NG2 is a target gene of MLL-AF4 and underlies glucocorticoid resistance in MLL-r B-ALL by regulating NR3C1 expression.

B-cell acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer, with long-term overall survival rates of ~85%. However, B-ALL harboring rearrangements of the MLL gene (also known as KMT2A), referred to as MLLr B-ALL, is common in infants and is associated with poor 5-year survival (<30%), frequent relapses, and refractoriness to glucocorticoids (GCs). GCs are an essential part of the treatment backbone for B-ALL and GC resistance is a major clinical predictor of poor outcome. Elucidating the mechanisms of GC resistance in MLLr B-ALL is, therefore, critical to guide therapeutic strategies that deepen the response after induction therapy. Neuron-glial antigen-2 (NG2) expression is a hallmark of MLLr B-ALL and is minimally expressed in healthy hematopoietic cells. We recently reported that NG2 expression is associated with poor prognosis and that anti-NG2 immunotherapy strongly reduces/delays relapse in MLLr B-ALL xenograft models. Despite its contribution to MLLr B-ALL pathogenesis and its diagnostic utility, the role of NG2 in MLLr-mediated leukemogenesis/chemoresistance remains elusive. Here we show that NG2 is an epigenetically regulated direct target gene of the leukemic MLL-AF4 fusion protein. NG2 negatively regulates the expression of the GC receptor NR3C1 and confers GC resistance to MLLr B-ALL cells in vitro and in vivo. Mechanistically, NG2 interacts with FLT3 to render ligand-independent activation of FLT3 signaling (a hallmark of MLLr B-ALL) and downregulation of NR3C1 via AP-1-mediated trans-repression. Collectively, our study elucidates the role of NG2 in GC resistance in MLLr B-ALL through FLT3/AP-1-mediated downregulation of NR3C1, providing novel therapeutic avenues for MLLr B-ALL.

Biochemical characterisation of a PL24 ulvan lyase from seaweed-associated Vibrio sp. FNV38.

Ulvan is a green macroalgal cell wall polysaccharide that has tremendous potential for valorisation due to its unique composition of sulphated rhamnose, glucuronic acid, iduronic acid and xylose. Several potential applications such as production of biofuels, bioplastics and other value-added products necessitate the breakdown of the polysaccharide to oligomers or monomers. Research on ulvan saccharifying enzymes has been continually increasing over the last decade, with the increasing focus on valorisation of seaweed biomass for a biobased economy. Lyases are the first of several enzymes that are involved in saccharifying the polysaccharide and several ulvan lyases have been structurally and biochemically characterised to enable their effective use in the valorisation processes. This study investigates the whole genome of Vibrio sp. FNV38, an ulvan metabolising organism and biochemical characteristics of a PL24 ulvan lyase that it possesses. The genome of Vibrio sp. FNV38 has a diverse CAZy profile with several genes involved in the metabolism of ulvan, cellulose, agar, and alginate. The enzyme exhibits optimal activity at pH 8.5 in 100 mM Tris-HCl buffer and 30 °C. However, its thermal stability is poor with significant loss of activity after 2 h of incubation at temperatures above 25 °C. Breakdown product analysis reveals that the enzyme depolymerised the polysaccharide predominantly to disaccharides and tetrasaccharides. Supplementary Information: The online version contains supplementary material available at 10.1007/s10811-023-03136-3.

Evolutionary analysis reveals the role of a non-catalytic domain of peptidyl arginine deiminase 2 in transcriptional regulation.

Peptidyl arginine deiminases (PADIs) catalyze protein citrullination, a post-translational conversion of arginine to citrulline. The most widely expressed member of this family, PADI2, regulates cellular processes that impact several diseases. We hypothesized that we could gain new insights into PADI2 function through a systematic evolutionary and structural analysis. Here, we identify 20 positively selected PADI2 residues, 16 of which are structurally exposed and maintain PADI2 interactions with cognate proteins. Many of these selected residues reside in non-catalytic regions of PADI2. We validate the importance of a prominent loop in the middle domain that encompasses PADI2 L162, a residue under positive selection. This site is essential for interaction with the transcription elongation factor (P-TEFb) and mediates the active transcription of the oncogenes c-MYC, and CCNB1, as well as impacting cellular proliferation. These insights could be key to understanding and addressing the role of the PADI2 c-MYC axis in cancer progression.

Chromosomal instability in aneuploid acute lymphoblastic leukemia associates with disease progression.

Chromosomal instability (CIN) lies at the core of cancer development leading to aneuploidy, chromosomal copy-number heterogeneity (chr-CNH) and ultimately, unfavorable clinical outcomes. Despite its ubiquity in cancer, the presence of CIN in childhood B-cell acute lymphoblastic leukemia (cB-ALL), the most frequent pediatric cancer showing high frequencies of aneuploidy, remains unknown. Here, we elucidate the presence of CIN in aneuploid cB-ALL subtypes using single-cell whole-genome sequencing of primary cB-ALL samples and by generating and functionally characterizing patient-derived xenograft models (cB-ALL-PDX). We report higher rates of CIN across aneuploid than in euploid cB-ALL that strongly correlate with intraclonal chr-CNH and overall survival in mice. This association was further supported by in silico mathematical modeling. Moreover, mass-spectrometry analyses of cB-ALL-PDX revealed a "CIN signature" enriched in mitotic-spindle regulatory pathways, which was confirmed by RNA-sequencing of a large cohort of cB-ALL samples. The link between the presence of CIN in aneuploid cB-ALL and disease progression opens new possibilities for patient stratification and offers a promising new avenue as a therapeutic target in cB-ALL treatment.

Integrative single-cell expression and functional studies unravels a sensitization to cytarabine-based chemotherapy through HIF pathway inhibition in AML leukemia stem cells.

Relapse remains a major challenge in the clinical management of acute myeloid leukemia (AML) and is driven by rare therapy-resistant leukemia stem cells (LSCs) that reside in specific bone marrow niches. Hypoxia signaling maintains cells in a quiescent and metabolically relaxed state, desensitizing them to chemotherapy. This suggests the hypothesis that hypoxia contributes to the chemoresistance of AML-LSCs and may represent a therapeutic target to sensitize AML-LSCs to chemotherapy. Here, we identify HIFhigh and HIFlow specific AML subgroups (inv(16)/t(8;21) and MLLr, respectively) and provide a comprehensive single-cell expression atlas of 119,000 AML cells and AML-LSCs in paired diagnostic-relapse samples from these molecular subgroups. The HIF/hypoxia pathway signature is attenuated in AML-LSCs compared with more differentiated AML cells but is more expressed than in healthy hematopoietic cells. Importantly, chemical inhibition of HIF cooperates with standard-of-care chemotherapy to impair AML growth and to substantially eliminate AML-LSCs in vitro and in vivo. These findings support the HIF pathway in the stem cell-driven drug resistance of AML and unravel avenues for combinatorial targeted and chemotherapy-based approaches to specifically eliminate AML-LSCs.