Childhood Acute Lymphoblastic Leukemia Research Articles

Predictors of low and very low bone mineral density in long-term childhood acute lymphoblastic leukemia survivors: Toward personalized risk prediction.

Cohorts of childhood acute lymphoblastic leukemia (cALL) survivors reaching adulthood are increasing. Approximately 30% of survivors meet criteria for low bone mineral density (BMD) 10years after diagnosis. We investigated risk factors for low BMD in long-term cALL survivors. We recruited 245 cALL survivors from the PETALE (Prévenir les effets tardifs des traitements de la leucémie aiguë lymphoblastique chez l'enfant) cohort, who were treated with the Dana Farber Cancer Institute protocols, did not experience disease relapse or hematopoietic stem cell transplants, and presented with more than 5years of event-free survival. Median time since diagnosis was 15.1years. Prevalence of low DXA-derived BMD (Z-score ≤-1) ranged between 21.9% and 25.3%, depending on site (lumbar spine (LS-BMD), femoral neck (FN-BMD), and total body (TB-BMD), and between 3.7% and 5.8% for very low BMD (Z-score ≤-2). Males had a higher prevalence of low BMD than females for all three outcomes (26%-32% vs. 18%-21%), and male sex acted as a significant risk factor for low BMD in all models. Treatment-related factors such as cumulative glucocorticoid (GC) doses and cranial radiation therapy (CRT) were associated with lower BMDs in the full cohort and in females at the FN-BMD site. Low and very low BMD is more prevalent in male cALL survivors. Male sex, high cumulative GC doses, CRT, risk group, and low body mass index (BMI) were identified as risk factors for low BMD. A longer follow-up of BMD through time in these survivors is needed to establish if low BMD will translate into a higher risk for fragility fractures through adulthood.

Investigation of inherited noncoding genetic variation impacting the pharmacogenomics of childhood acute lymphoblastic leukemia treatment.

Defining genetic factors impacting chemotherapy failure can help to better predict response and identify drug resistance mechanisms. However, there is limited understanding of the contribution of inherited noncoding genetic variation on inter-individual differences in chemotherapy response in childhood acute lymphoblastic leukemia (ALL). Here we map inherited noncoding variants associated with treatment outcome and/or chemotherapeutic drug resistance to ALL cis-regulatory elements and investigate their gene regulatory potential and target gene connectivity using massively parallel reporter assays and three-dimensional chromatin looping assays, respectively. We identify 54 variants with transcriptional effects and high-confidence gene connectivity. Additionally, functional interrogation of the top variant, rs1247117, reveals changes in chromatin accessibility, PU.1 binding affinity and gene expression, and deletion of the genomic interval containing rs1247117 sensitizes cells to vincristine. Together, these data demonstrate that noncoding regulatory variants associated with diverse pharmacological traits harbor significant effects on allele-specific transcriptional activity and impact sensitivity to antileukemic agents.

Exercise and QUality diet After Leukemia (EQUAL): A randomized weight loss trial among adult survivors of childhood leukemia in the Childhood Cancer Survivor Study.

Obesity is prevalent in childhood cancer survivors and interacts with cancer treatments to potentiate risk for cardiovascular (CV) death. We tested a remote weight-loss intervention that was effective among adults with CV risk factors in a cohort of adult survivors of childhood acute lymphoblastic leukemia (ALL) with overweight/obesity. In this phase 3 efficacy trial, survivors of ALL enrolled in the Childhood Cancer Survivor Study with body mass index (BMI)≥25 kg/m2 were randomized to a remotely-delivered weight-loss intervention versus self-directed weight loss, stratified by history of cranial radiotherapy (CRT). The primary endpoint was the difference in weight loss at 24-months in an intent-to-treat analysis. Analyses were performed using linear mixed effects models. Among 358 survivors (59% female, median attained age: 37 years, IQR: 33-43), baseline mean (SD) weight was 98.6 kg (24.0) for the intervention group (n=181) and 94.9 kg (20.3) for controls (n=177). Adherence to the intervention was poor; 15% of individuals in the intervention completed 24/30 planned coaching calls. Weight at 24-months was available for 274 (77%) participants. After controlling for CRT, sex, race/ethnicity, and age, the mean (SE) change in weight from baseline to 24-months was -0.4 kg (0.8) for intervention and 0.2 kg (0.6) for control participants (p=0.59). A remote weight-loss intervention that was successful among adults with CV conditions did not result in significant weight loss among adult survivors of childhood ALL. Future interventions in this population must be tailored to the unique needs of survivors to encourage engagement and adherence.

Impact of the COVID-19 pandemic on utilization and cost for care of pediatric and young adult ALL

ObjectiveAcute lymphoblastic leukemia (ALL) is the most common childhood malignancy and among the most common malignancies in young adults and requires a unique pattern of healthcare utilization including an acute/emergent presentation and an intensive initial 8 months of therapy followed by two years of outpatient treatment. The COVID-19 pandemic caused massive global disruptions in healthcare use and delivery. This report aims to examine the effects of the COVID-19 pandemic on the presentation, diagnosis and continued management of childhood and young adult ALL in regard to utilization and cost of care among commercially insured individuals in the United States.ResultsUtilizing a commercial insurance claims database, 529 pediatric and young adult patients were identified who were diagnosed with ALL between January 2016 and March 2021. New diagnoses were evaluated by era and demographics. Utilization was measured by COVID-related era as number of inpatient and outpatient encounters, inpatient days, and cumulative cost during the initial 8 months of therapy. None of these cost or utilization factors changed significantly during or shortly after the pandemic. These findings reinforce that the necessary care for pediatric and young adult ALL was unwavering despite the massive shifts in the healthcare system caused by the COVID-19 pandemic. This provides a valuable benchmark as we further examine the factors that influence the pandemic’s impact on health equity and access to care, especially in vulnerable pediatric and young adult populations. This is the first investigation of the effect of the COVID-19 pandemic on utilization and cost of care in pediatric and young adult cancer.

Quality of life in children and adolescents after treatment for acute lymphoblastic leukemia according to the NOPHO ALL2008 protocol.

The improved outcome of childhood acute lymphoblastic leukemia (ALL) over the last decades has increased the importance of assessing late effects and health-related quality of life (HRQoL), particularly when evaluating and comparing outcomes in clinical trials. This study aimed to assess HRQoL in children treated for ALL according to the NOPHO ALL2008 protocol. Children, aged 1 to less than 18years at diagnosis, alive in first remission, and their parents, were asked to complete PedsQL 4.0 Generic Core Scales (self- and proxy-report) at ≥6months after end of therapy. Data on socioeconomic factors and parent-reported toxicity were collected through a study-specific questionnaire, and the NOPHO ALL2008 database was used to identify eligible families and add additional disease- and treatment-related data. HRQoL data were collected during 2013-2019 in Sweden, Finland, and Denmark. A total of 299 children were included. The older children (8years and older) reported similar HRQoL scores compared to Finnish reference data, except lower scores for School Functioning in high-risk patients. Scores from the parent-proxy and self-reports from 5-7-year olds were notably lower than reference. Parent-reported toxicity was associated with lower total and physical HRQoL scores in adjusted models for younger as well as older children in the self-report and parent-proxy versions, and also with lower psychosocial score in the parent-proxy. Self-reported HRQoL was similar to reference population. The most important determinant for HRQoL after end of ALL treatment was parent-reported toxicity during treatment. Thus, minimizing complications is an obvious focus for future treatment protocols.

Adult survivors’ perceptions of their childhood and the influences of being treated for acute lymphoblastic leukaemia with allogeneic hematopoietic stem cell transplantation as a child: A phenomenographic study

PurposeAdults who had acute lymphoblastic leukaemia (ALL) as children and were treated with allogeneic hematopoietic stem cell transplantation (aHSCT) may have been affected in their lives due to several long-term complications. From a clinical point of view, it is of interest to study how survivors describe their perceptions of their childhood today. The aim was therefore to describe how adults perceived their childhood and the influences of being treated for ALL with aHSCT as a child. MethodSemi-structured telephone interviews were undertaken with 18 adults who had been treated for childhood ALL with aHSCT and were included in a national cohort of childhood ALL survivors, diagnosed between 1985 and 2007 at an age between 0 and 17 years. A phenomenographic analysis was used. ResultsThree categories emerged: Feeling different, Feeling security and Feeling guilty. The informants felt that they had been different from other children but had felt security with the healthcare professionals and in care. They felt guilty because both their siblings' and parents’ lives had been affected, but at the same time many perceived that they and their family members had become closer to one another. ConclusionsThe results emphasised that adults who had been treated for childhood ALL with aHSCT were affected both in negative and positive ways during their childhood. This indicates the importance for early psychosocial care interventions directed to children during their treatment, but also the need for person-centred psychological care in long-term outpatient clinics.

Bispecific monoclonal antibody blinatumomab in the first-line therapy of B-lineage acute lymphoblastic leukemia in children and adolescents: interim results of the Russian Ministry of Health approbation protocol

Bispecific monoclonal antibody blinatumomab in the first-line therapy of B-lineage acute lymphoblastic leukemia in children and adolescents: interim results of the Russian Ministry of Health approbation protocol

Comparison of Adaptive Functioning Between Survivors of Childhood Acute Lymphoblastic Leukemia and Their Healthy Peers; An Experience from a Low- and Middle-Income Country

Introduction: Acute Lymphoblastic Leukemia (ALL) is the most common cancer diagnosed in children. Advances in treatment increased the survival rates of these children. One important long term aspect of survivorship is adaptive functioning, which plays a pivotal role in a child’s quality of life. This study compares the adaptive functions of childhood leukemia survivors with their healthy peers. Methods: This descriptive case control study was conducted at the Pediatrics Hematology/ Oncology Department of Indus Hospital and Health Network (IHHN), from November 2022 to April 2023. Survivor children and adolescents aged 12 to 18 years old treated for ALL were included. Cases were divided in two groups: Group A, ALL survivors, and Group B, healthy adolescents matched in age to the ALL survivors. VABS assessment was conducted by a qualified psychologist, to evaluate the adaptive functioning. All the information was entered and analyzed by SPSS version 20. Results: A total of 50 adolescent children were examined along with 50 healthy controls. 72% of the survivors had B-cell ALL (B-ALL), followed by 22% had T-cell ALL (T-ALL) and 6%, had Mixed Phenotype ALL. Mean of adaptive behavior composite score, communication skills, daily living skills and the socialization skills standard score were significantly lower among patients compared to controls (p=0.0001). Conclusion: Overall adaptive skills were significantly affected among the survivors. These findings underscore the importance of tailored interventions and support services to enhance the adaptive functioning and overall well-being of these resilient survivors.

Neuropsychological task outcomes among survivors of childhood acute lymphoblastic leukemia in Malaysia

This study intended to explore the neuropsychological ramifications in childhood acute lymphoblastic leukemia (ALL) survivors in Malaysia and to examine treatment-related sequelae. A case-control study was conducted over a 2-year period. Seventy-one survivors of childhood ALL who had completed treatment for a minimum of 1 year and were in remission, and 71 healthy volunteers were enlisted. To assess alertness (processing speed) and essential executive functioning skills such as working memory capacity, inhibition, cognitive flexibility, and sustained attention, seven measures from the Amsterdam Neuropsychological Tasks (ANT) program were chosen. Main outcome measures were speed, stability and accuracy of responses. Mean age at diagnosis was 4.50 years (SD ± 2.40) while mean age at study entry was 12.18 years (SD ± 3.14). Survivors of childhood ALL underperformed on 6 out of 7 ANT tasks, indicating poorer sustained attention, working memory capacity, executive visuomotor control, and cognitive flexibility. Duration of treatment, age at diagnosis, gender, and cumulative doses of chemotherapy were not found to correlate with any of the neuropsychological outcome measures. Childhood ALL survivors in our center demonstrated significantly poorer neuropsychological status compared to healthy controls.

Changes in the Expression of DNMTs Before and After Treatment with Methotrexate (MTX)/Mercaptopurine (6-MP) in B-Cell ALL Children

Background: DNA methylation is catalyzed by DNA methyltransferases (DNMTs) which are encoded by DNMT1, DNMT3A, and DNMT3B. DNMTs play a major role in the abnormal methylation of tumor suppressors and cancer-related genes. Herein, this study explored the expression profile of DNMTs in pediatric patients with B-cell acute lymphoblastic leukemia (ALL), before and after methotrexate (MTX)/mercaptopurine (6-MP) treatment.
 Materials and Methods: This before-after prospective study included 30 matched children in sex and age (20 children with B-cell ALL and 10 healthy children used as a control or calibrator group). The expression profile of DNMTs was assessed at two-time points; at the diagnosis time and after MTX/6-MP treatment in the consolidation-maintenance phase of therapy. Notable, all pediatric patients included in this study continued the therapy without adverse events, except two children who were excluded from the study.
 Results: The average age of the patient group was 7.1 ± 1.3 years (in the range of 4-9 years), and the average age of the control group was 8.3 ± 1.7 years (6-10 years). The expression profile of DNMTs in B-cell ALL children was obtained completely different from that in the healthy group. After MTX/6-MP treatment of B-cell ALL children, the expression levels of DNMT1 and 3A were increased (p <0.01 & 0.04, respectively), and the expression level of DNMT3B was decreased (p <0.01), significantly.
 Conclusions: In ALL, the expression profile of DNMTs would be changed whereby contribute to abnormal growth and maturation capacity of leukemic stem cells and MTX/6-MP treatment could reverse this profile from a cancerous phenotype to the normal one.

The IKAROS Transcription Factor Gene IKZF1 as a Critical Regulator in the Pathogenesis of Childhood Acute Lymphoblastic Leukemia: Insights from a Bangladeshi Population

The IKAROS Transcription Factor Gene IKZF1 as a Critical Regulator in the Pathogenesis of Childhood Acute Lymphoblastic Leukemia: Insights from a Bangladeshi Population

Involvement of BCR::ABL1 in laminin adhesion of Philadelphia chromosome-positive acute lymphoblastic leukemia through upregulation of integrin α6.

Adhesion of cancer cells to extracellular matrix laminin through the integrin superfamily reportedly induces drug resistance. Heterodimers of integrin α6 (CD49f) with integrin β1 (CD29) or β4 (CD104) are major functional receptors for laminin. Higher CD49f expression is reportedly associated with a poorer response to induction therapy in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Moreover, a xenograft mouse model transplanted with primary BCP-ALL cells revealed that neutralized antibody against CD49f improved survival after chemotherapy. Considering the poor outcomes in Philadelphia chromosome (Ph)-positive ALL treated with conventional chemotherapy without tyrosine kinase inhibitors, we sought to investigate an involvement of the laminin adhesion. Ph-positive ALL cell lines expressed the highest levels of CD49f among the BCP-ALL cell lines with representative translocations, while CD29 and CD104 were ubiquitously expressed in BCP-ALL cell lines. The association of Ph-positive ALL with high levels of CD49f gene expression was also confirmed in two databases of childhood ALL cohorts. Ph-positive ALL cell lines attached to laminin and their laminin-binding properties were disrupted by blocking antibodies against CD49f and CD29 but not CD104. The cell surface expression of CD49f, but not CD29 and CD104, was downregulated by imatinib treatment in Ph-positive ALL cell lines, but not in their T315I-acquired sublines. Consistently, the laminin-binding properties were disrupted by the imatinib pre-treatment in the Ph-positive ALL cell line, but not in its T315I-acquired subline. BCR::ABL1 plays an essential role in the laminin adhesion of Ph-positive ALL cells through upregulation of CD49f.

Application of prime editing system to introduce TP53 R248Q hotspot mutation in acute lymphoblastic leukemia cell line.

In childhood acute lymphoblastic leukemia (ALL), TP53 gene mutation is associated with chemoresistance in a certain population of relapsed cases. To directly verify the association of TP53 gene mutation with chemoresistance of relapsed childhood ALL cases and improve their prognosis, the development of appropriate human leukemia models having TP53 mutation in the intrinsic gene is required. Here, we sought to introduce R248Q hotspot mutation into the intrinsic TP53 gene in an ALL cell line, 697, by applying a prime editing (PE) system, which is a versatile genome editing technology. The PE2 system uses an artificial fusion of nickase Cas9 and reverse-transcriptase to directly place new genetic information into a target site through a reverse transcriptase template in the prime editing guide RNA (pegRNA). Moreover, in the advanced PE3b system, single guide RNA (sgRNA) matching the edited sequence is also introduced to improve editing efficiency. The initially obtained MDM2 inhibitor-resistant PE3b-transfected subline revealed disrupted p53 transactivation activity, reduced p53 target gene expression, and acquired resistance to chemotherapeutic agents and irradiation. Although the majority of the subline acquired the designed R248Q and adjacent silent mutations, the insertion of the palindromic sequence in the scaffold hairpin structure of pegRNA and the overlap of the original genomic DNA sequence were frequently observed. Targeted next-generation sequencing reconfirmed frequent edit errors in both PE2 and PE3b-transfected 697 cells, and it revealed frequent successful edits in HEK293T cells. These observations suggest a requirement for further modification of the PE2 and PE3b systems for accurate editing in leukemic cells.

Prophylaxis with enoxaparin and antithrombin III in drug-induced coagulation alterations in childhood leukemia: a retrospective experience of 20 years

BackgroundThromboembolic complications are well known in the treatment of childhood acute lymphoblastic leukemia. Over the years it has not been possible to reach a consensus on a possible prophylaxis of thromboembolic events during intensive therapy. Only the administration of enoxaparin was able to achieve evidence in the literature to date.MethodsIn this retrospective study, 173 childhood leukemia patients were treated over 20 years with a thromboembolic prophylaxis including enoxaparin and AT III during induction therapy with L-asparaginase and cortisone.ResultsWe here report the effectiveness of administration of enoxaparin and AT III in childhood leukemia, showing a strikingly low prevalence of deep vein thrombosis (2.9%). Especially in adolescent patients, a particularly great need for AT III was demonstrated.ConclusionsWe recommend thromboembolic prophylaxis with enoxaparin and AT III substitution during induction/reinduction therapy with L-asparaginase and glucocorticosteroids, especially from adolescence onwards.

A noncoding regulatory variant in IKZF1 increases acute lymphoblastic leukemia risk in Hispanic/Latino children

Hispanic/Latino children have the highest risk of acute lymphoblastic leukemia (ALL) in the US compared to other racial/ethnic groups, yet the basis of this remains incompletely understood. Through genetic fine-mapping analyses, we identified a new independent childhood ALL risk signal near IKZF1 in self-reported Hispanic/Latino individuals, but not in non-Hispanic White individuals, with an effect size of ∼1.44 (95% confidence interval= 1.33-1.55) and a risk allele frequency of ∼18% in Hispanic/Latino populations and <0.5% in European populations. This risk allele was positively associated with Indigenous American ancestry, showed evidence of selection in human history, and was associated with reduced IKZF1 expression. We identified a putative causal variant in a downstream enhancer that is most active in pro-B cells and interacts with the IKZF1 promoter. This variant disrupts IKZF1 autoregulation at this enhancer and results in reduced enhancer activity in B cell progenitors. Our study reveals a genetic basis for the increased ALL risk in Hispanic/Latino children.

Exclusive Breastfeeding Duration and Risk of Childhood Cancers

Breastfeeding has been suggested to protect against childhood cancers, particularly acute lymphoblastic leukemia (ALL). However, the evidence stems from case-control studies alone. To investigate whether longer duration of exclusive breastfeeding is associated with decreased risk of childhood ALL and other childhood cancers. This population-based cohort study used administrative data on exclusive breastfeeding duration from the Danish National Child Health Register. All children born in Denmark between January 2005 and December 2018 with available information on duration of exclusive breastfeeding were included. Children were followed up from age 1 year until childhood cancer diagnosis, loss to follow-up or emigration, death, age 15 years, or December 31, 2020. Data were analyzed from March to October 2023. Duration of exclusive breastfeeding in infancy. Associations between duration of exclusive breastfeeding and risk of childhood cancer overall and by subtypes were estimated as adjusted hazard ratios (AHRs) with 95% CIs using stratified Cox proportional hazards regression models. A total of 309 473 children were included (51.3% boys). During 1 679 635 person-years of follow-up, 332 children (0.1%) were diagnosed with cancer at ages 1 to 14 years (mean [SD] age at diagnosis, 4.24 [2.67] years; 194 boys [58.4%]). Of these, 124 (37.3%) were diagnosed with hematologic cancers (81 [65.3%] were ALL, 74 [91.4%] of which were B-cell precursor [BCP] ALL), 44 (13.3%) with central nervous system tumors, 80 (24.1%) with solid tumors, and 84 (25.3%) with other and unspecified malignant neoplasms. Compared with exclusive breastfeeding duration of less than 3 months, exclusive breastfeeding for 3 months or longer was associated with a decreased risk of hematologic cancers (AHR, 0.66; 95% CI, 0.46-0.95), which was largely attributable to decreased risk of BCP-ALL (AHR, 0.62; 95% CI, 0.39-0.99), but not with risk of central nervous system tumors (AHR, 0.96; 95% CI, 0.51-1.88) or solid tumors (AHR, 0.87; 95% CI, 0.55-1.41). In this cohort study, longer duration of exclusive breastfeeding was associated with reduced risk of childhood BCP-ALL, corroborating results of previous case-control investigations in this field. To inform future preemptive interventions, continued research should focus on the potential biologic mechanisms underlying the observed association.

Long Non-coding RNA RP11-252C15.1 is a Potential Biomarker of Prognosis and Hallmark for Leukemogenesis in Children with B-cell Precursor Acute Lymphoblastic Leukemia.

Improved understanding of the prognostic biomarkers associated with childhood acute lymphoblastic leukemia (ALL) is needed for accurate risk group stratification. This study aimed to identify potential long-non coding RNA (lncRNA) markers and evaluate their prognostic value in children with ALL. We selected 50 children with newly diagnosed ALL and 20 age-matched patients with idiopathic immune thrombocytopenia (controls). RNA sequencing was performed to identify differentially expressed lncRNAs between the ALL and control groups. Correlation analysis was performed to determine the relationships between candidate lncRNAs, clinical features, and the risk of leukemogenesis. A total of 1,019 differentially expressed lncRNAs were identified between the ALL and control groups. Reverse-transcriptase (RT-qPCR) revealed that lncRNA RP11-252C15.1 and lncRNA RP11-701P16.2 were significantly upregulated in patients with ALL. Furthermore correlation analysis showed that lncRNA RP11-252C15.1 and lncRNA RP11-701P16.2 represent potential predictors of leukemogenesis; however, only lncRNA RP11-252C15.1 was associated with clinical features and outcome in children with B-cell precursor ALL (BCP-ALL). In vitro experiments confirmed that lncRNA RP11-252C15.1 was significantly overexpressed in BCP-ALL cell lines and promoted proliferation, and repressed apoptosis in MHH-CALL-3 cells. LncRNA RP11-252C15.1 is a potential oncogene in BCP-ALL pathogenesis and a prognostic biomarker in children with BCP-ALL.

Metabolic Fingerprint in Childhood Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy. Despite high cure rates, several questions remain regarding predisposition, response to treatment, and prognosis of the disease. The role of intermediary metabolism in the individualized mechanistic pathways of the disease is unclear. We have hypothesized that children with any (sub)type of ALL have a distinct metabolomic fingerprint at diagnosis when compared: (i) to a control group; (ii) to children with a different (sub)type of ALL; (iii) to the end of the induction treatment. In this prospective case-control study (NCT03035344), plasma and urinary metabolites were analyzed in 34 children with ALL before the beginning (D0) and at the end of the induction treatment (D33). Their metabolic fingerprint was defined by targeted analysis of 106 metabolites and compared to that of an equal number of matched controls. Multivariate and univariate statistical analyses were performed using SIMCAP and scripts under the R programming language. Metabolomic analysis showed distinct changes in patients with ALL compared to controls on both D0 and D33. The metabolomic fingerprint within the patient group differed significantly between common B-ALL and pre-B ALL and between D0 and D33, reflecting the effect of treatment. We have further identified the major components of this metabolic dysregulation, indicating shifts in fatty acid synthesis, transfer and oxidation, in amino acid and glycerophospholipid metabolism, and in the glutaminolysis/TCA cycle. The disease type and time point-specific metabolic alterations observed in pediatric ALL are of particular interest as they may offer potential for the discovery of new prognostic biomarkers and therapeutic targets.

Abstract 6595: HDAC8 targeting impairs Foxp3+Treg cell function and promotes anti-tumor immunity

Abstract HDAC8 is an evolutionarily distinct, X-linked, zinc-dependent class I histone/protein deacetylase implicated in developmental disorders, parasitic infections, myopathy, and cancers such as neuroblastoma, gastric cancer, hepatocellular carcinoma, oral squamous cell carcinoma, and childhood acute lymphoblastic leukemia. Here, we highlight a hitherto unknown role of HDAC8 in immune cells by presenting data on conditional targeting of HDAC8 in T cells and the effects of selective HDAC8 inhibitors (HDAC8i) on T cell-dependent immune responses. First, as a genetic approach, we produced C57BL/6 mice with floxed exon 3 of the HDAC8 gene suitable for targeted deletion of HDAC8 and, after crossing with syngeneic CD4cre mice, demonstrated conditional deletion of HDAC8 and enhanced acetylation of SMC3, a classic HDAC8 substrate, in their T-cells. Next, in a pharmacologic approach, we tested isoform-selective inhibitors of HDAC8 (OJ-1, PCI-34051) and assessed effects on Foxp3+ T-regulatory (Treg) cell suppressive function. Finally, we performed flow cytometry, RNAseq, CHIP-seq and mass spectrometry to investigate the underlying mechanisms by which HDAC8 knockout affected Treg function. Our data demonstrate that knocking down or inhibiting HDAC8: (i) impaired murine Treg suppressive function in vitro and in vivo, including inhibiting the ability of Tregs to promote mouse cardiac allograft survival (p&amp;lt;0.01); (ii) decreased Treg function in homeostatic proliferation assays (p&amp;lt;0.01), e.g., involving adoptive transfer of WT or HDAC8-/- Tregs plus WT conventional T cells into immunodeficient hosts; (iii) decreased intratumoral Tregs, increased CD4 and CD8 T cells, increased production of effectors such as IFN-g and granzyme-B, and limited growth of syngeneic mouse non-small cell lung cancer cell lines (p&amp;lt;0.01) (whereas no effect of Hdac8i on tumor growth was seen in immunodeficient hosts, indicating the key action was via immune cell targeting). Mechanistically, HDAC8 co-precipitated with Foxp3 and NFAT5 and promoted the deacetylation of Foxp3 and NFAT5, and knockout of HDAC8 enhanced the acetylation level of NFAT5 and promoted cytokine expression. Hence, our combined genetic and pharmacologic studies establish the importance of HDAC8 in Foxp3+ Treg cells and show that therapy with selective HDAC8 inhibitors can be a novel therapeutic approach in immuno-oncology. Citation Format: Fanhua Kong, Liqing Wang, Aaron Beeler, Wayne W. Hancock. HDAC8 targeting impairs Foxp3+Treg cell function and promotes anti-tumor immunity [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 6595.

Abstract 557: Clofarabine inhibits U87MG glioblastoma cell line proliferation through CD99 signaling pathway

Abstract The present work aimed to test the efficacy of clofarabine in glioblastoma (GBM) cells that express high levels of CD99. GBMs are the most malignant with the poorest prognosis among tumors of the central nervous system. Patients have a median survival of 12 to 15 months despite surgical, radio, and chemotherapy. CD99 is a cell surface receptor overexpressed in several types of tumors, including GBMs, justifying the interest of studies in CD99 as a possible therapeutic target. Our group has previously demonstrated that knockout of CD99 in the U87MG GBM cell line promoted a decrease in cell adhesion, migration, and invasion. Clofarabine is a third-line drug used in the treatment of refractory/relapsed childhood acute lymphoblastic leukemia. It is a purine analog that is transported into intracellular space, phosphorylated, and incorporated into DNA chain, inhibiting DNA synthesis and ultimately inducing cell death by apoptosis. The clofarabine effect has also been demonstrated in cells from solid tumors, such as breast, gastric, and bladder. Clofarabine action on the proliferation of Ewing sarcoma cells in vitro and in vivo was proved to be by direct binding to CD99 and independent of the drug internalization. In addition, clofarabine also had an effect on cells from other tumor types that express CD99. The present work demonstrated the significant action of clofarabine on the high CD99-expressing U87MG cells by inhibiting cell proliferation, adhesion, and migration, and by inducing cell cycle arrest and apoptosis. U87MG cells knocked out for CD99 expression were more resistant to clofarabine action on proliferation. RNAseq analysis of U87MG cells treated with clofarabine and compared to control showed downregulated genes involved in cell cycle regulation and mitosis, while upregulated genes were enriched in extracellular proteins, cell adhesion, locomotion, p53 signaling pathway, and apoptosis. Our data indicates clofarabine as a potential CD99 inhibitor to treat GBM patients. Citation Format: Camila Cristina de Souza, Roseli da Silva Soares, Antonio Marcondes Lerario, Suely Kazue Marie, Sueli Mieko Oba-Shinjo. Clofarabine inhibits U87MG glioblastoma cell line proliferation through CD99 signaling pathway [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 557.