Differentiation Arrest Research Articles

Role of Nodal signalling in testis development and initiation of testicular cancer.

Testicular development from the initially bipotential gonad is a tightly regulated process involving a complex signalling cascade to ensure proper sequential expression of signalling factors and secretion of steroid hormones. Initially, Sertoli cell specification facilitates differentiation of the steroidogenic fetal Leydig cells and establishment of the somatic niche, which is critical in supporting the germ cell population. Impairment of the somatic niche during fetal life may lead to development of male reproductive disorders, including arrest of gonocyte differentiation, which is considered the first step in the testicular cancer pathogenesis. In this review, we will outline the signalling pathways involved in fetal testis development focusing on the Nodal pathway, which has recently been implicated in several aspects of testicular differentiation in both mouse and human studies. Nodal signalling plays important roles in germ cell development, including regulation of pluripotency factor expression, proliferation and survival. Moreover, the Nodal pathway is involved in establishment of the somatic niche, including formation of seminiferous cords, steroidogenesis and Sertoli cell function. In our outline of fetal testis development, important differences between human and mouse models will be highlighted to emphasise that information obtained from mouse studies cannot always be directly translated to humans. Finally, the implications of dysregulated Nodal signalling in development of the testicular cancer precursor, germ cell neoplasia in situ, and testicular dysgenesis will be discussed - none of which arise in rodents, emphasising the importance of human models in the effort to increase our understanding of origin and early development of these disorders.

2036 – INFLAMMAGING AND T-ALL: ONCOGENE-CYTOKINE INTERACTIONS AND THEIR ROLE IN LEUKEMOGENESIS

T-cell acute lymphoblastic leukemia (T-ALL) can affect any age group; however, disease outcome is worse in older patients. Aging has been described to be accompanied by an increase in the body's proinflammatory status, a phenomenon referred as inflammaging. By analysing two T-ALL transcriptome datasets, we have found that inflammation pathways are upregulated in older patients, whereas younger patients present upregulation of cell cycle pathways, suggesting that inflammaging is also recapitulated in T-ALL. Here, we hypothesize that T-ALL oncogenes can cooperate with specific cytokines/stimuli and their signaling pathways to create distinct leukemic phenotypes. To test this, human cord blood CD34+ cells were transduced with different T-ALL oncogenes and grown in co-culture with stromal cells under different pro-inflammatory conditions. We observed that most of the transduced cells had their cell growth negatively affected by the proinflammatory conditions, although some oncogene combinations showed more tolerance to these conditions than others. Activated NOTCH1+TLX3 transduced cells represented the only gene combination that showed significantly higher cell growth in the presence of interferon-gamma (IFNγ) when compared to non-transduced cells in the same culture. IL-15 and IFNγ potentiated differentiation arrest observed with certain oncogene combinations. Normal uncommitted T-cell progenitors (CD7+CD5+CD44+CD1a-) responded to IL-15 via STAT5 and AKT phosphorylation, and this response was enhanced by chronic treatment with IL-15. Our results suggest that proinflammatory conditions can alter the effect of certain oncogenes on T-cell progenitors and influence leukemogenesis, potentially leading to distinct biological behaviors of resulting leukemias. These findings may lead to identification of cytokine dependencies associated with particular T-ALL genetic subtypes.

A charged multivesicular body protein (CHMP4B) is required for lens growth and differentiation

Charged multivesicular body protein 4B (CHMP4B) functions as a core component of the endosome sorting complex required for transport-III (ESCRT-III) machinery that facilitates diverse membrane remodeling and scission processes in eukaryotes. Mutations in the human CHMP4B gene underlie rare, inherited forms of early-onset lens opacities or cataract. Here we have characterized the lens phenotypes of mutant (knock-in) mice harboring a human cataract-associated mutation (p.D129V) in CHMP4B (Chmp4b-mutant) and conditional knockdown mice deficient in lens CHMP4B (Chmp4b-CKD). In situ hybridization localized Chmp4b transcripts to lens epithelial cells and elongating fiber cells at the lens equator. Heterozygous Chmp4b-mutant (D/V) mice were viable and fertile with lenses grossly similar to those of wild-type. However, homozygous Chmp4b-mutant (V/V) mice died by embryonic day 15.5 (E15.5) with grossly abnormal eye and brain histology. Chmp4b-CKD mice displayed variable degrees of lens dysmorphology including lens ablation. Immuno-localization of aquaporin-0 (AQP0) revealed lens fiber cell degeneration in homozygous Chmp4b-mutant (V/V) mouse embryos and in embryonic and postnatal Chmp4b-CKD mice. DNA fragmentation (TUNEL) analysis revealed global cell death in homozygous Chmp4b-mutant (V/V) embryos, whereas, cell death was confined to the lens of Chmp4b-CKD mice. Immuno-localization of the monocyte/macrophage marker macrosialin (CD68) suggested that severe lens degeneration in Chmp4b-CKD mice resulted in an ocular immune cell response. Collectively, these mouse data suggest that (1) heterozygous, germ-line mutations in Chmp4b may not manifest as cataract, (2) homozygous, germ-line mutations in Chmp4b are embryonic lethal, and (3) conditional loss of Chmp4b results in arrest of lens growth and differentiation.

Protein malnutrition impairs bone marrow endothelial cells affecting hematopoiesis

Protein malnutrition (PM) affects hematopoiesis leading to bone marrow (BM) hypoplasia and arrests hematopoietic stem cells (HSC) in G0/G1 cell cycle phases, which cause anemia and leukopenia. Hematopoiesis is mainly regulated by BM niches where endothelial cells (EC) present a key regulatory role. Thus, our objective is to evaluate whether PM affects the modulatory capacity of EC on hematopoiesis. C57BL/6 male mice received for 5 weeks a normal protein diet (12% casein) or a low protein diet (2% casein). MSC were isolated and differentiated invitro into EC and the synthesis of SCF, Ang-1, CXCL-12, IL-11, TGF-β and G-CSF were evaluated. The HSC and hematopoietic progenitors were quantified and the EC capacity to modulate the hematopoietic system was also evaluated. Moreover, the ability of PM bone marrow to support hematopoieisis was assessed by proliferation of infused leukemic myelo-monoblasts cells. PM decreases HSC and hematopoietic progenitor pool and promotes cell cycle arrest and a lower proliferation rate of leukemic myelo-monoblasts. PM also committed hematopoietic regulatory characteristics from EC, resulting in the modification of both cell cycle pattern and hematopoietic differentiation. BM microenvironment is compromised in PM, and since PM disturbs EC, it becomes one of the factors responsible for the hematopoietic cell cycle arrest and impairment of HSC differentiation.

Abstract 4647: Keratinocyte specific deletion of TWIST1 promotes differentiation and inhibits UVB-induced hyperproliferation

Abstract Non-melanoma skin cancers (NMSC) have one of the highest incidences in the United States with over 5 million diagnoses every year. The major risk factor for the development of NMSC is solar ultraviolet radiation consisting of both UVA and UVB. In epidermal keratinocytes, UV radiation results in DNA damage as well as deregulation of both proliferative and survival signaling pathways that contribute to tumorigenesis. The transcription factor TWIST1 has been reported to be essential for the formation and invasiveness of chemically-induced tumors in skin. TWIST1 deletion in mouse epidermis results in arrested cell cycle progression in response to treatment with the tumor promoter, TPA, which in turn prevents skin tumor growth in two-stage skin carcinogenesis assays. However, the impact of the keratinocyte specific TWIST1 deletion on skin carcinogenesis caused by UV radiation has not been clarified. In preliminary experiments, we found that primary mouse keratinocytes with TWIST1 knockout (KO) displayed an increase in the G2/M phase population after a single exposure to 20mJ/cm2 of UVB radiation. Accordingly, protein levels of cell cycle regulator p21 increased while those of pMDM2 decreased after UVB exposure. Moreover, we found that primary mouse keratinocytes with TWIST1 KO displayed an increase in apoptosis as shown by augmented Annexin V staining that corresponded with the reduction in levels of anti-apoptotic regulators Bcl-2 and Bcl-XL and concomitant upregulation of cleaved PARP and cleaved caspase-7 after UVB exposure. Furthermore, deletion of TWIST1 in skin keratinocytes in vivo (using K5-Cre x TWIST1flox/flox mice) led to a significant reduction in UVB-induced epidermal hyperproliferation compared to wild-type mice assessed by both Ki67 immunofluorescence staining as well as BrdU incorporation. Additionally, the protein level for p21 was increased while the levels of c-myc, and Cyclin B1 were reduced in the TWIST1 deficient keratinocytes in vivo. Interestingly, expression of differentiation markers such as Loricrin, Keratin-10, Sca-1, and transglutaminase-1 were also increased in the epidermis of TWIST1 deficient mice. These results from the in vivo model show that TWIST1 deletion results in an interruption of UVB-induced hyperproliferation via cell cycle arrest and induction of keratinocyte differentiation. Collectively, our findings indicate that the deficiency of TWIST1 has significant effects on the proliferation, survival and differentiation of keratinocytes; processes important for UVB skin tumor development. Future experiments will continue to assess the changes in oncogenic signaling in epidermal keratinocytes in response to UVB exposure and further investigate the impact of the deletion of TWIST1 on UV-induced skin carcinogenesis. Research supported by CPRIT 2018 Grant RP150247 and by CONACYT Fellowship 671661. Citation Format: Fernando Eguiarte-Solomon, Okkyung Rho, John DiGiovanni. Keratinocyte specific deletion of TWIST1 promotes differentiation and inhibits UVB-induced hyperproliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4647.

An investigation of methylation pattern changes in the IKZF1 promoter in patients with childhood B-cell acute lymphoblastic leukemia.

BackgroundIkaros family zinc finger 1 (IKZF1) is a transcription factor with an important role in controlling hematopoietic proliferation and function, particularly lymphoid cell differentiation. It was previously shown that various mechanisms and expression patterns of Ikaros are linked to a variety of cancers. We hypothesized that aberrant methylation (hypomethylation) of the IKZF1 promoter region might be one of the causes of B-cell acute lymphoblastic leukemia (B-ALL). In B-ALL patients, an increased expression of this gene is a potential cause of B-cell differentiation arrest and proliferation induction. Therefore, as more than 90% of patients with ALL are <15 years old, we investigated the methylation pattern of the IKZF1 promoter in childhood B-ALL.MethodsTwenty-five newly diagnosed B-ALL cases were included (all younger than 15 yr). In addition, we selected 25 healthy age- and sex-matched children as the control group. We collected the blood samples in EDTA-containing tubes and isolated lymphocytes from whole blood using Ficoll 1.077 Lymphosep. Next, we extracted genomic DNA with the phenol/chloroform method. Two microgram of DNA per sample was treated with sodium bisulfite using the EpiTect Bisulfite Kit, followed by an assessment of DNA methylation by polymerase chain reaction (PCR) analysis of the bisulfite-modified genomic DNA.ResultsOur data highlighted a hypomethylated status of the IKZF1 promoter in the ALL cases (96% of the cases were unmethylated). In contrast, the control group samples were partially methylated (68%).ConclusionThis study demonstrated a hypomethylated pattern of the IKZF1 promoter region in childhood B-ALL, which might underlie the aberrant Ikaros expression patterns that were previously linked to this malignancy.

S860 TARGETING ABERRANT DNA METHYLATION AS A NOVEL AND UNIFORM THERAPEUTIC STRATEGY FOR THE TREATMENT OF T‐CELL ACUTE LYMPHOBLASTIC LEUKEMIA AND LYMPHOMA

Background:Genome‐wide DNA methylation profiling studies have previously shown that T‐cell acute lymphoblastic leukemia (T‐ALL) patients can be classified into CpG island methylator phenotype (CIMP) positive and negative tumors. However, as these DNA methylation signatures have not been compared to corresponding profiles in subsets of normal human T cell precursors, it remains unclear if DNA methylation is uniformly perturbed in human T‐ALL. Therefore, and given that molecular basis for these CIMP signatures remains largely unknown, DNA methylation inhibitors have not been extensively evaluated as a potential novel therapeutic strategy for human T‐ALL.Aims:In this study, we aimed to directly compare DNA methylation signatures between human T‐ALLs and corresponding normal human T‐cell precursors. In addition, we evaluated the potential role of Decitabine as a new therapeutic strategy for the treatment of human T‐ALL and T‐LBL.Methods:DNA methylation profiling was performed by Illumina 850k EPIC arrays for human T‐ALL (n = 100) and normal T‐cell subsets (n = 22, 2 donors). T‐ALL/T‐LBL cell lines and patient derived xenografts in NSG mice were used for preclinical evaluation of Decitabine.Results:Our analysis confirmed that human T‐ALLs can be classified in CIMP+ and CIMP‐ T‐ALLs based on DNA methylation signatures, and that this classification is strongly associated with genetic subtypes. Indeed, CIMP+ T‐ALLs are enriched for immature, TLX and HOXA leukemias, whereas CIMP‐ cases mainly belong to the TAL‐LMO rearranged subtype of human T‐ALL. Given that each molecular genetic subtype is linked to a specific T‐cell differentiation arrest, we wondered whether CIMP status would merely be a reflection of T‐cell maturation and/or cell of origin. However, and most notably, integration of these data with DNA methylation signatures obtained from 11 different subsets along the T‐cell differentiation trajectory, revealed that DNA methylation profiles from both CIMP+ and CIMP‐ T‐ALLs are strongly perturbed as compared to all their normal T‐cell counterparts. Indeed, human T‐ALLs uniformly display a DNA hypermethylation phenotype as compared to their normal counterpart and putative cell of origin.Given this, we wondered if targeting aberrant DNA methylation could serve as a uniform therapeutic strategy for both CIMP+ and CIMP‐ human T‐ALLs. To evaluate this, we used a xenograft model of luciferase‐positive LOUCY cells and showed that Decitabine treatment caused a marked reduction in bioluminescence with a concomitant loss of leukemic blasts in peripheral blood and a significant increase in leukemia free survival. Next, we used patient derived xenograft models from 6 different T‐ALL/T‐LBL patients (different genetic subtypes, CIMP+ and CIMP‐ and including one relapse) to show that Decitabine treatment resulted in a significant increase in leukemia free survival for all samples analyzed (log‐rank test, p &lt; 0,05).Finally we used human T‐ALL cell lines to investigate the molecular effects of 48 h Decitabine treatment. As expected Decitabine treatment was associated with a decreased in total DNA 5mC levels and induced up‐regulation and down‐regulation of 1623 and 452 genes, respectively (padj &lt; 0,005, absolute Fc &gt; 1,25). Enrichment analysis revealed that Decitabine most prominently caused downregulation of MYC and E2F gene signatures.Summary/Conclusion:All together, our data suggest that human T‐ALL samples are uniformly characterized by aberrant DNA methylation signatures and that Decitabine might serve as a promising and uniform novel therapeutic strategy for all genetic subtypes of human T‐ALL and T‐LBL.

A Recurrent Activating Missense Mutation in Waldenström Macroglobulinemia Affects the DNA Binding of the ETS Transcription Factor SPI1 and Enhances Proliferation.

The ETS-domain transcription factors divide into subfamilies based on protein similarities, DNA-binding sequences, and interaction with cofactors. They are regulated by extracellular clues and contribute to cellular processes, including proliferation and transformation. ETS genes are targeted through genomic rearrangements in oncogenesis. The PU.1/SPI1 gene is inactivated by point mutations in human myeloid malignancies. We identified a recurrent somatic mutation (Q226E) in PU.1/SPI1 in Waldenström macroglobulinemia, a B-cell lymphoproliferative disorder. It affects the DNA-binding affinity of the protein and allows the mutant protein to more frequently bind and activate promoter regions with respect to wild-type protein. Mutant SPI1 binding at promoters activates gene sets typically promoted by other ETS factors, resulting in enhanced proliferation and decreased terminal B-cell differentiation in model cell lines and primary samples. In summary, we describe oncogenic subversion of transcription factor function through subtle alteration of DNA binding leading to cellular proliferation and differentiation arrest. SIGNIFICANCE: The demonstration that a somatic point mutation tips the balance of genome-binding pattern provides a mechanistic paradigm for how missense mutations in transcription factor genes may be oncogenic in human tumors.This article is highlighted in the In This Issue feature, p. 681.

S146 DECIPHERING THE ROLE OF RUNX1 ISOFORMS IN THE DEVELOPMENT OF TRANSIENT ABNORMAL MYELOPOIESIS

Background: Transient abnormal myelopoiesis (TAM) arises from fetal hematopoietic cells in 5–30% of neonates with Down syndrome (DS) and is characterized by the accumulation of immature megakaryoblasts with the triad of fetal origin, GATA1 s mutation and trisomy 21 being necessary and sufficient for its induction. Yet, molecular mechanisms underlying this cooperation are incompletely understood. Aims: We aimed to identify the oncogenic factors on human chromosome 21 (Hsa21) and unravel their molecular synergy with GATA1 s during the development of TAM/ML-DS. Methods: To study the mechanisms underlying the cooperativity between GATA1 s and trisomy 21 in TAM/ML-DS pathogenesis, we performed a CRISPR-Cas9 screening targeting the 218 currently annotated coding genes on Hsa21 with 1090 sgRNAs in both a ML-DS and control cell line. The top ML-DS-specific candidates were functionally and molecularly validated in vitro and in vivo. Results: Comprehensive CRISPR-Cas9 loss-of-function screening of the Hsa21 coding genes (n = 218) revealed a strong and specific RUNX1 dependency indicated by the depletion of the ML-DS cell line CMK with minor effect on non-DS leukemia cells (K562). RNA-sequencing followed by isoform-specific qRT-PCR validation in leukemic blasts from ML-DS patients demonstrated deregulation of the RUNX1 isoform equilibrium compared to other types of leukemia or hematopoietic stem and progenitor cells (HSPCs) from healthy donors. In an in vitro set up using Gata1s-mutated, pre-leukemic murine fetal HSPCs, we observed that ectopic deregulation of RUNX1 isoforms by overexpression synergized with Gata1 s, resulting in enhanced proliferation and accumulation of immature megakaryocytic progenitors. Accordingly, ectopic expression of the ML-DS-dominant RUNX1 isoform in human CD34+ HSPCs led to a loss of mature megakaryocytes and increased monocytic differentiation. Inversely, shifting the expression towards the main hematopoietic RUNX1 isoform enhanced megakaryocytic differentiation and impaired proliferation of HSPCs. The same was observed in ML-DS patient blasts. Restoring the expression of the main hematopoietic RUNX1 isoform induced differentiation and cell cycle arrest of ML-DS blasts, while further elevating the expression of the ML-DS-dominant RUNX1 isoform enhanced proliferation and accumulation of a megakaryocytic CD41+CD117+ population. Importantly, overexpression of the RUNX1 isoform in Gata1 s fetal HSPCs was sufficient to induce a leukemic phenotype upon transplantation into syngeneic C57BL/6J recipients after a short latency of 40 days and high penetrance (100%). The leukemic cells engrafted in secondary recipients and displayed a megakaryocytic progenitor-like phenotype (CD41+CD117+CD34−CD16/32low). Moreover, global gene expression profiling by RNA-sequencing confirmed a ML-DS-like RNA expression profile of the murine leukemias. Using ChIP-sequencing of tagged versions of GATA1 and GATA1 s, we could determine differential chromatin occupancy at the RUNX1 locus implicating a feed-forward loop driving the expression of specific RUNX1 isoforms in GATA1s-mutated cells. Similarly, pulldown assays of tagged GATA1/GATA1 s showed isoform specific interactions with RUNX1. Summary/Conclusion: Our Hsa21-wide CRIPSR-Cas9 screening in combination with functional validation in vitro and in vivo places a specific RUNX1 isoform in the center of an interaction network with mutated GATA1 s during the transformation of fetal HSPCs in the background of trisomy 21. Our data highlight the importance of alternative splicing in leukemias and will guide the development of truly specific and targeted therapies.

PS1014 GLYCOLYSIS VERSUS OXIDATIVE PHOSPHORYLATION – POTENTIAL THERAPEUTIC TARGETS IN AML

Background:Cancer cells alter their metabolism to sustain high levels of growth and proliferation. One of the most common alterations is the metabolic shift of cancer cells from oxidative phosphorylation (OXPHOS) to glycolysis, regardless of oxygen presence (aerobic glycolysis), resulting in lower ATP production. This alteration is associated with up‐regulation of glucose transporters (GLUTs) and glycolytic enzymes, such as hexokinases, and enhanced lactate fermentation. This shift is mainly regulated by HIF‐1, p53 and MYC. Hence, metabolism constitute a new target in acute myeloid leukemia (AML), where mutations in metabolism [isocitrate dehydrogenase 1/2 (IDH1/2)] were already describe. AML is a heterogeneous hematologic neoplasia characterized by myeloid differentiation arrest and uncontrolled proliferation.Aims:This study aimed to metabolically characterize AML cell lines and assess the potential of glycolysis and OXPHOS metabolism as a therapeutic target in AML using in vitro models.Methods:Six AML cell lines of different subtypes and with different genetic backgrounds were used (HEL, HL‐60, K‐562, KG‐1, NB‐4 and THP‐1). The presence of IDH1/2 exon 4 mutations was assessed using Sanger sequencing. Intracellular peroxides, superoxide anion and mitochondrial membrane potential levels were measured by fluorimetry using DCFH2‐DA, DHE and JC‐1 probes, respectively. Cells were incubated in presence or absence of 2‐deoxy‐D‐glucose (2‐DG; glycolysis inhibitor) or oligomycin complex (OXPHOS inhibitor). Metabolic activity was assessed by resazurin assay and cell death by flow cytometry (FC) using Annexin V and 7‐AAD double staining. Cell cycle was also assessed by FC, using propidium iodide/RNase. Glucose intake was measured by 18F‐FDG uptake and expression levels of 8 SLC2A, 2 hexokinases and HIF‐1α were assessed using RT‐PCR.Results:NB‐4 cell line has the highest 18F‐FDG uptake, whereas HL‐60 and THP‐1 cells showed the lowest. HEL, K‐562 and KG‐1 had similar 18F‐FDG uptake. THP‐1 cells expressed the 8 tested SLC2A genes. NB‐4 cell line also had the highest peroxide and superoxide anion levels. K‐562 and KG‐1 cells had similar ROS levels and IDH1 genotype (single nucleotide variant rs11554137 in heterozygosity). Glycolysis inhibition reduced metabolic activity in a dose‐ and cell line dependent manner and HEL and KG‐1 were the most sensitive cell lines to 2‐DG. OXPHOS inhibition led to a decrease in metabolic activity in a cell line‐dependent manner. K‐562 cells were the most sensitive to this inhibition and THP‐1 cells were the most resistant to both compounds (2‐DG and oligomycim). These inhibitors induce cell death by apoptosis and cell cycle arrest in G0/G1 phase. 2‐DG induced a more evident cytotoxic effect on K‐562 and NB‐4 and a cytostatic effect on KG‐1 and THP‐1 cells. Conversely, oligomycin had a cytotoxic effect on K‐562 and KG‐1 cells and a cytostatic effect on NB‐4, THP‐1 and K 562. Moreover, 2‐DG decreased 18F‐FDG uptake, mainly in KG‐1 and NB‐4 cell lines, inducing up‐regulation of SLC2A11 and down‐regulation of HK1 gene expression. Lastly, oligomycin increased 18F‐FDG uptake, particularly in K‐562 cells, with up‐regulation of SLC2A1 and HK2 gene expression.Summary/Conclusion:AML cell lines have different metabolic preferences, with HEL, KG‐1 and NB‐4 cells appearing to be more glycolytic, whereas K‐562 cells more dependent on OXPHOS. Contrary, THP‐1 cells seems to be OXPHOS independent. These cell lines seem to reprogram their metabolism in order to overcome OXPHOS inhibition, suggesting that glycolysis could be a better therapeutic target in AML.

The Profile of Immunophenotype and Genotype Aberrations in Subsets of Pediatric T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is a biologically heterogeneous malignancy, which reflects distinctive stages of T-cell differentiation arrest. We have revisited a cohort of pediatric T-ALL, in order to test if immunophenotypes associated with molecular alterations would predict the patient's outcome. Genetic mutations, translocations and copy number alterations were identified through Sanger sequencing, RT-PCR, FISH and multiplex ligation-dependent probe amplification (MLPA). We defined 8 immunophenotypic T-ALL subtypes through multiparametric flow cytometry: early T-cell precursor (ETP, n = 27), immature (n = 38), early cortical (n = 15), cortical (n = 50), late cortical (n = 53), CD4/CD8 double negative mature (n = 31), double positive mature (n = 35) and simple positive mature (n = 31) T-ALL. Deletions (del) or amplifications (amp) in at least one gene were observed in 87% of cases. The most frequent gene alterations were CDKN2A/Bdel (71.4%), NOTCH1mut (47.6%) and FBXW7mut (17%). ETP-ALL had frequent FLT3mut (22.2%) and SUZ12del (16.7%) (p < 0.001), while CDKN2A/Bdel were rarely found in this subtype (p < 0.001). The early cortical T-ALL subtype had high frequencies of NOTCH1mut and IL7Rmut (71%, 28.6%, respectively), whereas, mature T-ALL with double positive CD4/CD8 had the highest frequencies of STIL-TAL1 (36.7%), LEF1del (27.3%) and CASP8AP2del (22.7%). The co-existence of two groups of T-ALL with NOTCH1mut/IL7Rmut, and with TLX3/SUZ12del/NF1del/IL7Rmut, were characterized with statistical significance (p < 0.05) but only STIL-TAL1 (pOS 47.5%) and NOTCH1WT/FBXW7WT (pOS 55.3%) are predictors of poor T-ALL outcomes. In conclusion, we have observed that 8 T-ALL subgroups are characterized by distinct molecular profiles. The mutations in NOTCH1/FBXW7 and STIL-TAL1 rearrangement had a prognostic impact, independent of immunophenotype.

PARI (PARPBP) suppresses replication stress-induced myeloid differentiation in leukemia cells.

Hyperproliferative cancer cells face increased replication stress, which can result in accumulation of DNA damage. As DNA damage can arrest proliferation, and, in the case of myeloid leukemia, induce differentiation of cancer cells, understanding the mechanisms that regulate the replication stress response is paramount. Here, we show that PARI, a replisome protein involved in regulating DNA repair and replication stress, suppresses differentiation of myeloid leukemia cells. We show that PARI is overexpressed in myeloid leukemia cells, and its knockdown reduces leukemia cell proliferation in vitro and in vivo in xenograft mouse models. PARI depletion enhances replication stress and DNA-damage accumulation, coupled with increased myeloid differentiation. Mechanistically, we show that PARI inhibits activation of the NF-κB pathway, which can initiate p21-mediated differentiation and proliferation arrest. Finally, we show that PARI expression negatively correlates with expression of differentiation markers in clinical myeloid leukemia samples, suggesting that targeting PARI may restore differentiation ability of leukemia cells and antagonize their proliferation.

The novel dihydroorotate dehydrogenase (DHODH) inhibitor BAY 2402234 triggers differentiation and is effective in the treatment of myeloid malignancies

Acute myeloid leukemia (AML) is a devastating disease, with the majority of patients dying within a year of diagnosis. For patients with relapsed/refractory AML, the prognosis is particularly poor with currently available treatments. Although genetically heterogeneous, AML subtypes share a common differentiation arrest at hematopoietic progenitor stages. Overcoming this differentiation arrest has the potential to improve the long-term survival of patients, as is the case in acute promyelocytic leukemia (APL), which is characterized by a chromosomal translocation involving the retinoic acid receptor alpha gene. Treatment of APL with all-trans retinoic acid (ATRA) induces terminal differentiation and apoptosis of leukemic promyelocytes, resulting in cure rates of over 80%. Unfortunately, similarly efficacious differentiation therapies have, to date, been lacking outside of APL. Inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in the de novo pyrimidine synthesis pathway, was recently reported to induce differentiation of diverse AML subtypes. In this report we describe the discovery and characterization of BAY 2402234 - a novel, potent, selective and orally bioavailable DHODH inhibitor that shows monotherapy efficacy and differentiation induction across multiple AML subtypes. Herein, we present the preclinical data that led to initiation of a phase I evaluation of this inhibitor in myeloid malignancies.

Arsenic trioxide blocked proliferation and cardiomyocyte differentiation of human induced pluripotent stem cells: Implication in cardiac developmental toxicity

Arsenic trioxide (ATO) has been recommended as the first-line agent for the treatment of acute promyelocytic leukaemia (APL), due to its substantial anticancer effect. Numerous clinical reports have indicated that ATO is a developmental toxicant which can result in birth defects of human beings. But whether arsenic trioxide can lead to human cardiac developmental toxicity remains largely unknown. So the present study aims to explore the influence and mechanisms of ATO on human cardiac development by using a vitro cardiac differentiation model of human induced pluripotent stem cells (hiPSCs). Here we found that clinically achievable concentrations (0.1, 0.5 and 1 μM) of ATO resulted in a significant inhibition of proliferation during the whole process of cardiac differentiation of hiPSCs. Meanwhile, TUNEL assay revealed that ATO could cause cell apoptosis during cardiac differentiation in a concentration-dependent manner. Consistently, we found that ATO reduced the expressions of mesoderm markers Brachyury and EOMES, cardiac progenitor cell markers GATA-4, MESP-1 and TBX-5, and cardiac specific marker α-actinin in differentiated hiPSCs. Furthermore, ATO treatment had caused DNA damage which was shown in the upregulation of γH2AX, a sensitive marker for DNA double-strand breaks. Taken together, ATO blocked cardiomyocyte differentiation, induced apoptosis and cell growth arrest during cardiac differentiation of hiPSCs, which might be associated with DNA damage.

P53, stem cell biology and childhood blastomas

Childhood blastomas, unlike adult cancers, originate from developing organs in which molecular and cellular features exhibit differentiation arrest and embryonic characteristics. Conventional cancer therapies, which rely on the generalized cytotoxic effect on rapidly dividing cells, may damage delicate organs in young children, leading to multiple late effects. Deep understanding of the biology of embryonal cancers is crucial in reshaping the cancer treatment paradigm for children. p53 plays a major physiological role in embryonic development, by controlling cell proliferation, differentiation and responses to cellular stress. Tumor suppressor function of p53 is commonly lost in adult cancers through genetic alterations. However, both somatic and germline p53 mutations are rare in childhood blastomas, suggesting that in these cancers, p53 may be inactivated through other mechanisms than mutation. In this review, we summarize current knowledge about p53 pathway inactivation in childhood blastomas (specifically neuroblastoma, retinoblastoma and Wilms' tumor) through various upstream mechanisms. Laboratory evidence and clinical trials of targeted therapies specific to exploiting p53 upstream regulators are discussed. Despite the low rate of inherent TP53 mutations, p53 pathway inactivation is a common denominator in childhood blastomas. Exploiting p53 and its regulators is likely to translate into more effective targeted therapies with minimal late effects for children. (see Video Abstract, Supplemental Digital Content 1, http://links.lww.com/COON/A23).

MiR-183-5p Inhibits Occurrence and Progression of Acute Myeloid Leukemia via Targeting Erbin.

Erbin has been shown to have significant effects on the development of solid tumors. However, little is known about its function and regulatory mechanism in hematological malignancies. The biological function of Erbin on cell proliferation was measured in vitro and in vivo. The predicted target of Erbin was validated by dual-luciferase reporter assay and rescue experiment. We found that overexpression of Erbin could inhibit the cell proliferation and promote the cell differentiation of acute myeloid leukemia (AML) cells, whereas depletion of Erbin could enhance the cell proliferation and block the cell differentiation in AML cells in vitro and in vivo. Besides, miR-183-5p was identified as the upstream regulator that negatively regulated the Erbin expression. The results were confirmed by dual-luciferase reporter and RNA pull-down assay. Furthermore, we found that miR-183-5p negatively regulated Erbin, resulting in enhanced cell proliferation of AML cells via activation of RAS/RAF/MEK/ERK and PI3K/AKT/FoxO3a pathways. The activation of RAS/RAF/MEK/ERK and PI3K/AKT/FoxO3a pathways was mediated by Erbin interacting with Grb2. These results were also validated by rescue experiments in vitro and in vivo. All above-mentioned findings indicated that the miR-183-5p/Erbin signaling pathway might represent a novel prognostic biomarker or therapeutic target for treatment of AML.

Precursor B-cell development in bone marrow of Good syndrome patients

Good syndrome is an immunodeficiency presenting with thymoma, hypogammaglobulinemia and almost absent B cells. To investigate the origin of the B-cell lymphopenia in these patients, we studied B cell differentiation in the bone marrow of Good syndrome patients. We found very low numbers of precursor B cells in bone marrow of Good syndrome patients and a differentiation arrest after the pro-B-cell stage; this is different from other agammaglobulinemia patients with a defect in pre B-cell receptor signaling.

Pan-HDAC Inhibitors Restore PRDM1 Response to IL21 in CREBBP-Mutated Follicular Lymphoma.

Follicular lymphoma arises from a germinal center B-cell proliferation supported by a bidirectional crosstalk with tumor microenvironment, in particular with follicular helper T cells (Tfh). We explored the relation that exists between the differentiation arrest of follicular lymphoma cells and loss-of-function of CREBBP acetyltransferase.Experimental Design: The study used human primary cells obtained from either follicular lymphoma tumors characterized for somatic mutations, or inflamed tonsils for normal germinal center B cells. Transcriptome and functional analyses were done to decipher the B- and T-cell crosstalk. Responses were assessed by flow cytometry and molecular biology including ChIP-qPCR approaches. Conversely to normal B cells, follicular lymphoma cells are unable to upregulate the transcription repressor, PRDM1, required for plasma cell differentiation. This defect occurs although the follicular lymphoma microenvironment is enriched in the potent inducer of PRDM1 and IL21, highly produced by Tfhs. In follicular lymphoma carrying CREBBP loss-of-function mutations, we found a lack of IL21-mediated PRDM1 response associated with an abnormal increased enrichment of the BCL6 protein repressor in PRDM1 gene. Moreover, in these follicular lymphoma cells, pan-HDAC inhibitor, vorinostat, restored their PRDM1 response to IL21 by lowering BCL6 bound to PRDM1. This finding was reinforced by our exploration of patients with follicular lymphoma treated with another pan-HDAC inhibitor. Patients showed an increase of plasma cell identity genes, mainly PRDM1 and XBP1, which underline the progression of follicular lymphoma B cells in the differentiation process. Our data uncover a new mechanism by which pan-HDAC inhibitors may act positively to treat patients with follicular lymphoma through the induction of the expression of plasma cell genes.

The signaling axis atypical protein kinase C \u03bb/\u03b9-Satb2 mediates leukemic transformation of B-cell progenitors

Epigenetically regulated transcriptional plasticity has been proposed as a mechanism of differentiation arrest and resistance to therapy. BCR-ABL leukemias result from leukemic stem cell/progenitor transformation and represent an opportunity to identify epigenetic progress contributing to lineage leukemogenesis. Primary human and murine BCR-ABL+ leukemic progenitors have increased activation of Cdc42 and the downstream atypical protein kinase C (aPKC). While the isoform aPKCζ behaves as a leukemic suppressor, aPKCλ/ι is critically required for oncogenic progenitor proliferation, survival, and B-cell differentiation arrest, but not for normal B-cell lineage differentiation. In vitro and in vivo B-cell transformation by BCR-ABL requires the downregulation of key genes in the B-cell differentiation program through an aPKC λ/ι-Erk dependent Etv5/Satb2 chromatin repressive signaling complex. Genetic or pharmacological targeting of aPKC impairs human oncogenic addicted leukemias. Therefore, the aPKCλ/ι-SATB2 signaling cascade is required for leukemic BCR-ABL+ B-cell progenitor transformation and is amenable to non-tyrosine kinase inhibition.

Icariside II induces cell cycle arrest and differentiation via TLR8/MyD88/p38 pathway in acute myeloid leukemia cells

Acute myeloid leukemia (AML) is a devastating hematological malignancy, characterized by differentiation arrest and unscheduled proliferation of immature cells of the myeloid lineage. Inducing AML cell differentiation has emerged as a promising therapeutic strategy for the therapy of AML. Icariside II, an active component of Herba Epimedii, has been well defined to promote osteogenic differentiation. However, the differentiation-inducing effect of Icariside II on AML cells has not been explored. In this study, we investigated the differentiation-inducing effect and underlying mechanism of Icariside II in AML HL-60 and THP-1 cell lines. Icariside II induced G1 phase cell cycle arrest by down-regulating Cyclin-dependent kinases (CDK2, CDK4 and CDK6) and up-regulating Cyclin-dependent kinase inhibitor (p21 and p27). Importantly, Icariside II could induce differentiation of AML cells, accompanied by the up-regulation of Toll-like receptor 8 (TLR8), myeloid differentiation factor 88 (MyD88) and phosphorylated p38. Further study indicated the cell cycle arrest and differentiation induced by Icariside II could be abrogated by TLR8-specific inhibitor CU-CPT9a. Collectively, these findings firstly demonstrate Icariside II induces cell cycle arrest and differentiation of AML cells via activation of TLR8/MyD88/p38 pathway, suggesting Icariside II could be developed into a novel differentiation-inducing agent for AML.