pre-B Cells Research Articles

Abstract 5117: CK2 inhibiton exhibits synergistic effect in acute lymphoblastic leukemia

Abstract Survival for pre-B cell Acute lymphoblastic leukemia (B ALL) has improved greatly, however. High risk (HR) subgroups continue to result in significant mortality and morbidity of pediatric oncology patients. Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is a lethal disease, often associated with high-risk clinical features. T-ALL therapy has made less improvement than B ALL and continues to represent a significant number of high-risk outcomes. Novel treatment strategies are required to overcome chemotherapy resistance and improve morbidity for HR B ALL. The development of treatments that can overcome chemotherapy resistance is essential for improving survival in T-ALL. Deletions of IKZF1, resulting in decreased IKAROS function, are present in High-risk ALL patients. Casein kinase 2 (CK2), a protein kinase upregulated in ALL, phosphorylates IKAROS preventing DNA binding reducing transcriptional regulation in ALL patients. Past studies have shown that pharmacologic inhibition of CK2 with CX-4945 rescues IKAROS function as a transcriptional regulator. Receptor tyrosine kinase c-KIT is transcriptionally regulated by IKAROS and is often upregulated in cancer. The purpose of this study was to determine whether restoration of IKAROS via CX-4945 would act in a synergistic manner with c-KIT inhibitor imatinib in ALL.Methods/results B-ALL Nalm6 cells were treated with imatinib over 72-hours to establish the half-maximal inhibitory concentration (IC50). The IC50 was used to determine the concentration range of imatinib to be used in combination with CX-4945 in a 72-hour synergy analysis of cytotoxicity.. Cell viability was measured using WST-1 cell proliferation assay. The data were analyzed using Calcusyn and synergy was defined as a combination index below 0.85. The study was repeated to determine a more precise range of synergy. The cytotoxicity of single drug c-KIT inhibitor, Imatinib, in Nalm6 B-ALL yielded an IC50 of 25uM while CK2 inhibitor CX-4945 resulted in 4uM. When a combination was administered with a constant dose of CX-4945 at 4uM and varying concentrations of imatinib, synergy was observed at multiple dose combinations. Conclusion: The data shows that CK2 inhibitor, CX-4945, and c-KIT inhibitor, imatinib, exhibit a synergistic therapeutic effect. Imatinib is indicated for use in Philadelphia positive (Ph+) acute lymphoblastic leukemia so future studies will include testing CX-4945/imatinib synergy in Ph+ BALL where we expect to see more potent synergistic effect. Citation Format: Daniel Gary Bogush, Joseph Schramm, Katarina Dovat, Yali Ding, Chingakham Singh, Sinisa Dovat. CK2 inhibiton exhibits synergistic effect in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5117.

Antigen-independent, autonomous B cell receptor signaling drives activated B cell DLBCL.

Diffuse large B cell lymphoma of activated B cell type (ABC-DLBCL), a major cell-of-origin DLBCL subtype, is characterized by chronic active B cell receptor (BCR) signaling and NF-κB activation, which can be explained by activating mutations of the BCR signaling cascade in a minority of cases. We demonstrate that autonomous BCR signaling, akin to its essential pathogenetic role in chronic lymphocytic leukemia (CLL), can explain chronic active BCR signaling in ABC-DLBCL. 13 of 18 tested DLBCL-derived BCR, including 12 cases selected for expression of IgM, induced spontaneous calcium flux and increased phosphorylation of the BCR signaling cascade in murine triple knockout pre-B cells without antigenic stimulation or external BCR crosslinking. Autonomous BCR signaling was associated with IgM isotype, dependent on somatic BCR mutations and individual HCDR3 sequences, and largely restricted to non-GCB DLBCL. Autonomous BCR signaling represents a novel immunological oncogenic driver mechanism in DLBCL originating from individual BCR sequences and adds a new dimension to currently proposed genetics- and transcriptomics-based DLBCL classifications.

Targeting TNF/IL-17/MAPK pathway in hE2A-PBX1 leukemia: effects of OUL35, KJ-Pyr-9, and CID44216842.

t(1;19)(q23;p13) is one of the most common translocation genes in childhood acute lymphoblastic leukemia (ALL) and is also present in acute myeloid leukemia (AML) and mixed-phenotype acute leukemia (MPAL). This translocation results in the formation of the oncogenic E2A-PBX1 fusion protein, which contains a trans-activating domain from E2A and a DNA-binding homologous domain from PBX1. Despite its clear oncogenic potential, the pathogenesis of E2A-PBX1 fusion protein is not fully understood (especially in leukemias other than ALL), and effective targeted clinical therapies have not been developed. To address this, we established a stable and heritable zebrafish line expressing human E2A-PBX1 (hE2A-PBX1) for high-throughput drug screening. Blood phenotype analysis showed that hE2A-PBX1 expression induced myeloid hyperplasia by increasing myeloid differentiation propensity of hematopoietic stem cells (HSPC) and myeloid proliferation in larvae, and progressed to AML in adults. Mechanistic studies revealed that hE2A-PBX1 activated the TNF/IL-17/MAPK signaling pathway in blood cells and induced myeloid hyperplasia by upregulating the expression of runx1. Interestingly, through high-throughput drug screening, three small molecules targeting the TNF/IL-17/MAPK signaling pathway were identified, including OUL35, KJ-Pyr-9, and CID44216842, which not only alleviated the hE2A-PBX1-induced myeloid hyperplasia in zebrafish but also inhibited the growth and oncogenicity of human pre-B ALL cells with E2A-PBX1. Overall, this study provides a novel hE2APBX1 transgenic zebrafish leukemia model and identifies potential targeted therapeutic drugs, which may offer new insights into the treatment of E2A-PBX1 leukemia.

Single-cell analysis with childhood and adult systemic lupus erythematosus

Patients with systemic lupus erythematosus (SLE), a heterogeneous and chronic autoimmune disease, exhibit unique changes in the complex composition and transcriptional signatures of peripheral blood mononuclear cells (PBMCs). While the mechanism of pathogenesis for both childhood-onset SLE (cSLE) and adult-onset SLE (aSLE) remains unclear, cSLE patients are considered more unpredictable and dangerous than aSLE patients. In this study, we analysed single-cell RNA sequencing data (scRNA-seq) to profile the PBMC clusters of cSLE/aSLE patients and matched healthy donors and compared the PBMC composition and transcriptional variations between the two groups. Our analysis revealed that the PBMC composition and transcriptional variations in cSLE patients were similar to those in aSLE patients. Comparative single-cell transcriptome analysis between healthy donors and SLE patients revealed IFITM3, ISG15, IFI16 and LY6E as potential therapeutic targets for both aSLE and cSLE patients. Additionally, we observed that the percentage of pre-B cells (CD34-) was increased in cSLE patients, while the percentage of neutrophil cells was upregulated in aSLE patients. Notably, we found decreased expression of TPM2 in cSLE patients, and similarly, TMEM150B, IQSEC2, CHN2, LRP8 and USP46 were significantly downregulated in neutrophil cells from aSLE patients. Overall, our study highlights the differences in complex PBMC composition and transcriptional profiles between cSLE and aSLE patients, providing potential biomarkers that could aid in diagnosing SLE.

PBX1: a TALE of two seasons-key roles during development and in cancer.

Pre-B cell leukemia factor 1 (PBX1) is a Three Aminoacid Loop Extension (TALE) homeodomain-containing transcription factor playing crucial roles in organ pattering during embryogenesis, through the formation of nuclear complexes with other TALE class and/or homeobox proteins to regulate target genes. Its contribution to the development of several organs has been elucidated mainly through the study of murine knockout models. A crucial role for human development has been recently highlighted through the discovery of different de novo pathogenic PBX1 variants in children affected by developmental defects. In the adult, PBX1 is expressed in selected tissues such as in the brain, in the gastro-intestinal and urinary systems, or in hematopoietic stem and progenitor cells, while in other organs is barely detectable. When involved in the t(1;19) chromosomal translocation it acts as an oncogene, since the resulting fusion protein drives pre-B cell leukemia, due to the induction of target genes not normally targeted by the native protein. Its aberrant expression has been associated to tumor development, progression, or therapy-resistance as in breast cancer, ovarian cancer or myeloproliferative neoplasm (MPN). On the other hand, in colorectal cancer PBX1 functions as a tumor suppressor, highlighting its context-dependent role. We here discuss differences and analogies of PBX1 roles during embryonic development and in cancer, focusing mainly on the most recent discoveries.

Combinatorial targeting of telomerase and DNA-PK induces synergistic apoptotic effects against Pre-B acute lymphoblastic leukemia cells.

Due to the high demand for novel approaches for leukemia-targeted therapy, this study investigates the impact of DNA-PK inhibitor NU7441 on the sensitivity of pre-B ALL cells to the telomerase inhibitor MST-312. The study involved NALM-6 cells treated with MST-312 and NU7441, assessing their viability and metabolic activity using trypan blue and MTT assays. The study also evaluated apoptosis, gene expression changes, and DNA damage using flow cytometry, qRT-PCR, and micronucleus assays. The binding energy of MST-312 in the active site of telomerase was calculated using molecular docking. The study's findings revealed a synergistic decline in both cell viability and metabolic activity in NALM-6 cells when exposed to the combined treatment of MST-312 and NU7441, and this decrease occurred without any adverse effects on healthy PBMC cells. Furthermore, the combination treatment exhibited a significantly higher induction of apoptosis than treatment with MST-312 alone, as observed through flow cytometry assay. qRT-PCR analysis revealed that this enhanced apoptosis was associated with a notable downregulation of Bcl-2 expression and an upregulation of Bax gene expression. Moreover, the combination therapy decreased expression levels of hTERT and c-Myc genes. The micronucleus assay indicated that the combination treatment increased DNA damage in NALM-6 cells. Also, a good conformation between MST-312 and the active site of telomerase was revealed by docking data. The study suggests that simultaneous inhibition of telomerase and DNA-PK in pre-B ALL presents a novel targeted therapy approach.

Transcription factor PBX4 regulates limb development and haematopoiesis in mice.

The mammalian Pre-B cell leukaemia transcription factors 1-4 (PBX1-4) constitutes the PBC class of the homeodomain (HD)-containing proteins, which play important roles in diverse developmental processes. The functions and the underlying molecular mechanisms of PBX1-3 but not PBX4 have been extensively studied, and they have been reported to direct essential morphogenetic processes and organogenesis. In the present study, we generated knockin mice of FLAG-tagged PBX4 and the Pbx4 knockout (KO) mice and carried out in-depth characterisation of PBX4 expression and function. PBX4 was initially detected only in the testis among several organs of the adult mice and was expressed in spermatocytes and spermatids. However, no abnormality in spermatogenesis, but growth retardation and premature death after birth were observed in most adult Pbx4 KO mice. These animals were inactive and had shorter hindlimbs and lower numbers of reticulocytes and lymphocytes, probably caused by abnormalities at earlier developmental stages. Pbx4 mRNAs were indeed detected in several embryonic cell types related to limb development by in situ hybridisation and single-cell RNA-sequencing analysis. Pbx4 protein was also detected in the bone marrow of adult mice with a lower level compared with that in the testis. PBX4 preferentially binds to the promoters of a large number of genes including those for other HD-containing proteins and ribosomal proteins whose mutations are related to anaemia. PBX4-binding sites are enriched in motifs similar to those of other HD-containing proteins such as PKNOX1 indicating that PBX4 may also act as a co-transcription factor like other PBC proteins. Together, these results show that PBX4 participates in limb development and haematopoiesis while its function in spermatogenesis has not been revealed by gene KO probably due to the complementary effects of other genes.

IKZF3/Aiolos H195Y mutation identified in a mouse model of B cell leukemia results in altered DNA binding and altered STAT5-dependent gene expression

Precursor B cell acute lymphoblastic leukemia (Pre-B-ALL) arises from developing B cells and frequently involves mutations in genes encoding transcription factors. In this study, we investigated the function of mutations in the transcription factor IKZF3 (Aiolos), R137* and H195Y, discovered in a mouse model of pre-B-ALL. R137* IKZF3 mutation resulted in a truncated protein, while electrophoretic mobility shift assay showed that H195Y IKZF3 mutation resulted in a protein with altered DNA binding. 38B9 pre-B cell lines were generated expressing WT and H195Y IKZF3 proteins. Anti-IKZF3 ChIP-seq showed that H195Y IKZF3 interacted with a larger number of sites that were different than WT IKZF3. Treatment with interleukin-7 induced changes in gene expression in 38B9 cells expressing WT IKZF3, but did not induce any changes in gene expression in cells expressing H195Y IKZF3. Anti-STAT5 ChIP-seq showed that expression of H195Y IKZF3 resulted in redistribution of STAT5 binding sites in the genome. H195Y IKZF3 binding sites overlapped with a subset of STAT5 binding sites, including in the promoter of the Cish gene. These findings suggest that H195Y mutation of IKZF3 results in altered DNA binding specificity and altered binding of STAT5 to target genes.

Presence of Enteric Fever with Unusual Clinical Scenarios: A Case Series

Salmonella species are associated with bacteraemia, diarrhoea, as well as focal infections. Salmonella Typhi is the most pathogenic species. Salmonella Paratyphi B commonly affects immunocompromised hosts. The present case series showcases six cases of infection caused by typhoidal Salmonellae with unusual clinical scenarios. The first case report explains a case of neonatal meningitis where the causative agent was S. Paratyphi B. A case of lumbar spondylodiscitis is also reported where the aetiology was found to be Salmonella Typhi. Case three reports S. Typhi infection in a patient with Pre-B cell Acute Lymphoblastic Leukemia (ALL). S. Paratyphi B was isolated from a blood culture of a patient on haemodialysis and also from ascitic fluid from a case of Chronic Liver Disease (CLD). S. Typhi was isolated from the pleural fluid of a patient with carcinoma of the prostate.

The chemokine receptor CCR8 is not a high-affinity receptor for the human chemokine CCL18.

The primate-specific chemokine CCL18 is a potent chemoattractant for T cells and is expressed at elevated levels in several inflammatory diseases. However, the cognate receptor for CCL18 remains unconfirmed. Here, we describe attempts to validate a previous report that the chemokine receptor CCR8 is the human CCL18 receptor (Islam et al. J Exp Med. 2013, 210:1889-98). Two mouse pre-B cell lines (4DE4 and L1.2) exogenously expressing CCR8 exhibited robust migration in response to the known CCR8 ligand CCL1 but not to CCL18. Similarly, CCL1 but not CCL18 induced internalization of CCR8 on 4DE4 cells. CCR8 expressed on Chinese hamster ovarian (CHO) cells mediated robust G protein activation, inhibition of cAMP synthesis and β-arrestin2 recruitment in response to CCL1 but not CCL18. Several N- and C-terminal variants of CCL18 also failed to stimulate CCR8 activation. On the other hand, and as previously reported, CCL18 inhibited CCL11-stimulated migration of 4DE4 cells expressing the receptor CCR3. These data suggest that CCR8, at least in the absence of unidentified cofactors, does not function as a high affinity receptor for CCL18.

Association of Visfatin gene polymorphism with obesity related metabolic disorders among Pakistani population: a case control study

In recent years, the global prevalence of obesity and its associated metabolic disorders has reached alarming levels, presenting a significant challenge to public health worldwide. Visfatin, also known as pre-B cell colony-enhancing factor (PBEF) or nicotinamide phosphoribosyltransferase (NAMPT), is an adipokine that has been implicated in various physiological processes, including glucose homeostasis, lipid metabolism, and inflammation. The main objective of this proposed study is to find out the association between visfatin genetic variants and metabolic syndrome. The sample size of the study consisted of 300 blood samples (150 control and 150 cases). This study found that the genotypic frequency of visfatin SNPs, including rs2302559 (OD: 18.222; 95% CI 10.228–32.466; p-value < 0.001) and rs1215113036 (OD: 129.40; 95% CI 44.576–375.693; p-value < 0.001) were significantly associated with metabolic syndrome. Moreover, the frequency of the mutant alleles of both visfatin SNPs was found to be higher in patients with metabolic syndrome as compared to controls. Results of the current study indicate that people with any genetic variation of Visfatin, such as rs2302559 and rs1215113036, are more likely to develop metabolic syndrome. Visfatin genetic variants are linked to an increased risk of metabolic syndrome, implying it’s role in disease pathophysiology.

Constitutively active CaMKII Drives B lineage acute lymphoblastic leukemia/lymphoma in tp53 mutant zebrafish.

Acute lymphoblastic leukemia/lymphoma (ALL) is the most common pediatric cancer and is a malignancy of T or B lineage lymphoblasts. Dysregulation of intracellular Ca2+ levels has been observed in patients with ALL, leading to improper activation of downstream signaling. Here we describe a new zebrafish model of B ALL, generated by expressing human constitutively active CaMKII (CA-CaMKII) in tp53 mutant lymphocytes. In this model, B cell hyperplasia in the kidney marrow and spleen progresses to overt leukemia/lymphoma, with only 29% of zebrafish surviving the first year of life. Leukemic fish have reduced productive genomic VDJ recombination in addition to reduced expression and improper splicing of ikaros1, a gene often deleted or mutated in patients with B ALL. Inhibiting CaMKII in human pre-B ALL cells induced cell death, further supporting a role for CaMKII in leukemogenesis. This research provides novel insight into the role of Ca2+-directed signaling in lymphoid malignancy and will be useful in understanding disease development and progression.

MYSM1 attenuates DNA damage signals triggered by physiologic and genotoxic DNA breaks

Patients with deleterious variants in MYSM1 have an immune deficiency characterized by B-cell lymphopenia, hypogammaglobulinemia, and increased radiosensitivity. MYSM1 is a histone deubiquitinase with established activity in regulating gene expression. MYSM1 also localizes to sites of DNA injury but its function in cellular responses to DNA breaks has not been elucidated. This study sought to determine the activity of MYSM1 in regulating DNA damage responses (DDRs) to DNA double-stranded breaks (DSBs) generated during immunoglobulin receptor gene (Ig) recombination and by ionizing radiation. MYSM1-deficient pre- and non-B cells were used to determine the role of MYSM1 in DSB generation, DSB repair, and termination of DDRs. Genetic testing in a newborn with abnormal screen for severe combined immune deficiency, T-cell lymphopenia, and near absence of B cells identified a novel splice variant in MYSM1 that results in nearly absent protein expression. Radiosensitivity testing in patient's peripheral blood lymphocytes showed constitutive γH2AX, a marker of DNA damage, in B cells in the absence of irradiation, suggesting a role for MYSM1 in response to DSBs generated during Ig recombination. Suppression of MYSM1 in pre-B cells did not alter generation or repair of Ig DSBs. Rather, loss of MYSM1 resulted in persistent DNA damage foci and prolonged DDR signaling. Loss of MYSM1 also led to protracted DDRs in U2OS cells with irradiation induced DSBs. MYSM1 regulates termination of DNA damage responses but does not function in DNA break generation and repair.

Regulation of the STAT3 pathway by lupus susceptibility gene Pbx1 in T cells

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease in which poorly characterized genetic factors lead to the production of proinflammatory or autoreactive T cells. Pre-B cell leukemia homeobox 1 (PBX1) is a transcription factor whose dominant negative isoform (PBX1-D) is overexpressed in the CD4+ T cells of SLE patients and lupus-prone mice. Pbx1-D overexpression favors the expansion of proinflammatory T cells and impairs regulatory T (Treg) cell development. Here we show that Pbx1 deficiency and Pbx1-D overexpression decreased STAT3 expression and activation in T cells. Accordingly, Pbx1 deficiency in T cells and Pbx1-D overexpression reduced STAT3-dependent TH17 cell polarization in vitro, but it had no effect in vivo at steady state. STAT3-dependent follicular helper T (TFH) cell polarization in vitro and splenic TFH cell frequency were not affected by either Pbx1 deficiency or Pbx1-D overexpression. Pbx1 deficiency also increased the expression of cell cycle arrest and pro-apoptotic genes, with an increased apoptosis in T cells. Our results suggest a complex interplay between PBX1 and STAT3, which may contribute to lupus pathogenesis through dysregulation of the cell cycle and apoptosis.

Uridine Synthesis Is the Metabolic Vulnerability in Relapse-Associated B-ALL Cells with Active Pre-BCR Signaling

Relapse stubbornly continues to limit survival for children with B-cell progenitor acute lymphoblastic leukemia (B-ALL). B-ALL cells accumulate at the developmental checkpoint regulating the transition from pro-B to pre-B cells and express the pre-B cell receptor (preBCR). B-ALL samples have previously been categorized as preBCR positive and preBCR negative subtypes. We previously found that pre-B cells with active preBCR signaling are present at diagnosis and highly predictive of relapse, termed relapse predictive cells (RPCs) (Good et al. Nature Med 2018). Whole transcriptome sequencing of pre-B RPCs from patient samples demonstrated enrichment of gene signature pathways is involved in oxidative phosphorylation (OXPHOS), pyrimidine metabolism, and glycolysis pathways compared to pre-B cells from patients in complete remission. Thus, we hypothesized that RPCs have unique metabolic demands driven by their active signaling. Utilizing TARGET dataset, B-ALL patients were classified as preBCR high or low based on the sum of normalized counts of preBCR components and signaling molecules. Notably, preBCR high patients (top 25%ile) had inferior overall survival compared to preBCR low (bottom 75%ile; P=0.009). Further, preBCR high patients demonstrated enrichment for the metabolic gene signatures identified in RPCs. CyTOF analysis in primary B-ALL samples demonstrated increased expression of glycolysis (PFKFB4, ENO1, LDHA) and pentose phosphate pathway (G6PD, PGD) proteins in preB cells from patients destined to relapse compared to those in continued remission. These data support the link between a distinct metabolic state in preB cells associated with relapse. To investigate the metabolic state associated with the preBCR in B-ALL, we utilized cell line models (preBCR high: Nalm6, 697, Kasumi2; preBCR low: REH, Nalm16, RS4;11). We found that preBCR high cells are more metabolically active than preBCR low cells with significantly higher oxygen consumption rate and extracellular acidification rate. To identify their targetable dependencies, cells were starved of glucose or glutamine for 24 hours. Neither cell type was sensitive to glutamine starvation. However, preBCR high cells were uniquely vulnerable to glucose deprivation (killing effect 64% vs 25%, P=0.027) indicating a distinct need for glucose in preBCR high cells. To unravel the unique glucose dependency of preBCR high cells, we performed isotype tracing with U- 13C-glucose and U- 13C-glutamine. Both preBCR high and preBCR low cells use glutamine as the primary carbon source in the TCA cycle. However, glucose is primarily utilized for the pentose phosphate pathway and nucleotide synthesis in preBCR high cells, as evidenced by higher fractional 13C labeling in m+5 UTP, UDP and ATP. Uridine, but not other pyrimidines or purines, rescued preBCR high cells from glucose starvation, highlighting glucose-dependent uridine synthesis as the metabolic vulnerability of preBCR high cells. To target this dependency, we treated the cells with the dihydroorotate dehydrogenase (DHODH) inhibitor (BAY-2402234), which disrupts a crucial step in uridine synthesis. Remarkably, DHODH inhibition resulted in preferential killing in preBCR high but not preBCR low cells. Evaluation of the in vivo efficacy of DHODH inhibition in xenografts will be presented. Together, we found that preBCR high B-ALL cells are associated with relapse and are highly energetic, relying uniquely on glucose for survival to fuel the PPP and uridine synthesis. Inhibition of uridine synthesis is a novel strategy to target these treatment resistant and relapse-associated cells.

Genomic Determinants of Therapy Response in ETV6::RUNX1 Leukemia

ETV6::RUNX1 leukemia is the second most common childhood B-cell acute lymphoblastic leukemia subtype. Although it has a low overall relapse risk, a significant proportion of relapses occur within this subtype due to its relatively high incidence. No consistent genomic biomarkers have been identified that predict therapy response beyond early therapy response as measured by minimal residual disease at the end of induction. In this study, our aim was to use multiomic data from 175 ETV6::RUNX1 leukemias treated according to the NOPHO ALL2000 and ALL2008 protocols to identify genomic features that predict therapy response at presentation. Cases were classified into three responder groups based on MRD at end-of-induction (EOI) therapy: fast responders with negative MRD, intermediate responders with positive but &amp;lt;0.1% MRD, and slow responders with &amp;gt;0.1% MRD. Response data was also collected at mid-induction (day 15) and end-of-consolidation (EOC), when available. Diagnostic and remission DNA were analyzed using array-based copy number data (n=142) or whole genome sequencing (WGS) (n=33), and diagnostic bulk RNA sequencing (n=51) was complemented with single cell multiomics (RNA, surface protein and VDJ sequencing) data from both healthy (n=8) and leukemic bone marrow cells upon chemotherapy (n=8). Overall, the frequency of somatic genetic changes was not significantly associated with treatment response suggesting that genetic diversification through elevated random mutagenesis events may not underlie the drug resistant phenotype. However, analysis of mutational signatures revealed that fast-responding leukemic blasts frequently exhibited the APOBEC mutational signature and had higher cell cycle activity (Figure 1A). Accordingly, we show that expression of A3B is highly specific to cycling cells also in healthy bone marrow hematopoietic progenitor cells, pro- and pre-B cells. We also examined RAG-mediated genetic instability that manifests in focal structural variations and has highest activity in ETV6::RUNX1leukemia (Figure 1B). We confirmed several recurrent RAG off-target sites, including significant enrichment of the known risk locus TBX1LR1 in slow responding cases. Strikingly, slow-responding leukemias had also frequent rearrangements in the immunoglobulin kappa genes and the kappa-deleting elements, despite their pro-B-like transcriptome state upon diagnosis. Based on the single cell multiomics profiles, we found that upon treatment the blast phenotype shifts towards an immature-B-like transcriptome, with concomitant expression of Ig light chains with productive sequence, thereby permitting activation of BCR signaling that provides a survival advantage. At CNV level, we found that the chromosomal alterations related to the translocation event associated with treatment response. Specifically, slow responders had more often the amplification of der(21)t(12;21) chromosome, impacting expression of USP5, TIGAR and SLC2A3, associated in earlier studies to chemoresistance and tumorigenesis. In comparison, fast responders were often missing a region of chromosome 12p with lower expression of several genes including oncogenic KRAS. In summary, combining multiomic and response data, we characterized genetic alterations and mutational processes that affect the treatment response in ETV6::RUNX1leukemia. Our findings highlight the genetic and transcriptomic heterogeneity of this common pediatric leukemia subtype and its implications for treatment sensitivity, paving the way for better classification of the disease at presentation.

The Therapeutic Efficacy of CD19 Chimeric Antigen Receptor T Cells in Immune Thrombocytopenia Model

Abstract Introduction: ITP is a hemorrhagic autoimmune disease characterized by excessive platelet destruction and impaired platelet production,resulting in a high risk of bleeding. The pathogenesis of ITP involves immune intolerance towards platelet antigen, with platelet autoantibodies produced by plasma cells playing a significant role. Current treatments targeting this pathogenesis mainly rely on CD20 monoclonal antibodies, such as rituximab. However, as CD20 expression is present at the pre-B cell and mature B cell stages but absent on the surface of mature plasma cells. This can explain why a subset of autoantibody-positive patients do not respond to B-cell depletion therapy. CAR-T cell therapy, a form of adoptive cellular immunotherapy, has shown great potential in hematological malignancies and solid tumors. Recent studies have demonstrated its potential effectiveness of CAR-T cell therapy in various autoimmune diseases. CD19, expressed at all stages of B cell differentiation, presents an opportunity for CAR-T cell therapy in ITP. This study aims to evaluate the efficacy of CD19 CAR-T cell immunotherapy in an active murine ITP model. Methods: CD42-knockout mice were immunized with wild type (WT) C57BL/6 mice and splenocytes from these immunized mice were transplanted into irradiated WT mice to construct a murine model of active ITP. The ITP mice were then randomized into two groups, with the experimental group receiving an intraperitoneal injection with 1×10 6 CD19 CAR-T cells. Platelet counts were monitored weekly, and after 3 weeks, the ITP mice were euthanized and sacrificed. Single-cell suspensions were prepared from harvested spleens for analysis. Anti-murine antibodies were analyzed using flow cytometry (Beckmann Coulter, Brea, CA, USA). All data were analyzed by Statistical Package for the Social Sciences (SPSS) software, version 25.0 (IBM Corp., Armonk, NY, USA). P &amp;lt; 0.05 was considered statistically significance. Results: Our results demonstrated that the platelet counts of ITP mice reached their lowest level on day 7 after spleen cell transplantation, gradually recovering within a week. Platelet counts were significantly higher in the CD19 CAR-T cell therapy group compared to the control group on day 7 and persisted until day 21. There were no significant differences in weight between two groups, indicating no severe complication or CRS were identified. In the third week CD19 CAR-T cell therapy, the proportion of plasma cells in the spleen was determined, showing a marked decrease in the percentage of CD138+ plasma cells in the CAR-T group compared to the control group. Additionally, at day 14, the titers of anti-platelet CD42-specific antibodies were significantly lower in the CAR-T cell treated group compared to the control group. Furthermore, the mean fluorescence intensity (MFI) of CD19+ B cells in the spleen cells of the CD19 CAR-T cell immunotherapy group was significantly decreased compared to the control group. Conclusion: This study demonstrates the effectiveness of CD19 CAR-T cell therapy in treating thrombocytopenia by suppressing plasma and B cells in a mouse model of active ITP. These findings provide valuable insights into the potential therapeutic application of CD19 CAR-T cell immunotherapy for immune thrombocytopenia. Disclosure: No conflict of interest.

Optimizing Ex-Vivo Expanded NK Cell- Mediated Cellular Cytotoxicity By Obinutuzumab Combined with NKTR-255 in Burkitt Lymphoma (BL)

Background: We have previously reported the success of treating children with Burkitt lymphoma (BL) resulting in a ≥90% long-term EFS utilizing short intensive rituximab containing chemoimmunotherapy (Cairo et al, Blood, 2007; Goldman /Cairo et al, Leukemia, 2012, Goldman /Cairo et al, Br J Haematol., 2014; Goldman /Cairo et al, Leukemia, 2021). However, the prognosis is dismal in children who are refractory or progress after chemoimmunotherapy (Cairo et al Br J Haematol. 2018). The mechanisms associated with this poor prognosis are mainly due to chemoradioimmunotherapy resistance and an immunosuppressive BL microenvironment (TME) (Cairo et al Br J Haematol. 2016; Cairo et al Br J Haematol. 2019) and represent an urgent unmet need. The CD20 molecule is universally expressed by normal B cells in all stages of development, from the pre-B cell up to the mature plasma cell as well as by most B cell malignancies including CLL, FL and BL (Chu/Cairo, BJH, 2016). Obinutuzumab is a humanized, type II anti-CD20 monoclonal antibody glycoengineered to enhance Fc receptor affinity. It has lower complement-dependent cytotoxicity than rituximab but greater ADCC, phagocytosis and direct B-cell killing effects (Chu/Cairo, BJH, 2018). Obinutuzumab has been successfully utilized in front-line therapy in FLL (Marcus, et al, NEJM, 2017) and CLL (Goede, et al, NEJM, 2014; Moreno, et al, Lancet, 2019). Our group has successfully ex-vivo expanded functional and active peripheral blood NK cells (PBNK) with irradiated feeder cells APC (K562-IL21-41BBL) (Chu/Cairo, et al, JITC 2020). We previously demonstrated that obinutuzumab has significantly enhanced ex-vivo expanded PBNK mediated cytotoxicity against BL and pre-B-ALL cell lines compared to rituximab (Tiwari/Cairo et al, BJH, 2015). NKTR-255 is an IL-15 receptor agonist designed to activate the IL-15 pathway and NK cells and promote the survival and expansion of memory CD8+ T cells without inducing suppressive regulatory T cells (Kuo/Zalevsky, Cancer Res. 2017). NKTR-255 stimulates proliferation and survival of NK, CD8+ T cells, which may lead to sustained anti-tumor immune response. Objective: To investigate the anti-BL effects of NKTR-255 on the ADCC of ex-vivo expanded NK cells with obinutuzumab against rituximab-resistant BL in vitro and in vivo using human BL xenografted NSG mice. Methods: NK cells were expanded with lethally irradiated K562-mbIL21-41BBL cells as previously described (Denman/Lee, et al, PLoS One, 2012). Expanded PBNK cells were isolated using Miltenyi NK cell isolation kit. NKTR-255 was generously provided by Nektar Therapeutics. In vitro cytotoxicity was examined using luminescence reporter-based assays. IFN-g, granzyme B and perforin levels were examined by standard enzyme-linked immunosorbent assays as we previously described (Chu/Cairo, Front. Immunol, 2022). Rituximab-resistant BL cells Raji-2R and Raji-4RH were used as target cells. Luciferase expression Raji-4RH cells were xenografted to NSG mice as we previously described (Chu/Cairo, et al, JITC 2021). Results: NKTR-255 significantly enhanced the proliferation of ex-vivo expanded NK cells (p&amp;lt;0.0001) at day 7. NKTR-255 significantly enhanced the in vitro cytoxicity of expanded NK cells when combined with obinutuzumab against rituximab-resistant BL cells like Raji-2R (E:T=3:1, p &amp;lt;0.01), and Raji-4RH (E:T=3:1, p&amp;lt;0.01) as compared to the control groups. NKTR-255 also significantly enhanced IFN-g, granzyme and perforin release from expanded NK cells when combined with obinutuzumab against Raji-2R (E:T=3:1, IFN-g: p&amp;lt;0.001, granzyme: p&amp;lt;0.001 and perforin: p&amp;lt;0.001) and Raji-4RH (E:T=3:1, IFN- g: p&amp;lt;0.001, granzyme: p&amp;lt;0.01 and perforin: p&amp;lt;0.01) as compared to controls. Our in vivo study demonstrated that the combination of NKTR-255, Obinutuzumab and expanded NK cells significantly improved the survival of mice xenografted with Raji-4RH compared to controls (Fig.1). Conclusion: We found that NKTR-255 significantly enhanced the ADCC of expanded NK cells with Obinutuzumab against rituximab-resistant BL cells in vitro with enhanced IFN- g, granzyme B and perforin release. The in vivo effects of NKTR-255 with expanded NK cells and Obinutuzumab against rituximab-resistant BL cells using humanized NSG models are very promising. Mechanisms studies of BL relapsed from the combination therapy are under investigation. (Supported by HHOW and St Baldrick grants).

Targeting Endothelial PERK-DLL4 Axis to Enhance Hematopoietic Stem Cell and Lymphoid Progenitor Regeneration

Despite our current information on hundreds of genes implicated in hematopoietic stem cells (HSC) biology, major gaps still exist in our understanding of HSC physiology and their regeneration mechanism under disease and stress conditions, hindering the development of strategies to enhance HSC regeneration. HSCs reside in a tightly controlled bone marrow HSC niche that regulates and maintains hematopoietic homeostasis. HSC niche is composed of a heterogeneous population of specialized stroma cells. Among these, the endothelial cells and the perivascular stroma cells provide critical pro-hematopoietic factors to support HSC pool and committed lineage progenitors particularly lymphoid progenitors. In addition, evidence suggests that endothelium-expressing Notch ligand may promote Notch-dependent long-term HSC proliferation. Recent single cell transcriptome profiling revealed that Delta-like 4 (DLL4) is expressed by the vascular endothelial cells at a higher level than other Notch ligands while its expression down-regulates in response to myeloablation. However, the molecular mechanism regulating the dynamic DLL4 expression during hematopoietic regeneration in response to myeloablative stimuli is unknown. Unfolded protein response (UPR) (also known as endoplasmic reticulum stress, ER stress) has been increasingly recognized as crucial for HSC maintenance. During hematopoietic regeneration, UPR is critical for meeting the increased demand for protein synthesis required for rapid cellular proliferation and for adapting to pathological conditions such as increased levels of reactive oxygen species following irradiation or chemotherapy. Unlike HSCs, how HSC niche endothelial cells deal with redox imbalance and higher requirements of vessel regeneration to regulate stress hematopoietic regeneration is not clear. In this study, using genetically engineered mouse models, we examined the dynamic expression and function of PERK, one arm of UPR, and its potential downstream target, Dll4, during the stress hematopoiesis triggered by transplantation. We found that PERK is activated in the mouse bone marrow endothelial cells following irradiation. Ablation of endothelial Perk in mice (referred as Perk iΔEC mice) promoted post-transplantation immediate recovery of blood cells derived from wild type donor mice and enhanced HSC and lymphoid progenitor regeneration with the expansion of B lineage progenitors including pre-proB, pro-B, and pre-B cells. In addition, by whole-mount immunostaining and intravital microscopy, we found that endothelial Perk deletion restrained disorganized angiogenesis and vascular leakage provoked by irradiation. Although irradiation itself had no effect on endothelial Dll4, Perk ablation markedly increased endothelial Dll4 expression. We found that Dll4 is essential for the post-irradiation HSC and lymphoid progenitor rejuvenation as ablation of endothelial Dll4 in mice (referred as Dll4 iΔEC mice) led to impaired short-term and long-term hematopoietic regeneration and markedly decreased wild type donor derived peripheral B cell and lymphoid progenitor recovery. We found that endothelial Dll4 is required for HSC quiescence maintenance, HSC niche retention and HSC self-renewal. Further, ablation of endothelial Dll4 led to disorganized bone marrow angiogenesis and increased marrow vascular leakage. Finally, we showed that deletion of Dll4 in Perk iΔEC mice completely abrogated the increased numbers of HSC and lymphoid progenitors mediated by Perk deletion, suggesting that up-regulation of endothelial Dll4 is likely responsible for Perk ablation-mediated enhanced HSC and lymphoid progenitor regeneration. Taken together, our results revealed a novel regulatory mechanism by which endothelial Dll4 expression is suppressed by PERK of UPR during transplantation while ablating Perk increases Dll4 expression. Endothelial DLL4 in the bone marrow endothelial cells is critical for supporting vascular integrity and myeloablation-induced HSC and lymphoid progenitor regeneration. Our findings suggest that targeting endothelial PERK-DLL4 axis could be a viable option to improve post-irradiation regeneration of HSC and lymphoid progenitors.

Single-Cell RNA Sequencing Identifies Distinct Immune Microenvironment in Patients with Primary Plasma Cell Leukemia

Background: Primary plasma cell leukemia (PCL) is a highly aggressive form of multiple myeloma (MM). While recent advances in cellular immunotherapy have significantly improved the outcome of patients with MM, these benefits have been limited for PCL patients, and their survival remains poor. This is in partly attributable to our limited understanding of the dysregulated immune microenvironment in PCL. Furthermore, distinct biological features of PCL compared with MM have not been clearly defined. In this regard, we performed single-cell RNA sequencing of bone marrow (BM) immune cells and tumor cells from patients with MM and PCL at diagnosis and compared them with BM immune cells from healthy donors. Methods: Single-cell analysis was performed using 10X Genomics with BM mononuclear cells (BMMCs) isolated from healthy donors (n=3), MM patients (n=2), PCL patients with lower circulating plasma cells (5-20%) (PCL-1) (n=2), and PCL patients with higher circulating plasma cells (&amp;gt;20%) (PCL-2) (n=2) at diagnosis. Sequencing files were processed with CellRanger (10x Genomics) and the data were analyzed with the Seurat toolkit. Cell types were annotated with SingleR based on the Human Primary Cell Atlas built-in references. Pre-B cells, B cells, plasmablasts, plasma cells, granulocyte/macrophage progenitors (GMPs), hematopoietic stem and progenitor cells (HSPCs), myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs), classical monocytes, non-classical monocytes, T cells, natural killer (NK) cells, and stromal cells between healthy donors, MM, PCL-1, and PCL-2 were annotated. Results: We sequenced a total of 85,268 BMMCs to analyze the heterogeneity and transcriptomic features of BMMCs from healthy, MM, and PCL patients. We characterized changes in the transcriptomic features of T cells, NK cells, monocytes, and DCs between healthy, MM, PCL-1, and PCL-2. We examined CD8 + and CD4 + T cell subsets based on differentiation status, including CCR7 + CD45RA + naïve (TN), CCR7 + CD45RA − central memory T (TCM), CCR7 − CD45RA − effector memory (TEM), and CCR7 − CD45RA + terminally differentiated effector memory T (TEMRA) cells. The proportion of CD8 + TN was decreased in MM, PCL-1, and PCL-2 compared with healthy, while the proportion of TEM was increased in MM and PCL-2 compared with healthy. Of note, the proportion of CD8 + NKG7 + cells was highest in PCL-2, followed by PCL-1 and MM, which were minimally detected in healthy. Among CD4 + T cells, the proportion of regulatory T cells did not differ between the groups. However, cytotoxic CD4 + T cells was increased in MM, PCL-1, and PCL-2 compared with healthy. Analysis of NK cells revealed that the proportion of immature NK cells was decreased, while the proportion of mature NK cells was increased in patients with MM, PCL-1, and PCL-2 compared with healthy. We also examined the plasma cells and observed that diversity in plasma cell types is increased in PCL-1 and PCL-2 compared with MM, indicating that the PCL clone is heterogeneous. Conclusion: We demonstrate the characteristics of BM immune cells in PCL by single-cell RNA sequencing. Additional analyses are ongoing to uncover the association between PCL burden and BM immune cell composition and to gain insight into PCL heterogeneity and define high-risk PCL. Our work should improve our understanding to decipher therapeutic options for PCL.