebrafish.


2021 ◽  
Author(s):  
Joan So ◽  
Alexander C. Lewis ◽  
Lorey K. Smith ◽  
Kym Stanley ◽  
Lizzy Pijpers ◽  
...  

AbstractThe mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) catalyzes one of the rate-limiting steps in de novo pyrimidine biosynthesis, a pathway that provides essential metabolic precursors for nucleic acids, glycoproteins and phospholipids. DHODH inhibitors (DHODHi) are clinically used for autoimmune diseases and are emerging as a novel class of anti-cancer agents, especially in acute myeloid leukemia (AML) where pyrimidine starvation was recently shown to reverse the characteristic differentiation block in AML cells. Herein we show that DHODH blockade rapidly shuts down protein translation in leukemic stem cells (LSCs) by down-regulation of the multi-functional transcription factor YY1, has potent activity against AML in vivo and is well tolerated with minimal impact on normal blood development. Moreover, we find that ablation of CDK5, a gene that is recurrently deleted in AML and related disorders, increases the sensitivity of AML cells to DHODHi. Our studies provide important molecular insights and identify a potential biomarker for an emerging strategy to target AML.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Takuyo Kanayama ◽  
Mitsuru Miyachi ◽  
Yohei Sugimoto ◽  
Shigeki Yagyu ◽  
Ken Kikuchi ◽  
...  

AbstractB7-H3 (also known as CD276) is associated with aggressive characteristics in various cancers. Meanwhile, in alveolar rhabdomyosarcoma (ARMS), PAX3-FOXO1 fusion protein is associated with increased aggressiveness and poor prognosis. In the present study, we explored the relationship between PAX3-FOXO1 and B7-H3 and the biological roles of B7-H3 in ARMS. Quantitative real time PCR and flow cytometry revealed that PAX3-FOXO1 knockdown downregulated B7-H3 expression in all the selected cell lines (Rh-30, Rh-41, and Rh-28), suggesting that PAX3-FOXO1 positively regulates B7-H3 expression. Gene expression analysis revealed that various genes and pathways involved in chemotaxis, INF-γ production, and myogenic differentiation were commonly affected by the knockdown of PAX3-FOXO1 and B7-H3. Wound healing and transwell migration assays revealed that both PAX3-FOXO1 and B7-H3 were associated with cell migration. Furthermore, knockdown of PAX3-FOXO1 or B7-H3 induced myogenin expression in all cell lines, although myosin heavy chain induction varied depending on the cellular context. Our results indicate that PAX3-FOXO1 regulates B7-H3 expression and that PAX3-FOXO1 and B7-H3 are commonly associated with multiple pathways related to an aggressive phenotype in ARMS, such as cell migration and myogenic differentiation block.


Blood ◽  
2021 ◽  
Author(s):  
Amanda G Davis ◽  
Daniel T. Johnson ◽  
Dinghai Zheng ◽  
Ruijia Wang ◽  
Nathan D. Jayne ◽  
...  

Post-transcriptional regulation has emerged as a driver for leukemia development and an avenue for therapeutic targeting. Among post-transcriptional processes, alternative polyadenylation (APA) is globally dysregulated across cancer types. However, limited studies have focused on the prevalence and role of APA in myeloid leukemia. Furthermore, it is poorly understood how altered poly(A) site (PAS) usage of individual genes contributes to malignancy or whether targeting global APA patterns might alter oncogenic potential. In this study, we examined global APA dysregulation in acute myeloid leukemia (AML) patients by performing 3' Region Extraction And Deep Sequencing (3'READS) on a subset of AML patient samples along with healthy hematopoietic stem and progenitor cells (HSPCs) and by analyzing publicly available data from a broad AML patient cohort. We show that patient cells exhibit global 3' untranslated region (UTR) shortening and coding sequence (CDS) lengthening due to differences in PAS usage. Among APA regulators, expression of FIP1L1, one of the core cleavage and polyadenylation factors, correlated with the degree of APA dysregulation in our 3'READS dataset. Targeting global APA by FIP1L1 knockdown reversed the global trends seen in patients. Importantly, FIP1L1 knockdown induced differentiation of t(8;21) cells by promoting 3'UTR lengthening and downregulation of the fusion oncoprotein AML1-ETO. In non-t(8;21) cells, FIP1L1 knockdown also promoted differentiation by attenuating mTORC1 signaling and reducing MYC protein levels. Our study provides mechanistic insights into the role of APA in AML pathogenesis and indicates that targeting global APA patterns can overcome the differentiation block of AML patients.


2021 ◽  
Author(s):  
Diane Yang ◽  
Sanjeet Patel ◽  
Wojciech J. Szlachcic ◽  
Jolanta Chmielowiec ◽  
Diane Scaduto ◽  
...  

Genetic analysis of an adult patient with an unusual course of Ketosis-Prone Diabetes (KPD) and lacking islet autoantibodies demonstrated a nucleotide variant in the<i> </i>5’-UTR of <i>PDX1</i>, a beta-cell development gene. When differentiated to the pancreatic lineage, his induced pluripotent stem cells stalled at the definitive endoderm stage. Metabolomic analysis of the cells revealed that this was associated with leucine hypersensitivity during transition from the definitive endoderm to the pancreatic progenitor stage, and RNA-sequencing showed defects in leucine-sensitive mTOR pathways contribute to the differentiation deficiency. CRISPR-Cas9 manipulation of the <i>PDX1</i> variant demonstrated that it is necessary and sufficient to confer leucine sensitivity and the differentiation block, likely due to disruption of binding of the transcriptional regulator NFY to the <i>PDX1</i> 5’-UTR, leading to decreased PDX1 expression at the early pancreatic progenitor stage. Thus, the combination of an underlying defect in leucine catabolism characteristic of KPD with a functionally relevant heterozygous variant in a critical beta-cell gene that confers increased leucine sensitivity and inhibits endocrine cell differentiation resulted in the phenotype of late-onset beta-cell failure in this patient. We define the molecular pathogenesis of a diabetes syndrome and demonstrate the power of multi-omics analysis of patient-specific stem cells for clinical discovery.


2021 ◽  
Author(s):  
Diane Yang ◽  
Sanjeet Patel ◽  
Wojciech J. Szlachcic ◽  
Jolanta Chmielowiec ◽  
Diane Scaduto ◽  
...  

Genetic analysis of an adult patient with an unusual course of Ketosis-Prone Diabetes (KPD) and lacking islet autoantibodies demonstrated a nucleotide variant in the<i> </i>5’-UTR of <i>PDX1</i>, a beta-cell development gene. When differentiated to the pancreatic lineage, his induced pluripotent stem cells stalled at the definitive endoderm stage. Metabolomic analysis of the cells revealed that this was associated with leucine hypersensitivity during transition from the definitive endoderm to the pancreatic progenitor stage, and RNA-sequencing showed defects in leucine-sensitive mTOR pathways contribute to the differentiation deficiency. CRISPR-Cas9 manipulation of the <i>PDX1</i> variant demonstrated that it is necessary and sufficient to confer leucine sensitivity and the differentiation block, likely due to disruption of binding of the transcriptional regulator NFY to the <i>PDX1</i> 5’-UTR, leading to decreased PDX1 expression at the early pancreatic progenitor stage. Thus, the combination of an underlying defect in leucine catabolism characteristic of KPD with a functionally relevant heterozygous variant in a critical beta-cell gene that confers increased leucine sensitivity and inhibits endocrine cell differentiation resulted in the phenotype of late-onset beta-cell failure in this patient. We define the molecular pathogenesis of a diabetes syndrome and demonstrate the power of multi-omics analysis of patient-specific stem cells for clinical discovery.


Cancers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 3858
Author(s):  
Adriana C. Pliego Zamora ◽  
Hansini Ranasinghe ◽  
Jessica E. Lisle ◽  
Chun Ki Ng ◽  
Stephen Huang ◽  
...  

We recently characterised the NUP98-HOXD13 (NHD13) mouse as a model of T-cell pre-leukaemia, featuring thymocytes that can engraft in recipient animals and progress to T-cell acute lymphoblastic leukaemia (T-ALL). However, loss of this engraftment ability by deletion of Lyl1 did not result in any loss of leukemogenesis activity. In the present study, we observe that NHD13 thymocytes overexpress EPHA3, and we characterise thymocyte behaviour in NHD13 mice with deletion of EphA3, which show a markedly reduced incidence of T-ALL. Deletion of EphA3 from the NHD13 mice does not prevent the abnormal accumulation or transplantation ability of these thymocytes. However, upon transplantation, these cells are unable to block the normal progression of recipient wild type (WT) progenitor cells through the normal developmental pathway. This is in contrast to the EphA3+/+ NHD13 thymocytes, which block the progression of incoming WT progenitors past the DN1 stage. Therefore, EphA3 is not critical for classical self-renewal, but is essential for mediating an interaction between the abnormally self-renewing cells and healthy progenitors—an interaction that results in a failure of the healthy cells to differentiate normally. We speculate that this may orchestrate a loss of healthy cell competition, which in itself has been demonstrated to be oncogenic, and that this may explain the decrease in T-ALL incidence in the absence of EphA3. We suggest that pre-leukaemic self-renewal in this model is a complex interplay of cell-intrinsic and -extrinsic factors, and that multiple redundant pathways to leukaemogenesis are active.


Cancers ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1968
Author(s):  
Camilla Bertuzzo Veiga ◽  
Erin M. Lawrence ◽  
Andrew J. Murphy ◽  
Marco J. Herold ◽  
Dragana Dragoljevic

The development of myelodysplasia syndromes (MDS) is multiphasic and can be driven by a plethora of genetic mutations and/or abnormalities. MDS is characterized by a hematopoietic differentiation block, evidenced by increased immature hematopoietic cells, termed blast cells and decreased mature circulating leukocytes in at least one lineage (i.e., cytopenia). Clonal hematopoiesis of indeterminate potential (CHIP) is a recently described phenomenon preceding MDS development that is driven by somatic mutations in hemopoietic stem cells (HSCs). These mutant HSCs have a competitive advantage over healthy cells, resulting in an expansion of these clonal mutated leukocytes. In this review, we discuss the multiphasic development of MDS, the common mutations found in both MDS and CHIP, how a loss-of-function in these CHIP-related genes can alter HSC function and leukocyte development and the potential disease outcomes that can occur with dysfunctional HSCs. In particular, we discuss the novel connections between MDS development and cardiovascular disease.


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