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BackgroundAcute 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.MethodsGene 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.ResultsWe 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.ConclusionOur 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.

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.

Latent class analysis (LCA) is a useful statistical approach for understanding heterogeneity in a population. This article provides a pedagogical introduction to LCA modeling and provides an example of its use to understand youths' daily coping strategies. The analytic procedures are outlined for choosing the number of classes and integration of the LCA variable within a structural equation model framework, specifically a latent class moderation model, and a detailed table provides a summary of relevant modeling steps. This applied example demonstrates the modeling context when the LCA variable is moderating the association between a covariate and two outcome variables. Results indicate that students' coping strategies moderate the association between social stress and negative mood; however, they do not moderate the social stress-positive mood association. Online supplemental materials include R (MplusAutomation) code to automate the enumeration procedure, ML three-step auxiliary variable integration, and the generation of figures for visually depicting LCA results.

Anabolic metabolism of carbon in mammals is mediated via the one- and two-carbon carriers S-adenosyl methionine and acetyl-coenzyme A. In contrast, anabolic metabolism of three-carbon units via propionate has not been shown to extensively occur. Mammals are primarily thought to oxidize the three-carbon short chain fatty acid propionate by shunting propionyl-CoA to succinyl-CoA for entry into the TCA cycle. Here, we found that this may not be absolute as, in mammals, one nonoxidative fate of propionyl-CoA is to condense to two three-carbon units into a six-carbon trans-2-methyl-2-pentenoyl-CoA (2M2PE-CoA). We confirmed this reaction pathway using purified protein extracts provided limited substrates and verified the product via LC-MS using a synthetic standard. In whole-body in vivo stable isotope tracing following infusion of 13C-labeled valine at steady state, 2M2PE-CoA was found to form via propionyl-CoA in multiple murine tissues, including heart, kidney, and to a lesser degree, in brown adipose tissue, liver, and tibialis anterior muscle. Using ex vivo isotope tracing, we found that 2M2PE-CoA also formed in human myocardial tissue incubated with propionate to a limited extent. While the complete enzymology of this pathway remains to be elucidated, these results confirm the in vivo existence of at least one anabolic three- to six-carbon reaction conserved in humans and mice that utilizes propionate.