Hypoxia increases the biogenesis of IGF2BP3-bound circular RNAs.

BACKGROUND: Insulin-like Growth Factor 2 Binding Protein 3 (IGF2BP3) promotes cancer migration and invasion by binding to several coding and non-coding RNAs. Hypoxia stimulates tumor progression by upregulating Hypoxia Inducible Factors and downstream signaling. Quaking (QKI) gene, which is upregulated in hypoxia and promotes epithelial to mesenchymal transition (EMT), induces circular RNAs. Therefore, the axis between IGF2BP3, QKI, circular RNAs and their respective host genes under hypoxia was studied. METHODS AND RESULTS: Several IGF2BP3-bound circular RNAs were previously identified in HepG2. There were 13 circRNAs originating from 8 host genes bound to IGF2BP3. We confirmed their binding to IGF2BP3 in U87MG using an RNA Immunoprecipitation assay. MALAT1, an oncogenic lncRNA was also found to be associated with IGF2BP3. Three adherent cell lines expressing high levels of IGF2BP3 viz., HeLa, HepG2 and U87MG were cultured under normoxia (20%O2) and hypoxia (<0.2%O2) for 48-168 h. Expression of IGF2BP3, QKI, EMT markers, IGF2BP3-bound circRNAs and their host mRNAs expression were assessed by quantitative real-time PCR (qRT-PCR) in both normoxia and hypoxia. The hypoxia markers viz., VEGF and CA9 were upregulated in all the cell lines in hypoxia at all time points along with an increase in SNAIL. We found 6 genes, viz., PHC3, CDYL, ANKRD17, ARID1A, NEIL3 and FNDC3B with increased expression both at the mRNA and circRNA level indicating their synergistic role in tumor initiation. Overall, we found that circRNA to mRNA expression was observed to be increased for most of the genes and time points of hypoxia in all the cell lines. IGF2BP3 and QKI were also upregulated in hypoxia indicating their role in circRNA biogenesis and stability. CONCLUSION: Our data implies that hypoxia augments circRNA biogenesis which might subsequently play a role in tumor progression.

Anti-factor B antibodies in atypical hemolytic uremic syndrome.

BACKGROUND: The etiology of atypical hemolytic uremic syndrome (aHUS) is unknown in 30-40% of patients. Anti-factor B (FB) antibodies are reported in C3 glomerulopathy (C3G) and immune-complex membranoproliferative glomerulonephritis (IC-MPGN), though not in aHUS. METHODS: We screened patients < 18-year-old from cohorts of aHUS and C3G/idiopathic IC-MPGN. Anti-FB IgG antibodies were measured by ELISA and confirmed by Western blot. Normative levels were based on antibody levels in 103 healthy blood donors. RESULTS: Prevalence of anti-FB antibodies was 9.7% (95% CI 6.1-14.5%; n = 21) in 216 patients with aHUS, including 11.5% (95% CI 6.4-18.5%; n = 14) in anti-FH associated aHUS and 11.8% (95% CI 4.4-23.9%; n = 6) in patients without a definitive genetic or autoimmune etiology. Patients with significant genetic variants did not show anti-FB antibodies. In patients with concomitant anti-FB and anti-FH antibodies, median anti-FH titers were higher (11,312 AU/mL vs. 4920 AU/mL; P = 0.04). Anti-FB antibody titer correlated with disease severity (hemoglobin and platelets; P < 0.05), declined following plasma exchange and increased during relapse. While 4/64 patients with C3G (6.3%) and 1/17 with IC-MPGN showed anti-FB antibodies, titers were higher in aHUS (544.8 AU/mL vs. 1028.8 AU/mL; P = 0.003). CONCLUSION: Anti-FB antibodies are present in 6-10% of patients with aHUS and C3G/IC-MPGN, with higher titers in the former. The diagnostic and therapeutic implication of anti-FB antibodies in aHUS needs confirmation and further studies. The study shows propensity for autoantibody generation and co-existence of multiple risk factors for aHUS in Indian children.

Chronic hyperglycemia drives alterations in macrophage effector function in pulmonary tuberculosis.

Diabetes mellitus (DM) alters immune responses and given the rising prevalence of DM in tuberculosis (TB) endemic countries; hyperglycemia can be a potential risk factor for active TB development. However, the impact of hyperglycemia on TB-specific innate immune response in terms of macrophage functions remains poorly addressed. We assessed macrophage effector functions in uncontrolled DM patients with or without TB infection (PTB+DM and DM), non-diabetic TB patients (PTB), and non-diabetic-uninfected controls. Phagocytic capacity against BCG and surface expression of different pattern recognition receptors (PRRs) (CD11b, CD14, CD206, MARCO, and TLR-2) were measured via flow cytometry. Effector molecules (ROS and NO) required for bacterial killing were assessed via DCFDA and Griess reaction respectively. A systematic dysregulation in phagocytic capacity with concurrent alterations in the expression pattern of key PRRs (CD11b, MARCO, and CD206) was observed in PTB+DM. These altered PRR expressions were associated with decreased phagocytic capacity of macrophages. Similarly, ROS was aberrantly higher while NO was lower in PTB+DM. These altered macrophage functions were positively correlated with increasing disease severity. Our results highlight several key patterns of immune dysregulation against TB infection under hyperglycemic conditions and highlight a negative impact of hyperglycemia with etiology and progression of TB.

Cross-neutralization of SARS-CoV-2 by HIV-1 specific broadly neutralizing antibodies and polyclonal plasma.

Cross-reactive epitopes (CREs) are similar epitopes on viruses that are recognized or neutralized by same antibodies. The S protein of SARS-CoV-2, similar to type I fusion proteins of viruses such as HIV-1 envelope (Env) and influenza hemagglutinin, is heavily glycosylated. Viral Env glycans, though host derived, are distinctly processed and thereby recognized or accommodated during antibody responses. In recent years, highly potent and/or broadly neutralizing human monoclonal antibodies (bnAbs) that are generated in chronic HIV-1 infections have been defined. These bnAbs exhibit atypical features such as extensive somatic hypermutations, long complementary determining region (CDR) lengths, tyrosine sulfation and presence of insertions/deletions, enabling them to effectively neutralize diverse HIV-1 viruses despite extensive variations within the core epitopes they recognize. As some of the HIV-1 bnAbs have evolved to recognize the dense viral glycans and cross-reactive epitopes (CREs), we assessed if these bnAbs cross-react with SARS-CoV-2. Several HIV-1 bnAbs showed cross-reactivity with SARS-CoV-2 while one HIV-1 CD4 binding site bnAb, N6, neutralized SARS-CoV-2. Furthermore, neutralizing plasma antibodies of chronically HIV-1 infected children showed cross neutralizing activity against SARS-CoV-2 pseudoviruses. Collectively, our observations suggest that human monoclonal antibodies tolerating extensive epitope variability can be leveraged to neutralize pathogens with related antigenic profile.

Mosaic variegated aneuploidy syndrome 2 with biallelic novel CEP57 splice site variation in Indian siblings: Expanding the clinical and molecular spectrum.

Mosaic variegated aneuploidy syndrome 2 (MVA2) (MIM# 614114) is a rare autosomal recessive condition caused by biallelic loss of function variants in the CEP57 gene. MVA2 is characterized by a variable phenotype ranging from poor growth to facial dysmorphism, short stature and congenital heart defects. Only 11 families and 5 pathogenic variants of MVA2 have been described so far. Intragenic duplication of 11 nucleotides (c.915_925dup11) in homozygous or compound heterozygous state is the commonest genetic aberration (10/13). We describe the first Indian family with two siblings with a novel homozygous splice site variant (c.382+2T>C) in CEP57. Molecular characterization demonstrated skipping of exon 3 due to the variant with protein modeling predicting subsequent complete loss of function. This is the first report of a splice site variation in CEP57 leading to MVA2.

BAALC gene expression tells a serious patient outcome tale in NPM1-wild type/FLT3-ITD negative cytogenetically normal-acute myeloid leukemia in adults.

Acute myeloid leukemia with normal cytogenetics (CN-AML) is the largest group of AML patients which is associated with a variegated patient outcome. Multiple molecular markers have been used to risk-stratify these patients. Estimation of expression of BAALC gene (Brain and Acute Leukemia, Cytoplasmic) mRNA level is one of the predictive markers which has been identified in multiple studies. In this study, we examined the clinical and prognostic value of BAALC gene expression in 149 adult CN-AML patients. We also utilized multi-omics databases to ascertain the association of BAALC gene expression with comprehensive molecular and clinicopathologic features in AML. BAALC overexpression was associated with CD34 positivity on leukemic blasts (p = 0.0026) and the absence of NPM1 gene mutation (p < 0.0001), presence of RUNX1 gene mutation (p < 0.001) and poor patient outcomes, particularly in NPM1-wild type/FLT3-ITD negative adult CN-AML patients. Additionally, BAALC expression was associated with the alteration of methylation of its promoter. Further, pathway analysis revealed that BAALC expression is correlated with MYC targets and Ras signalling. We conclude that high BAALC expression associates with poor patient outcome in NPM1-wild type/FLT3-ITD negative adult CN-AML patients.

Prognostic utility of key copy number alterations in T cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease, characterized by an abnormal transformation of T cells into highly proliferative leukemic lymphoblasts. Identification of common genetic alterations has provided promising opportunities for better risk stratification in T-ALL. Current treatment in T-ALL still poses the major challenge of integrating the knowledge of molecular alterations in the clinical setting. We utilized the Multiplex Ligation Dependent Probe Amplification (MLPA) method to determine the frequency of common copy number alterations (CNAs) in 128 newly diagnosed T-ALL patients. We also studied the association of these CNAs with patient's clinical characteristics and survival. The highest frequency of deletion was observed in CDKN2A (59.38%), followed by CDKN2B (46.88%), LMO1 (37.5%), and MTAP (28.12%). PTPN2 (22.66%), PHF6 (14.06%), and MYB (14.06%) had the highest number of duplication events. A total of 89.06% patients exhibited CNAs. STIL::TAL1, NUP214::ABL1, and LMO2::RAG2 fusions were observed in 5.47%, 3.12%, and 0.78% of patients, respectively. CDKN2A, CDKN2B, and PTPN2 gene deletions were mainly observed in pediatric patients, while CNAs of NF1 and SUZ12 were observed more frequently in adults. In pediatric patients, alterations in CDKN2B, CASP8AP2, and AHI1 were associated with poor prognosis, while SUZ12 and NF1 CNAs were associated with favorable prognosis. In adult patients, ABL1 CNA emerged as an independent indicator of poor prognosis. The observed molecular heterogeneity in T-ALL may provide the basis for variations observed in clinical response in T-ALL and MLPA based CNA detection may help in risk stratification of these patients.

Biogenesis of intronic miRNAs located in clusters by independent transcription and alternative splicing.

miRNAs are generally classified as "intergenic" or "intronic" based upon their genomic location. Intergenic miRNAs are known to be transcribed as independent transcription units, while intronic miRNAs are believed to be processed from the introns of their hosting transcription units and hence share common regulatory mechanisms and expression patterns with its host gene. Recent reports in the literature suggest that some intronic miRNAs, which do not show concordance in expression with their respective host genes, might be transcribed and regulated as independent transcription units. However, there is no direct evidence for the existence of independently transcribed intronic miRNA in humans to date. We have characterized the full-length primary transcripts (pri-miRNAs) of three human intronic miRNAs-miR 106b, miR 93, and miR 24-1-by RNA ligase-mediated RACE and show that human intronic miRNA can indeed be transcribed as independent transcription units. Also, clustered miRNAs are generally believed to arise from a common primary transcript and are expected to have similar expression profiles. However, we have identified several novel alternatively spliced transcripts by RT-PCR, each of which harbors a single pre-miRNA from a cluster of closely located intronic miRNAs. We show that these transcripts represent unique pri-miRNAs for each of these clustered miRNAs. We also report the identification of conserved splice acceptor signals which are responsible for maturation of these novel splice variants. Our results suggest that alternative splicing might play a role in uncoupling the expression of clustered miRNAs from each other, which otherwise are generally believed to be co-transcribed and co-expressed.

BALR-6 regulates cell growth and cell survival in B-lymphoblastic leukemia.

BACKGROUND: A new class of non-coding RNAs, known as long non-coding RNAs (lncRNAs), has been recently described. These lncRNAs are implicated to play pivotal roles in various molecular processes, including development and oncogenesis. Gene expression profiling of human B-ALL samples showed differential lncRNA expression in samples with particular cytogenetic abnormalities. One of the most promising lncRNAs identified, designated B-ALL associated long RNA-6 (BALR-6), had the highest expression in patient samples carrying the MLL rearrangement, and is the focus of this study. RESULTS: Here, we performed a series of experiments to define the function of BALR-6, including several novel splice forms that we identified. Functionally, siRNA-mediated knockdown of BALR-6 in human B-ALL cell lines caused reduced cell proliferation and increased cell death. Conversely, overexpression of BALR-6 isoforms in both human and mouse cell lines caused increased proliferation and decreased apoptosis. Overexpression of BALR-6 in murine bone marrow transplantation experiments caused a significant increase in early hematopoietic progenitor populations, suggesting that its dysregulation may cause developmental changes. Notably, the knockdown of BALR-6 resulted in global dysregulation of gene expression. The gene set was enriched for leukemia-associated genes, as well as for the transcriptome regulated by Specificity Protein 1 (SP1). We confirmed changes in the expression of SP1, as well as its known interactor and downstream target CREB1. Luciferase reporter assays demonstrated an enhancement of SP1-mediated transcription in the presence of BALR-6. These data provide a putative mechanism for regulation by BALR-6 in B-ALL. CONCLUSIONS: Our findings support a role for the novel lncRNA BALR-6 in promoting cell survival in B-ALL. Furthermore, this lncRNA influences gene expression in B-ALL in a manner consistent with a function in transcriptional regulation. Specifically, our findings suggest that BALR-6 expression regulates the transcriptome downstream of SP1, and that this may underlie the function of BALR-6 in B-ALL.

Inflammation as a driver of hematological malignancies.

Hematopoiesis is a tightly regulated process that produces all adult blood cells and immune cells from multipotent hematopoietic stem cells (HSCs). HSCs usually remain quiescent, and in the presence of external stimuli like infection or inflammation, they undergo division and differentiation as a compensatory mechanism. Normal hematopoiesis is impacted by systemic inflammation, which causes HSCs to transition from quiescence to emergency myelopoiesis. At the molecular level, inflammatory cytokine signaling molecules such as tumor necrosis factor (TNF), interferons, interleukins, and toll-like receptors can all cause HSCs to multiply directly. These cytokines actively encourage HSC activation, proliferation, and differentiation during inflammation, which results in the generation and activation of immune cells required to combat acute injury. The bone marrow niche provides numerous soluble and stromal cell signals, which are essential for maintaining normal homeostasis and output of the bone marrow cells. Inflammatory signals also impact this bone marrow microenvironment called the HSC niche to regulate the inflammatory-induced hematopoiesis. Continuous pro-inflammatory cytokine and chemokine activation can have detrimental effects on the hematopoietic system, which can lead to cancer development, HSC depletion, and bone marrow failure. Reactive oxygen species (ROS), which damage DNA and ultimately lead to the transformation of HSCs into cancerous cells, are produced due to chronic inflammation. The biological elements of the HSC niche produce pro-inflammatory cytokines that cause clonal growth and the development of leukemic stem cells (LSCs) in hematological malignancies. The processes underlying how inflammation affects hematological malignancies are still not fully understood. In this review, we emphasize the effects of inflammation on normal hematopoiesis, the part it plays in the development and progression of hematological malignancies, and potential therapeutic applications for targeting these pathways for therapy in hematological malignancies.

Using Human Fetal Neural Stem Cells to Elucidate the Role of the JAK-STAT Cell Signaling Pathway in Oligodendrocyte Differentiation In Vitro.

Oligodendrocytes (OL) are the myelinating cells of the central nervous system that mediate nerve conduction. Loss of oligodendrocytes results in demyelination, triggering neurological deficits. Developing a better understanding of the cell signaling pathways influencing OL development may aid in the development of therapeutic strategies. The primary focus of this study was to investigate and elucidate the cell signaling pathways implicated in the developmental maturation of oligodendrocytes using human fetal neural stem cells (hFNSCs)-derived primary OL and MO3.13 cell line. Successful differentiation into OL was established by examining morphological changes, increased expression of mature OL markers MBP, MOG and decreased expression of pre-OL markers CSPG4 and O4. Analyzing transcriptional datasets (using RNA sequencing) in pre-OL and mature OL derived from hFNSCs revealed the novel and critical involvement of the JAK-STAT cell signaling pathway in terminal OL maturation. The finding was validated in MO3.13 cell line whose differentiation was accompanied by upregulation of IL-6 and the transcription factor STAT3. Increased phosphorylated STAT3 (pY705) levels were demonstrated by western blotting in hFNSCs-derived primary OL as well as terminal maturation in MO3.13 cells, thus validating the involvement of the JAK-STAT pathway in OL maturation. Pharmacological suppression of STAT3 phosphorylation (confirmed by western blotting) was able to prevent the increase of MBP-positive cells as demonstrated by flow cytometry. These novel findings highlight the involvement of the JAK-STAT pathway in OL maturation and raise the possibility of using this as a therapeutic strategy in demyelinating diseases.

Glial cholesterol redistribution in hypoxic injury in vitro influences oligodendrocyte maturation and myelination.

Hypoxic insult to the fetal brain causes loss of vulnerable premyelinating oligodendrocytes and arrested oligodendrocyte differentiation. Astrocytes influence oligodendrocyte differentiation and the astrocytic response to hypoxia could affect oligodendrocyte maturation under hypoxia. To identify pathways by which astrocytes influence oligodendroglial maturation in hypoxic injury, human fetal neural stem cell-derived astrocytes were exposed to 0.2 % oxygen for 48 hours. Transcriptomic analysis revealed the upregulation of the cholesterol-biosynthesis pathway in hypoxia-exposed astrocytes. Hypoxia-exposed primary astrocytes and astrocytic cell line (SVG) showed increased expression of hydroxy-methyl-glutaryl-CoA reductase (HMGCR), squalene epoxidase (SQLE), apolipoprotein E (apoE) and ATP-binding cassette transporter 1 (ABCA1) on qPCR and Western blot. Hypoxic SVG also showed increased cholesterol content in cells and culture supernatants and increased cell surface expression of ABCA1. Interestingly hypoxia-exposed premyelinating oligodendrocytes (Mo3.13) showed reduced cholesterol along with decreased expression of HMGCR and SQLE on qPCR and Western blot. Exogenous cholesterol increased the differentiation of Mo3.13 as measured by increased expression of myelin basic protein (MBP) on flow cytometry. Hypoxia exposure resulted in increased cholesterol transport from astrocytes to oligodendrocytes in cocultures with BODIPY-cholesterol labelled SVG and membrane-labelled Mo3.13. As exogenous cholesterol enhanced oligodendrocyte differentiation, our findings indicate that increased cholesterol synthesis by astrocytes and transport to oligodendrocytes could supplement oligodendroglial maturation in conditions of hypoxic brain injury in neonates.

Decoding the genetic symphony: Profiling protein-coding and long noncoding RNA expression in T-acute lymphoblastic leukemia for clinical insights.

T-acute lymphoblastic leukemia (T-ALL) is a heterogeneous malignancy characterized by the abnormal proliferation of immature T-cell precursors. Despite advances in immunophenotypic classification, understanding the molecular landscape and its impact on patient prognosis remains challenging. In this study, we conducted comprehensive RNA sequencing in a cohort of 35 patients with T-ALL to unravel the intricate transcriptomic profile. Subsequently, we validated the prognostic relevance of 23 targets, encompassing (i) protein-coding genes-BAALC, HHEX, MEF2C, FAT1, LYL1, LMO2, LYN, and TAL1; (ii) epigenetic modifiers-DOT1L, EP300, EML4, RAG1, EZH2, and KDM6A; and (iii) long noncoding RNAs (lncRNAs)-XIST, PCAT18, PCAT14, LINC00202, LINC00461, LINC00648, ST20, MEF2C-AS1, and MALAT1 in an independent cohort of 99 patients with T-ALL. Principal component analysis revealed distinct clusters aligning with immunophenotypic subtypes, providing insights into the molecular heterogeneity of T-ALL. The identified signature genes exhibited associations with clinicopathologic features. Survival analysis uncovered several independent predictors of patient outcomes. Higher expression of MEF2C, BAALC, HHEX, and LYL1 genes emerged as robust indicators of poor overall survival (OS), event-free survival (EFS), and relapse-free survival (RFS). Higher LMO2 expression was correlated with adverse EFS and RFS outcomes. Intriguingly, increased expression of lncRNA ST20 coupled with RAG1 demonstrated a favorable prognostic impact on OS, EFS, and RFS. Conclusively, several hitherto unreported associations of gene expression patterns with clinicopathologic features and prognosis were identified, which may help understand T-ALL's molecular pathogenesis and provide prognostic markers.

Refinement of Risk-Stratification of Cytogenetically Normal Acute Myeloid Leukemia Adult Patients by MN1 Expression.

INTRODUCTION: Acute myeloid leukemia with normal cytogenetics (CN-AML) represents a heterogeneous group having diverse genetic mutations. Understanding the significance of each of these mutations is necessary. In this study, we evaluated the prognostic role of MN1 expression in adult CN-AML patients. METHOD: One hundred and sixty-three de-novo adult AML patients were evaluated for MN1 expression by real-time PCR. MN1 expression was correlated with the clinical characteristics of the patients and their outcomes. RESULTS: Higher MN1 expression was associated with NPM1 wild-type (p<0.0001), CD34 positivity (p=0.006), and lower clinical remission rate (p=0.027). FLT3-ITD and CEBPA mutations had no association with MN1 expression. On survival analysis, a high MN1 expression was associated with poor event-free survival (Hazard Ratio 2.47, 95% Confidence Interval: 1.42-4.3; p<0.0001) and overall survival (Hazard Ratio 4.18, 95% Confidence Interval: 2.17-8.08; p<0.0001). On multivariate analysis, the MN1 copy number emerged as an independent predictor of EFS (p<0.0001) and OS (p<0.0001). CONCLUSION: MN1 expression is an independent predictor of outcome in CN-AML.

Mitochondrial gene expression signature predicts prognosis of pediatric acute myeloid leukemia patients.

Introduction: Gene expression profile of mitochondrial-related genes is not well deciphered in pediatric acute myeloid leukaemia (AML). We aimed to identify mitochondria-related differentially expressed genes (DEGs) in pediatric AML with their prognostic significance. Methods: Children with de novo AML were included prospectively between July 2016-December 2019. Transcriptomic profiling was done for a subset of samples, stratified by mtDNA copy number. Top mitochondria-related DEGs were identified and validated by real-time PCR. A prognostic gene signature risk score was formulated using DEGs independently predictive of overall survival (OS) in multivariable analysis. Predictive ability of the risk score was estimated along with external validation in The Tumor Genome Atlas (TCGA) AML dataset. Results: In 143 children with AML, twenty mitochondria-related DEGs were selected for validation, of which 16 were found to be significantly dysregulated. Upregulation of SDHC (p<0.001), CLIC1 (p=0.013) and downregulation of SLC25A29 (p<0.001) were independently predictive of inferior OS, and included for developing prognostic risk score. The risk score model was independently predictive of survival over and above ELN risk categorization (Harrell's c-index: 0.675). High-risk patients (risk score above median) had significantly inferior OS (p<0.001) and event free survival (p<0.001); they were associated with poor-risk cytogenetics (p=0.021), ELN intermediate/poor risk group (p=0.016), absence of RUNX1-RUNX1T1 (p=0.027), and not attaining remission (p=0.016). On external validation, the risk score also predicted OS (p=0.019) in TCGA dataset. Discussion: We identified and validated mitochondria-related DEGs with prognostic impact in pediatric AML and also developed a novel 3-gene based externally validated gene signature predictive of survival.

Hypoxia Induces Early Neurogenesis in Human Fetal Neural Stem Cells by Activating the WNT Pathway.

Fetal neural stem cells (FNSCs) present in the human fetal brain differentiate into cells of neuronal and glial lineages. The developing fetus is exposed to lower oxygen concentrations than adults, and this physiological hypoxia may influence the growth and differentiation of the FNSCs. This study aimed to evaluate the effect of hypoxia on the differentiation potential of human FNSCs isolated from the subventricular zone of aborted fetal brains (n = 5). FNSCs were isolated, expanded, and characterized by Nestin and Sox2 expression using immunocytochemistry and flow cytometry, respectively. These FNSCs were exposed to 20% oxygen (normoxia) and 0.2% oxygen (hypoxia) concentrations for 48 h, and hypoxia exposure (n = 5) was validated. Whole transcriptome analyses (Genespring GX13) of FNSCs exposed to hypoxia (Agilent 4 × 44 K human array slides) highlighted that genes associated with neurogenesis were enriched upon exposure to hypoxia. The pathway analysis of these enriched genes (using Metacore) showed the involvement of the WNT signaling pathway. Microarray analyses were validated using neuronal and glial lineage commitment markers, namely, NEUROG1, NEUROG2, ASCL1, DCX, GFAP, OLIG2, and NKX2.2, using qPCR (n = 9). DCX, ASCL1, NGN1, and GFAP protein expression was analyzed by Western blotting (n = 3). This demonstrated upregulation of the neuronal commitment markers upon hypoxia exposure, while no change was observed in astrocytic and oligodendrocyte lineage commitment markers. Increased expression of downstream targets of the WNT signaling pathway, TCF4 and ID2, by qPCR (n = 9) and increased protein expression of CTNNB1 (ß-catenin) and ID2 by Western blot (n = 3) indicated its involvement in mediating neuronal differentiation upon exposure to hypoxia.

Prognostic relevance of NPM1, CEBPA, and FLT3 mutations in cytogenetically normal adult AML patients.

BACKGROUND: Acute myeloid leukemia with normal cytogenetics (CN-AML) is the largest group of AML patients with very heterogenous patient outcomes. The revised World Health Organization classification of the hematolymphoid tumours, 2022, has incorporated AML with Nucleophosphmin1 (NPM1) and CCAAT/enhancer binding protein-alpha (CEBPA) mutations as distinct entities. Despite the existing evidence of the prognostic relevance of FMS-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD) in AML, it has not been included in the revised classification. METHOD: In this prospective study, we determined the prevalence of NPM1, CEBPA, and FLT3 gene mutations in 151 de novo CN-AML adult patients (age ≥18 years) in a tertiary care hospital in north India. Additionally, the prognostic relevance of these mutations was also evaluated. RESULTS: NPM1, FLT3-ITD, and CEBPA mutations were found in 33.11%, 23.84%, and 15.77% of CN-AML patients, respectively. CEBPA mutations were found at 3 domains: transactivation domain 1 (TAD1) in 10 (6.62%), transactivation domain 2 (TAD2) in 5 (3.31%), and basic leucine zipper domain (bZIP) in 11 (7.82%) patients. Patients with NPM1 mutation had better clinical remission rate (CR) (P=0.003), event-free survival (P=0.0014), and overall survival (OS) (P=0.0017). However, FLT3-ITD and CEBPA mutations did not show any association with CR (P=0.404 and 0.92, respectively). Biallelic CEBPA mutations were found in 12 (7.95%) patients and were associated with better OS (P=0.043). CONCLUSIONS: These findings indicate that NPM1 and CEBPA mutations can be precisely used for risk stratification in CN-AML patients.

Distinct oncogenic phenotypes in hematopoietic specific deletions of Trp53.

Loss of function in the tumor suppressor gene TP53 is the most common alteration seen in human cancer. In mice, P53 deletion in all cells leads predominantly to the development of T-cell lymphomas, followed by B-cell lymphomas, sarcomas and teratomas. In order to dissect the role of P53 in the hematopoietic system, we generated and analyzed two different mouse models deficient for P53. A pan-hematopoietic P53 deletion mouse was created using Vav1-Cre based deletion; and a B-cell-specific deletion mouse was created using a CD19-Cre based deletion. The Vav1-P53CKO mice predominantly developed T-cell malignancies in younger mice, and myeloid malignancies in older mice. In T-cell malignancies, there was accelerated thymic cell maturation with overexpression of Notch1 and its downstream effectors. CD19-P53CKO mice developed marginal zone expansion in the spleen, followed by marginal zone lymphoma, some of which progressed to diffuse large B-cell lymphomas. Interestingly, marginal zone and diffuse large B-cell lymphomas had a unique gene expression signature characterized by activation of the PI3K pathway, compared with wild type marginal zone or follicular cells of the spleen. This study demonstrates lineage specific P53 deletion leading to distinct phenotypes secondary to unique gene expression programs set in motion.

RNA binding protein IGF2BP1 synergizes with ETV6-RUNX1 to drive oncogenic signaling in B-cell Acute Lymphoblastic Leukemia.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common pediatric hematological malignancy, with ETV6::RUNX1 being the most prevalent translocation whose exact pathogenesis remains unclear. IGF2BP1 (Insulin-like Growth Factor 2 Binding Protein 1) is an oncofetal RNA binding protein seen to be specifically overexpressed in ETV6::RUNX1 positive B-ALL. In this study, we have studied the mechanistic role of IGF2BP1 in leukemogenesis and its synergism with the ETV6::RUNX1 fusion protein. METHODS: Gene expression was analyzed from patient bone marrow RNA using Real Time RT-qPCR. Knockout cell lines were created using CRISPR-Cas9 based lentiviral vectors. RNA-Seq and RNA Immunoprecipitation sequencing (RIP-Seq) after IGF2BP1 pulldown were performed using the Illumina platform. Mouse experiments were done by retroviral overexpression of donor HSCs followed by lethal irradiation of recipients using a bone marrow transplant model. RESULTS: We observed specific overexpression of IGF2BP1 in ETV6::RUNX1 positive patients in an Indian cohort of pediatric ALL (n=167) with a positive correlation with prednisolone resistance. IGF2BP1 expression was essential for tumor cell survival in multiple ETV6::RUNX1 positive B-ALL cell lines. Integrated analysis of transcriptome sequencing after IGF2BP1 knockout and RIP-Seq after IGF2BP1 pulldown in Reh cell line revealed that IGF2BP1 targets encompass multiple pro-oncogenic signalling pathways including TNFα/NFκB and PI3K-Akt pathways. These pathways were also dysregulated in primary ETV6::RUNX1 positive B-ALL patient samples from our center as well as in public B-ALL patient datasets. IGF2BP1 showed binding and stabilization of the ETV6::RUNX1 fusion transcript itself. This positive feedback loop led to constitutive dysregulation of several oncogenic pathways. Enforced co-expression of ETV6::RUNX1 and IGF2BP1 in mouse bone marrow resulted in marrow hypercellularity which was characterized by multi-lineage progenitor expansion and strong Ki67 positivity. This pre-leukemic phenotype confirmed their synergism in-vivo. Clonal expansion of cells overexpressing both ETV6::RUNX1 and IGF2BP1 was clearly observed. These mice also developed splenomegaly indicating extramedullary hematopoiesis. CONCLUSION: Our data suggest a combined impact of the ETV6::RUNX1 fusion protein and RNA binding protein, IGF2BP1 in activating multiple oncogenic pathways in B-ALL which makes IGF2BP1 and these pathways as attractive therapeutic targets and biomarkers.

Prognostic relevance of surface expression of cytokine receptor-like factor 2 in pediatric B-lineage acute lymphoblastic leukemia.

Overexpression of cytokine receptor-like factor 2 (CRLF2) resulting from its genomic rearrangement is the most frequent genetic alteration found in Philadelphia chromosome-like (Ph-like) B-cell acute lymphoblastic leukemia (B-ALL), a high-risk leukemia. Detection of CRLF2 expression by multiparameter flow cytometry has been proposed as a screening tool for the identification of Ph-like B-ALL. However, the prognostic relevance of flow cytometric expression of CRLF2 in pediatric B-ALL is not very clear. Additionally, its association with common copy number alterations (CNA) has not been studied in detail. Hence, in this study, we prospectively evaluated the flow cytometric expression of CRLF2 in 256 pediatric B-ALL patients and determined its association with molecular features such as common CNAs detected using Multiplex ligation-dependent probe amplification and mutations in CRLF2, JAK2 and IL7RA genes. Further, its association with clinicopathological features including patient outcome was assessed. We found that 8.59% (22/256) pediatric B-ALL patients were CRLF2-positive at diagnosis. Among CNAs, CRLF2 positivity was associated with presence of PAX5 alteration (P=0.041). JAK2 and IL-7R mutations were found in 9% and 13.6% CRLF2-positive patients, respectively. IGH::CRLF2 or P2RY8::CRLF2 fusions were each found in 1/22 individuals. CRLF2-positive patients were found to have inferior overall (hazard ratio (HR) =4.39, P=0.006) and event free survival (HR=2.62, P=0.045), independent to other clinical features. Furthermore, concomitant CNA of IKZF1 in CRLF2 positive patients was associated with a greater hazard for poor overall and event free survival, compared to patients without these alterations or presence of any one of them. Our findings demonstrate that the surface CRLF2 expression in association with IKZF1 copy number alteration can be used to risk stratify pediatric B-ALL patients.