Background: T cells equipped with chimeric antigen receptors (CAR) have shown remarkable efficacy in targeting B lineage malignancies. Improvement of the CAR structure is needed, however, with a view to developing flexibly modifiable spacers that are inert in interactions with unwanted cells. Specifically, binding to cells carrying receptors for IgG\'s crystallizable fragment (FcR), that recognize IgG-derived domains in CARs is to be avoided. Methods: Two novel CARs targeting the CD19 antigen where the IgG1-CH2 and -CH3 domains were replaced with Ig-like domains from signal-regulatory protein α (SIRPα) were designed in silico. An IgG1-based CAR and a CAR lacking both SIRPα and IgG1 domains were used as comparators. The phenotype and memory phenotype of the expanded cells were analyzed by flow cytometry, and CAR T cell activation and cytotoxic efficacy were assessed in co-culture experiments in response to CD19+ target cells. Unwanted interactions with FcR-expressing myeloid cells were interrogated in co-culture assays with THP-1 monocytic cells. Results: T cells carrying the novel SIRPα-based CARs enacted potent in vitro cytotoxicity against CD19 positive B-lineage leukemia cells, comparable to traditional IgG1-based CAR T cells. Co-culture of IgG1-based CAR T cells with FcR-expressing THP-1 monocytic cells led to prominent cell surface expression of CD69 on T cells together with production of Interleukin (IL)-2 and Interferon-γ, and production of IL-1β, indicating activation of the T cells and monocytes, respectively. Longer co-culture led to killing of the monocytes. No signs of T cell nor monocyte activation were detected in co-cultures of SIRPα-based CAR T cells with THP-1 cells. Arming T cells with the SIRPα-based CARs favored differentiation towards CD4+ phenotype during expansion, while the effects on memory phenotype of the T cells were equivalent between the SIRPα- and IgG1-based CARs. In a pilot experiment, T cells modified with one of the SIRPα-based CARs showed dose dependent leukemia cell control. Conclusion: The novel SIRPα based spacers offer a suitable backbone for developing chimeric antigen receptors that evade the off-target binding to FcR while the cells retain a favorable memory phenotype and efficient cytotoxicity, establishing a promising candidate for future in vivo and clinical testing.

Lenalidomide, a thalidomide derivative, is prescribed as maintenance therapy for multiple myeloma (MM). Patients with MM receiving lenalidomide were found to develop a distinct therapy-related B cell acute lymphoblastic leukemia (B-ALL). However, the molecular mechanism by which lenalidomide drives B-ALL is unknown. We show that thalidomide treatment of B cell lines increased CD34 expression and fibronectin adhesion. This resembled the effects of Ikzf1 loss of function mutations in B-ALL. IKZF1 is a transcription factor that can act as both a transcriptional activator and a repressor depending upon the target loci. In our experiments, thalidomide-induced degradation of IKZF1 increased the expression of its transcriptional repression targets Itga5 and CD34 explaining the increased adhesion and stemness. Strikingly, withdrawal of thalidomide lead to the mis-localization of IKZF1 to the cytoplasm. Moreover, chromatin immunoprecipitation data showed a long-term effect of thalidomide treatment on IKZF1 target loci. This included decreased chromatin occupancy at early B cell factor 1 (EBF1) and Spi1 (PU.1). Consequently, B-cell lineage specifying transcription factors including Pax5, Spi1 and EBF1 were downregulated even after 7 days of thalidomide withdrawal. Our study thus provides a molecular mechanism of thalidomide-induced B-ALL whereby thalidomide alters the chromatin occupancy of IKZF1 at key B-cell lineage transcription factors leading to a persistent block in B-cell differentiation.

Myocyte enhancer factor 2 (MEF2) is a key transcription factor (TF) in skeletal, cardiac, and neural tissue development and includes four isoforms: MEF2A, MEF2B, MEF2C, and MEF2D. These isoforms significantly affect embryonic development, nervous system regulation, muscle cell differentiation, B- and T-cell development, thymocyte selection, and effects on tumorigenesis and leukemia. This chapter describes the multifaceted roles of MEF2 family proteins, covering embryonic development, nervous system regulation, and muscle cell differentiation. It further elucidates the contribution of MEF2 to various blood and immune cell functions. Specifically, in B-cell precursor acute lymphoblastic leukemia (BCP-ALL), MEF2D is aberrantly expressed and forms a fusion protein with BCL9, CSF1R, DAZAP1, HNRNPUL1, and SS18. These fusion proteins are closely related to the pathogenesis of leukemia. In addition, it specifically introduces the regulatory effect of MEF2D fusion protein on the proliferation and growth of B-cell acute lymphoblastic leukemia (B-ALL) cells. Finally, we detail the positive feedback loop between MEF2D and IRF8 that significantly promotes the progression of acute myeloid leukemia (AML) and the importance of the ZMYND8-BRD4 interaction in regulating the IRF8 and MYC transcriptional programs. The MEF2D-CEBPE axis is highlighted as a key transcriptional mechanism controlling the block of leukemic cell self-renewal and differentiation in AML. This chapter starts with the structure and function of MEF2 family proteins, specifically summarizing and analyzing the role of MEF2D in B-ALL and AML, mediating the complex molecular mechanisms of transcriptional regulation and exploring their implications for human health and disease.

Genetic alterations of the repressive ETS family transcription factor gene ETV6 are recurrent in several categories of hematopoietic malignancy, including subsets of B-cell and T-cell acute lymphoblastic leukemias (B-ALL and T-ALL), myeloid neoplasms, and mature B-cell lymphomas. ETV6 is essential for adult hematopoietic stem cells (HSCs), contributes to specific functions of some mature immune cells, and plays a key role in thrombopoiesis as demonstrated by familial ETV6 mutations associated with thrombocytopenia and predisposition to hematopoietic cancers, particularly B-ALL. ETV6 appears to have a tumor suppressor role in several hematopoietic lineages, as demonstrated by recurrent somatic loss-of-function (LoF) and putative dominant-negative alterations in leukemias and lymphomas. ETV6 rearrangements contribute to recurrent fusion oncogenes such as the B-ALL-associated transcription factor (TF) fusions ETV6::RUNX1 and PAX5::ETV6, rare drivers such as ETV6::NCOA6, and a spectrum of tyrosine kinase gene fusions encoding hyperactive signaling proteins that self-associate via the ETV6 N-terminal pointed domain. Another subset of recurrent rearrangements involving the ETV6 gene locus appear to function primarily to drive overexpression of the partner gene. This review surveys what is known about the biochemical and genome regulatory properties of ETV6 as well as our current understanding of how alterations in these functions contribute to hematopoietic and nonhematopoietic cancers.

Pegylated (PEG)-asparaginase is used during the induction and intensification phases of treatment for B-cell acute lymphoblastic leukemia (B-ALL). It works by depleting the external sources of asparagine, causing the death of lymphoblasts. It has several adverse effects, including pancreatitis and hypertriglyceridemia; however, the simultaneous occurrence of both is uncommon. We present the case of an 18-year-old man with B-ALL who developed acute epigastric pain radiating to the back and non-bloody, non-bilious emesis following treatment with PEG-asparaginase. He was diagnosed with acute interstitial pancreatitis and severe hypertriglyceridemia. Conservative management was used for the pancreatitis, while hypertriglyceridemia was treated with an insulin infusion. Pancreatic toxicity and hypertriglyceridemia can necessitate the discontinuation of PEG-asparaginase, limiting treatment options and potentially increasing the risk of relapse. Therefore, further studies are needed to identify the factors contributing to hypertriglyceridemia and pancreatitis, aiding clinicians in monitoring and prevention.

UNASSIGNED: Paediatric acute B-cell lymphoblastic leukaemia is the most common cancer of the paediatric age. Although the advancement of scientific and technological knowledge has ensured a huge step forward in the management of this disease, there are 15%-20% cases of recurrence leading to serious complications for the patient and sometimes even death. It is therefore necessary to identify new and increasingly personalised biomarkers capable of predicting the degree of risk of B-ALL in order to allow the correct management of paediatric leukaemia patients.
UNASSIGNED: Starting from our previously published results, we validate the expression level of LINC00958 in a cohort of 33 B-ALL and 9 T-ALL childhood patients, using in-silico public datasets as support. Expression levels of LINC00958 in B-ALL patients stratified by risk (high risk vs. standard/medium risk) and who relapsed 3 years after the first leukaemia diagnosis were also evaluated.
UNASSIGNED: We identified the lncRNA LINC00958 as a biomarker of B-ALL, capable of discriminating B-ALL from T-ALL and healthy subjects. Furthermore, we associated LINC00958 expression levels with the disease risk classification (high risk and standard risk). Finally, we show that LINC00958 can be used as a predictor of relapses in patients who are usually stratified as standard risk and thus not always targeted for marrow transplantation.
UNASSIGNED: Our results open the way to new diagnostic perspectives that can be directly used in clinical practice for a better management of B-ALL paediatric patients.

Targeted therapies exploiting vulnerabilities of cancer cells hold promise for improving patient outcome and reducing side-effects of chemotherapy. However, efficacy of precision therapies is limited in part because of tumor cell heterogeneity. A better mechanistic understanding of how drug effect is linked to cancer cell state diversity is crucial for identifying effective combination therapies that can prevent disease recurrence.
Here, we characterize the effect of G2/M checkpoint inhibition in acute lymphoblastic leukemia (ALL) and demonstrate that WEE1 targeted therapy impinges on cell fate decision regulatory circuits. We find the highest inhibition of recovery of proliferation in ALL cells with KMT2A-rearrangements. Single-cell RNA-seq and ATAC-seq of RS4;11 cells harboring KMT2A::AFF1, treated with the WEE1 inhibitor AZD1775, reveal diversification of cell states, with a fraction of cells exhibiting strong activation of p53-driven processes linked to apoptosis and senescence, and disruption of a core KMT2A-RUNX1-MYC regulatory network. In this cell state diversification induced by WEE1 inhibition, a subpopulation transitions to a drug tolerant cell state characterized by activation of transcription factors regulating pre-B cell fate, lipid metabolism, and pre-BCR signaling in a reversible manner. Sequential treatment with BCR-signaling inhibitors dasatinib, ibrutinib, or perturbing metabolism by fatostatin or AZD2014 effectively counteracts drug tolerance by inducing cell death and repressing stemness markers.
Collectively, our findings provide new insights into the tight connectivity of gene regulatory programs associated with cell cycle and cell fate regulation, and a rationale for sequential administration of WEE1 inhibitors with low toxicity inhibitors of pre-BCR signaling or metabolism.

BACKGROUND: Cellular immunotherapy, represented by the chimeric antigen receptor T cell (CAR-T), has exhibited high response rates, durable remission, and safety in vitro and in clinical trials. Unfortunately, anti-CD19 CAR-T (CART-19) treatment alone is prone to relapse and has a particularly poor prognosis in relapsed/refractory (r/r) B-ALL patients. To date, addressing or reducing relapse remains one of the research priorities to achieve broad clinical application.
METHODS: We manufactured second generation CART-19 cells and validated their efficacy and safety in vitro and in vivo. Through co-culture of Nalm-6 cells with short-term cultured CART-19 cells, CD19-negative Nalm-6 cells were detected by flow cytometry, and further investigation of the relapsed cells and their resistance mechanisms was evaluated in vitro.
RESULTS: In this study, we demonstrated that CART-19 cells had enhanced and specific antileukemic activities, and the survival of B-ALL mouse models after CART-19 treatment was significantly prolonged. We then shortened the culture time and applied the serum-free culture to expand CAR-T cells, followed by co-culturing CART-19 cells with Nalm-6 cells. Surprisingly, we observed the proliferation of CD19-negative Nalm-6 cells around 28 days. Identification of potential resistance mechanisms showed that the relapsed cells express truncated CD19 proteins with decreased levels and, more importantly, CAR expression was detected on the relapsed cell surface, which may ultimately keep them antigen-negative. Furthermore, it was validated that CART-22 and tandem CART-22/19 cells could effectively kill the relapsed cells, but neither could completely eradicate them.
CONCLUSIONS: We successfully generated CART-19 cells and obtained a CD19-negative refractory relapsed B-ALL cell line, providing new insights into the underlying mechanisms of resistance and a new in vitro model for the treatment of r/r B-ALL patients with low antigen density.

Blinatumomab is efficacious in patients with B-cell acute lymphoblastic leukemia (B-ALL), yet limited real-world data exists in this context. This retrospective study provided real-world data on the treatment pattern, effectiveness, and safety of blinatumomab in Chinese patients with newly diagnosed (ND) and relapsed/refractory (R/R) B-ALL. Patients with B-ALL who received at least one dose of blinatumomab in frontline or R/R settings between August 2021 and June 2023 were included. The primary outcome was the treatment pattern of blinatumomab. Key secondary outcomes included complete remission (CR)/CR with incomplete blood cell recovery (CRi) rate, minimal residual disease (MRD) negativity, median event-free survival (EFS), and safety. The study included 96 patients with B-ALL; 53 (55.2%) patients were in the ND group and 43 (44.8%) patients were in the R/R group. The median treatment duration was one cycle (range: 1-5). Most patients underwent chemotherapies, allo-HSCT, or experimental CAR-T following blinatumomab. The ND patients using blinatumomab induction therapy achieved 100% CR/CRi rate; 87.2% achieved MRD negativity within two cycles of blinatumomab. In R/R re-induction patients, the CR/CRi rate was 50%; MRD negativity rate was 64.2%. In R/R patients using blinatumomab for consolidation, MRD negativity rate was 90.9%. The median EFS was not reached in both ND and R/R patients; 1-year EFS rate was 90.8% (95% CI: 67%, 97%) and 55.1% (95% CI: 30%, 74%), respectively. Grade ≥ 3 adverse events were observed in 12.5% patients. Blinatumomab was found to be effective with a tolerable safety profile in real world setting.

Structural variants drive tumorigenesis by disrupting normal gene function through insertions, inversions, translocations, and copy number changes, including deletions and duplications. Detecting structural variants is crucial for revealing their roles in tumor development, clinical outcomes, and personalized therapy. Presently, most studies rely on short-read data from next-generation sequencing that aligns back to a reference genome to determine if and, if so, where a structural variant occurs. However, structural variant discovery by short-read sequencing is challenging, primarily because of the difficulty in mapping regions of repetitive sequences. Optical genome mapping (OGM) is a recent technology used for imaging and assembling long DNA strands to detect structural variations. To capture the structural variant landscape more thoroughly in the human genome, we developed an integrated pipeline that combines Bionano OGM and Illumina whole-genome sequencing and applied it to samples from 29 pediatric B-ALL patients. The addition of OGM allowed us to identify 511 deletions, 506 insertions, 93 duplications/gains, and 145 translocations that were otherwise missed in the short-read data. Moreover, we identified several novel gene fusions, the expression of which was confirmed by RNA sequencing. Our results highlight the benefit of integrating OGM and short-read detection methods to obtain a comprehensive analysis of genetic variation that can aid in clinical diagnosis, provide new therapeutic targets, and improve personalized medicine in cancers driven by structural variation.