AbstractAcute Lymphoblastic Leukemia Research Articles

Abstract 5116: Stress-induced AMPK interactome analysis reveals novel protein-protein interactions associated with chromatin remodeling in acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) is a leading cause of cancer-related death among children, adolescents and young adults. We previously reported that ALL cells are vulnerable to metabolic stress conditions following AMP-activated protein kinase (AMPK) activation, leading to cell death. AMPK is the master metabolic regulator, and we and others have reported it interacts with chromatin-associated proteins to regulate gene expression in response to metabolic stress. To investigate AMPK’s mechanistic role in epigenetic gene regulation under metabolic stress, and identify new AMPK partners, we performed a comprehensive interactome analysis using TurboID-based proximity labeling proteomics. We generated HEK293T cells stably expressing TurboID fused to the AMPKα1, AMPKα2, AMPKβ1 or AMPKγ1. Following treatment with the allosteric AMPK activator PF-06409577 (PF064) for 1h, to mimic the cell’s rapid response to energy/metabolic stress, the samples underwent mass spectrometry-based proteomics. Proteomics data analysis identified novel altered (enhanced or reduced) AMPK protein-protein interactions (PPI) following PF064 treatment. The GO analysis of proteins interacting with AMPK showed that metabolic stress enhances PPIs involved in regulating gene expression, cellular metabolic processes, DNA repair, chromatin organization, histone modifications, etc. More importantly, we identified that AMPK interacted with members of the non-canonical Polycomb repressive complex 1 (PRC1.1) such as PCGF1, RING1, KDM2B, BCORL1, USP7. PRC1 is a known epigenetic regulator that monoubiquitinylates histone H2A at Lys119 (H2AK119ub) to control transcription and regulate gene expression. Using Co-IP, we confirmed these interactions in KASUMI-2 (Bp-ALL) and KE-37 (T-ALL) cells. When cells were treated with PF064 or 991 (another AMPK activator), we found that the interactions of AMPK with the catalytic core of PCR1.1 (PCGF1, RING1) were increased compared to untreated cells. To examine the effect of activated AMPK on PCR1.1 activity, we determined the level of H2AK119ub in ALL cells treated with PF064 and 991 for 1h. We found that the level of H2AK119ub protein expression was increased in ALL-treated cells indicating the PRC1.1 was activated. Together, these data suggest that stress-induced AMPK activation increased the catalytic activity of the PRC1.1 via PPI in ALL. Our findings are consistent with our previous published RNA-Seq and ChIP-Seq data indicating an epigenetic role for AMPK in altering gene expression examined under metabolic stress (two thirds exhibited downregulation), and provide insights into the AMPK-driven mechanism(s) involved. Further elucidation of the uncovered PPIs and its mechanistic role(s) may lead to potential epigenetic-based strategies to overcome resistance/refractoriness in ALL and other malignancies. Citation Format: Anna Shvab, Guy J. Leclerc, Julio C. Barredo. Stress-induced AMPK interactome analysis reveals novel protein-protein interactions associated with chromatin remodeling 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 5116.

Abstract 7111: Homoharringtonine is effective in treating acute lymphoblastic leukaemia via WEE1 downregulation and inducing DNA damage

Abstract Acute lymphoblastic leukaemia (ALL) is an aggressive haematolymphoid malignancy that originated from clonal mutations in lymphoid precursors. The prognosis is poor in adult patients and relapse/refractory disease is not uncommon which carries dismal prognosis. There is an urgent need to develop novel therapeutics. Homoharringtonine (HHT) is a plant alkaloid which can bind to ribosomal A-site and thus inhibit protein synthesis. It was used to treat acute myeloid leukaemia in China during 1980s. Herein, we conducted a study to explore the therapeutic efficacy and mechanism of action in ALL. T-ALL (DND-41, PEER) and B-ALL (PALL-2, SUP-B15) cell lines were treated with HHT for 24 and 72 hours. Cell viability was reduced at a time and dose-dependent manner in all cell lines we tested, with increased in percentage of apoptosis. Western blot showed upregulation of p53, with PARP and caspase 3 cleavages upon treating with HHT for 24 hours. Moreover, we observed WEE1 downregulation which allowed cell cycle progression through G2/M with damaged DNA. Comet assay showed evidence of DNA damage. Proteomics study by label-free protein quantitation showed downregulation of proteins involving in homologous recombination-mediated DNA repairing machinery, including BRCA1 and RAD51. The findings not only showed therapeutic efficacy of HHT in ALL, but also demonstrated the first evidence of DNA damage induced by HHT which might account for its therapeutic effects. Further studies will be conducted for delineating the mechanisms of DNA damage and its relationship with therapeutic efficacy. Citation Format: Chun-fung Sin, Kei-ching Yuen, Anan Jiao, Bai-lin Li, Hau-wai Yu. Homoharringtonine is effective in treating acute lymphoblastic leukaemia via WEE1 downregulation and inducing DNA damage [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 7111.

Abstract 141: A human induced pluripotent stem cell model of KMT2A rearranged leukemia

Abstract Acute lymphoblastic leukemia in infants (iALL) is a high-risk subtype of childhood leukemia with poor survival outcomes despite intensive therapies. Rearrangement of KMT2A (KMT2A-r) occurs in 70% of cases and is associated with refractoriness to therapy, early relapse, and rapid leukemia progression. KMT2A-r generates a driver fusion oncogene, most commonly KMT2A::AFF1 in iALL, which leads to epigenetic dysregulation of target gene transcription. iALL with KMT2A::AFF1 is an unusual cancer in that other somatic mutations are uncommon, there are no known genetic risk factors, and phenotypic switch from lymphoid to myeloid, mixed, or undifferentiated phenotype is common during treatment and particularly resistant to therapies. The cell of origin is thought to be a very early hematopoietic precursor, with transcriptomic studies of iALL blasts showing similarities to hematopoietic stem and progenitor cells (HSPCs), multipotent progenitors and early lymphoid progenitors (ELPs). Unfortunately, research into this rare but devastating disease has been hindered by a lack of appropriate, representative models. Interestingly, KMT2A-r originates in HSPCs in utero and the age of onset of iALL is a strong prognostic factor, with age younger than six months associated with the highest risk of relapse. However, little is known regarding how KMT2A-r subverts early hematopoiesis or drives the severe disease phenotype. In an effort to understand the role of the developmental state of the cell of origin in iALL, we have created a highly controlled induced pluripotent stem (iPS) cell model system of KMT2A::AFF1 leukemia. Specifically, we engineered human iPS cell lines with doxycycline regulatable expression of KMT2A::AFF1 fusion and we are employing CRISPR gene editing technology to introduce clinically identified variants of interest. Utilizing directed differentiation, we have produced functional HSPCs from iPS cells. Notably, this model recapitulates hematopoietic ontogeny, with the ability to control iALL induction at specific developmental stages. Following doxycycline treatment, we demonstrate that the cells express KMT2A::AFF1 fusion transcripts. We are utilizing single cell genomics to investigate transcriptomic changes during hematopoietic differentiation of our KMT2A::AFF1 iPS cells. We will compare these results to published data sets of single cell RNA sequencing data from healthy fetal HSPCs and to our existing iALL and pediatric KMT2A-r ALL single cell sequencing data sets. Our iPS cell based iALL model system provides the opportunity to investigate a range of critical and outstanding questions of iALL disease initiation, progression, and treatment. Citation Format: Erin Guest, Meagan Vacek, Jacqelyn Nemechek, Irina Pushel, Bradley T. Thornton, Molly Leyda, Midhat S. Farooqi, John M. Perry, Jay L. Vivian. A human induced pluripotent stem cell model of KMT2A rearranged 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 141.

A Curious Case of Jaw Swelling due to Precursor B Cell Acute Lymphoblastic Leukemia

ABSTRACT Acute lymphoblastic leukemia (ALL) is a rare leukemia in adults but is the most common type in children. We present a case of a 40-year-old female presenting with jaw pain and swelling with fever which appeared to be an abscess. Further evaluation revealed the presence of blast cells in the peripheral blood smear. Flow cytometry was performed which further revealed pre-B cell ALL. It underwent spontaneous tumor lysis and despite all the resuscitative measures, she succumbed to sepsis and multiorgan dysfunction. The purpose of this report is to document an unusual manifestation of an adult B cell acute lymphoblastic leukemia (ALL) presenting as a jaw mass masquerading as an abscess undergoing spontaneous tumor lysis.

Abstract A012: Exploring the role of adaptive amino acid responses in Ras-driven leukemia progression and therapy

Abstract Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, affecting the lymphoid cells of both B and T lineages. Most ALL cells are auxotrophic for asparagine, a nonessential amino acid for protein synthesis, due to the low expression of asparagine synthetase (ASNS), a rate limiting enzyme for de novo biosynthesis of asparagine. As a result, standard ALL treatment takes advantage of this vulnerability by giving patients L-asparaginase, a bacterial enzyme that depletes the circulating asparagine. However, previous work from our lab and others have shown that some ALL cells become resistance to L-asparaginase treatment through the induction of ASNS expression. Mechanistically, amino acid starvation actives the general control nonderepressible 2 (GCN2) kinase, leading to the accumulation of ATF4 transcriptional factor. ATF4, in turn, is recruited to the promoter of the ASNS gene to activate its transcription. However, the role of GCN2 kinase in the process of leukemogenesis under nutrient limiting environment has not been established. In this study, our goal is to use a mouse model of T-ALL driven by mutant KRas to determine the role of GCN2 in leukemogenesis and the therapeutic response to L-asparaginase treatment. Citation Format: Ji Zhang, Rodney Claude, Sankalp Srivastava, Sandeep Batra, Minghua Zhong, Utpal Dave, Ronald C. Wek. Exploring the role of adaptive amino acid responses in Ras-driven leukemia progression and therapy [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A012.

Profiling cGAS-STING pathway in T-Acute Lymphoblastic Leukemia

Abstract Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy, accounting for over one-quarter of all childhood malignancies. Approximately 15% of ALL derive from the T-cell lineage (T-ALL). Patients with newly diagnosed T-ALL undergo intensive multidrug combination chemotherapy resulting in many side effects. Despite chemotherapy regimens including steroids, and allogeneic transplantation, relapse remains the main challenge in children with T-ALL. Moreover, genetic alterations have been extensively examined in recent years; however, there are still currently few prognostic markers for therapy response. Therefore, alternative treatments are needed to treat this disease and improve overall survival. The cyclic GMP-AMP synthase - Stimulator of Interferon Genes (cGAS-STING) pathway has emerged as a possible target in treatment of cancers, aiming to increase anti-tumor immunity. Most clinical trials to-date have focused on STING agonists used for solid tumors. Interestingly, the high activation of this pathway in normal T cells can lead to ER stress and cell death via the activation of the BH3-family. The status of cGAS-STING pathway in T-ALL is largely unknown. We hypothesize that the cGAS-STING pathway is defective in T-ALL, thus protecting these malignant cells from apoptosis, and that increasing cGAS-STING activity may be a way of enhancing the treatment of this disease. Our preliminary data shows that some T-ALL cell lines do not produce IFN-β after stimulation to STING agonist. Also, some of these cell lines have a decreased cGAS expression, and have a decreased rate of apoptosis compared to healthy T-cells. Our project will hopefully lay the foundation for future targeted immunotherapies for treating T-ALL. Center for Pediatric Immunotherapy at Rainbow

Differential expression of immune receptors in pediatric Acute Lymphoblastic Leukemia (ALL)

Abstract Acute Lymphoblastic Leukemia (ALL) represents the most common pediatric cancer. Significant improvements in chemotherapy regimens caused a leap in the 5-year survival rates to surpass 90%. However, relapse significantly lowers the chances of survival to less than 50% in some cases due to resistance developed by malignant cells to chemotherapy. Natural Killer (NK) cells are a promising immunotherapy alternative for their ability to recognize and kill malignant cells directly through activation or inhibition signals or through cytokine release. We have previously identified 2B4 (SLAMF4), CS1 (SLAMF7) and LLT1 that can activate or inhibit NK cells upon the interaction with their ligands. In this study, the expression of 2B4, CS1, LLT1, NKp30 and NKp46 was determined in pediatric ALL and healthy subjects. Whole blood was collected from 42 pediatric ALL subjects before and after induction chemotherapy and the surface expression of receptors/ligand on T-cells, B-cells, monocytes, and NK-cells was compared with blood samples from 20 healthy subjects. The flow cytometry data showed a significant increase in CS1 and NKp46 expression on monocytes and LLT1 in T cells and monocytes of ALL subjects before treatment. A decrease of LLT1, 2B4, CS1 and NKp46 on T-cells of ALL subjects was also observed after treatment along with elevated expression of LLT1 on ALL subjects’ NK-cells before and after treatment. Furthermore, mRNA data showed altered expression of receptors in ALL subjects pre and post induction chemotherapy treatment. The results indicate that the differential expression of the receptors/ligand may play a role in the T cell and NK-cell mediated immune surveillance of pediatric ALL. Supported by funds from Cancer Research Foundation of North Texas and Team Connor Childhood Cancer Foundation

Abstract 5221: Identifying acute lymphoblastic leukemia risk loci in latino children via admixture mapping

Abstract Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and accounts for 25% of malignancies before the age of 20. Despite decades of inquiry into ALL causes, Latino children have up to 1.4 times the rate of ALL compared to their non-Latino White counterparts. The etiology of ALL is complex and this disparity in risk has not been fully explained by social factors alone, suggesting the role of genetic variants and ancestry. Previous literature has implicated variants in IKZF1, ARID5B, GATA3, PIP42KA, and ERG with greater risk allele frequencies or population-specific risk effects in Latinos. Indigenous American ancestry has also been previously associated with ALL risk, relapse, and poor prognosis. To elucidate the genetic and ancestral etiology of ALL, we conducted the first admixture mapping analysis of 1930 childhood ALL cases and 8520 controls of self-identified Latino ethnicity. We uncovered putative admixture associations on chromosomes 2, 7, 10, and 15 (P = 3.21 x 10−5 to 7.32 x 10−8 among top associations across the four loci). Two of the four loci, on chromosomes 2 and 15, were previously not known to be associated with ALL. Following imputation with the TOPMed reference panel, we fine-mapped each putative locus by testing the ALL risk association of each variant with a minor allele frequency greater than 1%. The top associated variants at each locus showed substantial frequency differences between ancestries from the gnomAD or 1000Genomes project. For example, the top associated variant on chromosome 2 has a risk allele frequency of 43% in Native Americans from the Human Genome Diversity Panel, but a frequency of 0.10% in Non-Finnish Europeans in gnomAD. Including these variants in the admixture mapping model greatly attenuated the admixture signals at each locus, suggesting that they are likely the causal SNPs or close proxies of the causal SNPs. The top associated variants from three out of the four loci passed regional multiple testing burden and mapped to genes MGAT5, IKFZ1 and ARID5B on chromosomes 2, 7 and 10, respectively. The top two SNPs on chromosome 15, which narrowly missed the regional threshold, mapped to genes RAB11A and MEGF11. We are pursuing replication of putative novel loci on chromosomes 2 and 15 in additional Latino GWAS datasets. Our results suggest that ALL risk variants with higher frequencies in individuals with Native American ancestry may contribute to the observed increased risk of ALL in Latino children. Investigation of such risk loci can contribute to the identification of new target genes for ALL prediction and therapeutics and new insights for precision medicine, which will reduce the burden of ALL. Citation Format: Jalen Langie, Soyoung Jeon, Xiaomei Ma, Catherine Metayer, Adam J. de Smith, Joseph L. Wiemels, Charleston W. Chiang. Identifying acute lymphoblastic leukemia risk loci in latino children via admixture mapping. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5221.

Abstract 2957: CD127 is expressed by acute lymphoblastic leukemias and is efficiently targeted by the IL7R-antagonist OSE-127 through macrophage-mediated antibody dependent phagocytosis

Abstract Acute lymphoblastic leukemia (ALL) arises from the uncontrolled proliferation of precursor B or T cells (B- or T-ALL). Novel immunotherapies are urgently needed to reduce polychemotherapy-related toxicities and to target relapsed/refractory disease. The Interleukin 7 receptor (IL7R) plays a pivotal role in the development of ALL through mutations leading to constitutive activation of the oncogenic IL7R pathway or through overexpression of the receptor. The IL7Rα chain (CD127)-targeting antibody OSE-127 is a full antagonist of the IL7R pathway with an excellent safety profile (NCT03980080), currently evaluated in phase 2 trials in inflammatory and autoimmune diseases (NCT04882007, NCT04605978). We previously reported in vivo efficacy of OSE-127 in CD127+ ALL patient-derived xenografts (PDXs) and synergy with standard of care polychemotherapy, but we had not evaluated CD127 protein expression in ALL patient cells. Moreover, the mechanism of action underlying OSE-127’s anti-leukemic activity remained to be fully elucidated. Here we show that CD127 is expressed by >84% ALL patients and that OSE-127 efficiently targets CD127 not only via its IL7R antagonist activity but also through macrophage-mediated antibody dependent phagocytosis (ADCP). First, CD127 surface expression was prospectively measured by flow cytometry in 372 diagnostic ALL patient samples. We detected CD127-positivity (defined as ≥10% CD127+ blasts) in 84.4% (314/372) cases, of which 39.5% (147/372) were CD127hi (≥ 50% CD127+ blasts). CD127 expression was higher in T- than in B-ALL. Among B-ALL, highest expression was detected in E2A-PBX1+ and BCR-ABL-like ALLs. Mechanistically, OSE-127 blocked STAT5 phosphorylation in IL7-responsive ALL-PDX cells, leading to decreased cell survival and proliferation. Surprisingly, we detected high in vivo efficacy of OSE-127 therapy in IL7-unresponsive PDXs (e.g., unable to induce phosphorylation of STAT5 upon ex vivo IL7 stimulation or with IL7R mutation-induced constitutive phosphorylation of STAT5). We uncovered that OSE-127 induced macrophage-mediated ADCP of CD127+ ALL cells in vitro. Accordingly, treatment of IL7-unresponsive, but OSE-127-sensitive models with an OSE-127-LALAPG variant (conserving IL7R-antagonistic properties but lacking ADCP activity due to Fc-inactivating mutations) did not impact leukemia development, thereby substantiating that ADCP contributes to the anti-leukemic efficacy of OSE-127. In fact, ADCP levels induced by OSE-127 treatment in vitro correlated with its capacity to reduce ALL blasts in the blood of PDX mice in vivo. Altogether, through its dual mode of action, OSE-127 may represent a powerful novel immunotherapy option for ALL patients, including cases with dysregulated IL7R signaling, particularly in combination with standard of care polychemotherapy. Citation Format: Irene Baccelli, Lennart Lenk, Anna Laqua, Dorothee Winterberg, Anna Dietterle, Frederique Corallo, Julien Taurelle, Emma Narbeburu, Beat Bornhauser, Jean-Pierre Bourquin, Fotini Vogiatzi, Simon Raffel, Martin Schrappe, Gunnar Cario, Monika Brüggemann, Denis M Schewe, Nicolas Poirier. CD127 is expressed by acute lymphoblastic leukemias and is efficiently targeted by the IL7R-antagonist OSE-127 through macrophage-mediated antibody dependent phagocytosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2957.

Abstract 4758: SETD4 transcription levels correlates with leukemic burden and SMYD2 transcription in acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy worldwide. It has a history of great rates of success in treatment, but adults and infants still share a dismal prognosis. This condition makes the development of new prognostic markers linked to effective therapeutic strategies a matter of pressing concern. While cancer has traditionally been viewed as a genetic disease derived from alterations in oncogenes and tumor suppressors, it is well known nowadays that epigenetic changes also play a fundamental role in tumorigenesis. The SET family of lysine methyltransferases (KMT) has been implicated in a number of cancers, and SETD4 is a member that is still poorly characterized. In the present study we used qPCR to analyze the expression pattern of SETD4 among 83 pediatric ALL patients and non-neoplastic bone marrow (BM) samples and investigated the correlation between SETD4 transcription changes with the leukemic burden in ALL patients during chemotherapy. We found that SETD4 transcription levels are significantly upregulated in BM samples derived from ALL patients compared to non-neoplastic BM (8,5-fold higher, p < 0,001) and its expression correlated with leukemic burden. Importantly, levels of SETD4 decreased in patients that responded to chemotherapy treatment. We further investigated whether SETD4 transcription levels associates with that of SMYD2, another KMT previously identified as a prognostic marker in ALL. Transcription levels of SETD4 and SMYD2 were examined on day 15th and 29th of chemotherapy. Surprisingly, a high level of correlation (Spearman r = 0.925, p < 0.01) between both genes was observed in both treatment time points. Finally, survival outcomes were worst in ALL patients with high levels of SETD4 transcription (log-rank test, p < 0,05), evidencing its dysregulated expression is associated with an unfavorable disease prognosis. Together, these results point to SETD4 as a useful prognostic marker, a possible tool to assess response to therapy and an attractive target for drug development. Citation Format: Luis Augusto Muniz Telles, Luis Henrique Sakamoto, Alan Jhones Assis, Doralina de Amaral Rabello, Andrea Barretto Motoyama, Fabio Pittella-Silva. SETD4 transcription levels correlates with leukemic burden and SMYD2 transcription in acute lymphoblastic leukemia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4758.

Abstract 1574: Homoharringtonine is effective in treating acute lymphoblastic leukaemia via modulation of apoptotic pathway

Abstract Acute lymphoblastic leukaemia (ALL) is an aggressive and highly lethal malignancy caused by clonal mutations in lymphoid progenitor cells. The prognosis for adult ALL remains poor, with only 30-40% of adult patients achieving long-term remission. Therefore, novel therapies are needed. Homoharringtonine (HHT) is a natural-derived plant alkaloid from tree of Cephalotaxus. FDA approved the use of HHT in the treatment of chronic myeloid leukaemia (CML) in 2012. Limited pre-clinical or clinical studies were conducted for therapeutic efficacy and molecular mechanisms of HHT on ALL to the best of our knowledge. To fill this knowledge gap, here we investigated the antileukemic activity of HHT in T lymphoblastic leukaemia (T-ALL) and B lymphoblastic leukaemia (B-ALL). In this study, T-ALL cell lines namely JURKAT, MOLT-4, LOUCY, CCRF-CEM and B-ALL cell clines namely PALL-2, SUP-B15 were used. Cell viability was reduced upon treatment of HHT at time and dose-dependent manner. Percentage of apoptotic cells that determined by flow cytometric analysis of PI and Annexin V double staining was increased upon treatment of HHT. Moreover, HHT was able to induce apoptosis in both T-ALL and B-ALL cell lines. Western blot analysis showed that MCL-1 protein level was reduced upon treating with HHT for 24 hours. The above findings provide conclusive evidence for the therapeutic effect of HHT on various ALL cell lines by modulating the apoptotic pathway. Citation Format: Anan Jiao, Kei Ching Yuen, Rahman Ud DIN, On Yi Phoebe Wong, Sin Chun-Fung. Homoharringtonine is effective in treating acute lymphoblastic leukaemia via modulation of apoptotic pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1574.

Abstract 2058: Identifications of novel variants using system biology-based approaches in NT5C2, its association to relapse acute lymphoblastic leukaemia

Abstract Acute Lymphoblastic Leukaemia (ALL) patients experience relapse despite having a good prognosis; approximately 90% of children and 75% to 85% of adolescents and young adults survive for five years. Chemotherapy resistance and a poor prognosis are linked to relapsed acute lymphoblastic Leukaemia (R-ALL). Evidence suggests that the cytosolic 5 nucleotidase II (NT5C2) gene mutations play a crucial role in the emergence of recurrent ALL; The early R-ALL is usually accompanied by 6-MP resistance, which is typically brought on by gain-of-function mutations impacting NT5C2-driven mutation in relapse mutations by different mechanisms is still unknown. The study aims to identify novel top missense variants of NT5C2 that play a crucial role in the resistance of chemotherapy drugs, altered biological pathways and their impact on the treatment of relapse leukaemia. NT5C2 (5'-Nucleotidase, Cytosolic II) is a Protein Coding gene. This gene induces resistance to chemotherapeutic drug 6 mercaptopurine Which causes leukaemia cell growth and leukaemia-initiating cell activity. The NT5C2 gene variants are extracted from Genome Aggregation Database (gnomAD) and ExAC. The extracted data contained 2280 variants with chromosome number, position reference allele, and altered allele and these were functionally annotated using the ANNOVAR tool. The variants are screened through various computational tools to narrow down based on non-synonymous and stopgain mutations in the exonic region. The predicted 519 variants narrow down by scoring GERP (>2.0) and CADD PHRED (cut-off score >10), ClinVar, SIFT score, Polyphen, PhyloP, PhastCons, LRT score predictions and which indicates the top 10% deleterious variants including reported and unknown variants. The SNPs, insertions, deletions, CNVs or structural variants of top missense variants on genes, transcripts, and protein sequence determined on Variants Effect Predictor on the ensemble. These variants were verified on dbSNP and Clinvar through their variant IDs. The functional prediction of these variants was made to detect their significant role in R-ALL. These findings examine the future role of NT5C2 mutations in the chemoresistance of relapse ALL. This may help to find a new therapeutic combination of drugs to treat relapse acute lymphoblastic leukaemia to improve overall survival. Citation Format: Sugunakar V, Obul Reddy Bandapalli, Ramita Sharma. Identifications of novel variants using system biology-based approaches in NT5C2, its association to relapse acute lymphoblastic leukaemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2058.

Abstract 3924: Cooperation between mutant IL7R and TLX3 generates mixed myeloid lymphoid leukemia from thymocyte progenitors

Abstract Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Despite the current pediatric treatment protocols have resulted in 5-year overall survival rates of up to 90%, treatments have substantial side effects. Therefore, it is important to identify more specific therapeutic targets and develop more effective and less toxic drugs and drug combinations. We have shown that overexpression of TLX3 in association with mutant IL7R (mutIL7R) is sufficient to generate aggressive leukemia from transduced CD4-CD8- (DN) thymocyte progenitors. To better understand mutIL7R/TLX3-mouse leukemia, we combined single cell RNA sequencing (scRNAseq), Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), DNA Next Generation Sequencing (NGS) Chromatin Immunoprecipitation Sequencing (ChIP-Seq) and flow cytometry to explore the cooperation of mutIL7R and TLX3 in orchestrating leukemogenesis. We found that mutIL-7R+TLX3 induced murine mixed T/myeloid leukemia. Myeloid cell clones (CD11b+) expressed identical TCR rearrangements as the T cell clones (CD8+), indicating that mutant IL-7R+TLX3 generated mixed leukemia from T cell progenitors. Moreover, the passaged leukemia lost the T-cell phenotype and acquired a pro-B-like phenotype, suggesting preferential expansion of pro-B cells (CD19+, B220+ IgM-), which were not detected in the founder leukemia presumably due to its low frequency. We next sought to determine whether the oncogene combination is sufficient to drive a similar immunophenotype profile in vitro. We showed that ectopic expression of mutIL7R and TLX3 in DN primary thymocytes growing on OP9-DL4 pushed the cells to myeloid (CD11b+, Gr1+) and B-cell (B220) phenotype over a time course of 4, 8 and 20 days, while T-cell development was blocked, suggesting that TLX3 expression prevented differentiation into CD4+/CD8+ T-cells. To better understand the genes regulated by TLX3 on driving a mixed phenotype leukemia we performed ChiP-seq of D1 cell line ectopically expressing mutIL7R and TLX3. We identified RUNX as a possible key mediator of the mixed-phenotype-leukemia induced by TLX3 in combination with mutIL7R. Additionally, scRNAseq showed upregulation of PU.1 in mice leukemia, suggesting PU.1 as a potential therapeutic target. To assess whether targeted therapy could successfully treat mutIL7R/TLX3-mouse leukemia, we treated mice that had been injected with the thymocytes transduced with mutIL-7R+TLX3 genes. We found that PU.1 inhibition reduced spleen weights, indicating decreasing of leukemia engraftment. Taken together, we generated a high-resolution dataset with detailed information about the transcriptional landscape and immunophenotype of leukemia generated by mutIL-7R+TLX3, a combination that is observed in patient samples, and suggests new therapeutic drug strategies. Citation Format: Gisele Rodrigues. Cooperation between mutant IL7R and TLX3 generates mixed myeloid lymphoid leukemia from thymocyte progenitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3924.

Abstract 6625: Mathematical modeling of single-cell data reveals the key role of stochasticity - not fitness - in determining the clonal origins of relapse in childhood leukemia

Abstract Acute lymphoblastic leukemia (cALL) is the most common pediatric cancer, and since up to 20% of children relapse after initially responding to chemotherapy treatment, the largest cause of cancer-related childhood deaths in the western world. With the idea that dissecting the evolutionary population dynamics leading to relapse would help explain treatment failure from a mechanistic standpoint, several studies have performed comparisons of genetic heterogeneity at diagnosis and relapse. These showed that as many as 70% of relapses are dominated by a single diagnostic subclone or one of its evolutionary progeny; leading to the prevailing view that treatment selection in cALL primarily operates at the genotype level. However, a crucial caveat of these studies is their reliance, due primarily to technical and economic constraints, on very limited data timepoints. While the dynamics linking said timepoints are often interpreted based on simple intuition. To overcome this sparse data challenge and reconstruct the entire spatial and temporal evolutionary trajectories of individual cALL cells from diagnosis to relapse, we have integrated experimental data with an in silico model. Single-cell WGS and RNAseq data were obtained from primary and xenografted leukemic cells before and after chemotherapy. These were then used to parameterize an agent-based mathematical model allowing tracking of the genotype and evolving phenotype of individual cells over time. When short treatment courses akin to those we delivered in vivo were used, the in silico model closely recapitulated the experimental results. Since cALL treatment lasts several years, it cannot be entirely modelled in vivo. Due to the rarity of post-treatment residual cells and the lack of universal cell surface markers to isolate them, direct analysis of clinical samples is also challenging. However, based on the initial treatment data, our calibrated model provided a unique opportunity to simulate longer remission-relapse dynamics of several leukemias. Mechanistic analysis of the collected data showed that, strikingly, in most cases, clonal dominance at relapse results from stochastic sweeps rather than deterministic differences in clonal fitness. Indeed, independent treatment of the same leukemia with an identical drug regimen more frequently ended with a considerably different clonal landscape (either polyclonal disease or a different dominant clone) than with the same outcome initially observed. Rather than genotype differences, the size of residual disease and a founder effect linked to the stochastic exit, post-treatment, of individual cells from quiescence were the key determinants of the relapse genetic landscape. Altogether, our data highlight the misinterpretation risk associated with snapshot analysis and might inform promising alternative treatment schedules. Citation Format: Virginia Turati, Javier Herrero, Mark Robertson-Tessi, Tariq Enver, Andriy Marusyk, Alexander RA Anderson. Mathematical modeling of single-cell data reveals the key role of stochasticity - not fitness - in determining the clonal origins of relapse in childhood leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6625.

Abstract 4797: β3-AR blockade induces differentiation of malignant leukemia T cells in mature T lymphocytes

Abstract Acute lymphoblastic leukemia (ALL) is a genetically heterogeneous cancer that arises from diverse genetic alterations during early lymphocyte development. About 10-15% of T-cell acute lymphoblastic leukemia (T-ALL) cases occur in childhood; despite having an event-free survival (EFS) rate of 85%, relapse is the main problem of this type of cancer. Mitochondria metabolic reprogramming plays a crucial role in the development of lymphocytes both in physiological and pathological processes such as differentiation and neoplastic transformation respectively. Mitochondria are the main actors of cellular homeostasis. They use glycolysis products exploiting them in the Krebs cycle and in oxidative phosphorylation to produce energy and plays key roles in reactive oxygen species (ROS) generation, Ca2+ signaling, cell death induction, and cellular differentiation. In particular, the increase of ROS is important to reduce tumor cell viability and at the same time is involved in induction of hematopoiesis. β3-adrenoreceptors (β3-Ars) is a class of receptors associated with pro-tumoral signaling in different tumors. In this study, we first have observed the expression of β3-Ars in leukemia T cells and then we studied how the modulation of this receptors affected tumor-related pathways. The treatment with an antagonist of β3-Ars (SR59230A) induced an increase of ROS leading to lymphoblastic cells differentiation, decrease of proliferation and enhanced induction of apoptosis signaling. Overall, these results suggest that β3-Ars could be a possible target for treatment of T-ALL. Citation Format: Francesco Carrozzo, Gennaro Bruno, Claudia Masi, Alessia Boaretto, Claudio Favre, Maura Calvani. β3-AR blockade induces differentiation of malignant leukemia T cells in mature T lymphocytes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4797.

A New Case of Childhood Acute Lymphoblastic B-Cell Leukemia from Pristina

Abstract Acute lymphoblastic leukemia (ALL) is a malignant disease caused by mutations in B- or T-cell precursors of bone marrow cells. Childhood acute lymphoblastic leukemia (ALL) is a subtype of pediatric cancer with a 1 in 2000 incidence. Here we present a new childhood ALL in a 3-year-old girl. As CD45/19, CD10/19, CD3, CD8, CD10, and CD19 were positive in immunohistochemically analyses of blast cells, a B-ALL was diagnosed with a causative ETV6-RUNX1 gene fusion. The patient was treated based on standard protocols BMF-ALL 2009. Interestingly, an aunt and a grandfather of the patient had experienced malignancies as well, which may be carefully interpreted as a hint on a familial cancer syndrome.

S100A8 inhibition in leukemic lymphoblasts induces sensitivity to chemotherapy and inhibition of disease relapse.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children and relapsed B-ALL is the leading cause of mortality in children with leukemia due to a lack of response to treatment. S100A8 is a low molecular weight calcium-binding intracellular protein that is expressed in certain cells, and its increased expression is seen in most tumors as well as in relapsed childhood B-ALL cases. The present study indicates the important role of S100A8 in improving viability and resistance to chemotherapy in relapsed B-ALL lymphoblasts. S100A8 levels were compared in B-ALL and relapsed B-ALL lymphoblasts that were sensitive and resistant to Vincristine, respectively. S100A8 was inhibited in the lymphoblasts of two patients by antisense locked nucleic acid (LNA) GapmeRs and the decreased expression of S100A8 was evaluated using quantitative real-time PCR and ELISA. Then, the S100A8 antisense LNA GapmeRs-transfected cells were treated with Vincristine and the expression levels of S100A8 mRNA and S100A8 protein were re-determined. At all of these stages, cell viability and LC50 were assessed by MTT assay. The results showed that S100A8 levels in relapsed B-ALL lymphoblasts were significantly higher than B-ALL lymphoblasts. Moreover, the increase in S100A8 expression was proportionate to the increase in Vincristine resistance in these cells. The S100A8 knockdown procedure using antisense LNA GapmeRs decreased the cell viability and increased vincristine sensitivity in lymphoblasts of two patients, and it also increased the sensitivity to chemotherapy in relapsed B-ALL lymphoblasts. According to the findings of the present study, S100A8 is effective in developing lymphoblast resistance to chemotherapy, and its enhanced expression may contribute to shifting B-ALL into the relapse phase of the illness. As a result, S100A8 may be a valuable target for managing and improving relapses B-ALL.

P198 ACUTE CORONARY SYNDROME COMPLICATED BY LEFT VENTRICULAR THROMBOSIS AND ISCHEMIC STROKE IN YOUNG WOMAN WITH ACUTE LYMPHOBLASTIC LEUKEMIA IN CHEMOTHERAPY TREATMENT

Abstract Acute lymphoblastic leukemia (ALL) is the most common cancer in children and accounts for 75% of all leukemias. In ALL, very immature leukemia cells build up in the bone marrow, destroying and replacing normal cells. Thrombotic events are a complication of acute leukemia induction therapy, particularly acute lymphoblastic leukemia (ALL) treated with L – asparaginase. We present the case of a 23–year–old girl suffering from acute lymphoblastic leukemia from pediatric age, undergoing chemotherapy treatment. In August 2021 she was admitted to ICU for acute myocarditis in shock which resolved after specific therapy. In September 2021 he came to our observation again for chest pain, fever, pulmonary thickening, but with normal echocardiography and echocardiography. The next day, the patient presented a worsening of symptoms, with the appearance of an elevated ST in the anterior to the ecg trace and apical, SIV and anterior wall akinesia with severely reduced FE on the echocardiogram. After collegial discussion also with fellow haematologists for the presence of thrombocytopenia, antithrombotic therapy was administered and an urgent coronary examination was carried out. The examination showed acute thrombotic occlusion of the middle–distal VAT. In the same session we proceeded to thrombus aspiration and implantation of medicated stents, procedure concluded in the absence of complications. The following morning the patient complained of paresthesia and weakness in the right side. On the echocardiogram, the presence of a thrombus in the apical area of about 2 cm. Skull CT was performed in urgency which showed cortical–subcortical hypodensity area in the left frontal–parietal site, referring to a recent ischemic lesion. In agreement with colleagues from Stroke Unite and hematology, heparin was infused intravenously in an attempt to dissolve the thrombus, but three episodes of VF were treated with effective DC shock during the hospital stay. On the same day, the girl was transferred intubated to resuscitation for respiratory arrest; in the following hours we witnessed the exitus. Our patient presented acute thrombosis both in the heart and in the brain, although she was being treated with antiplatelet and antithrombotic drugs and in the presence of thrombocytopenia. This case highlights the strong procoagulative role of haematological disease and of the chemotherapy indicated for it.

Somatic FOXC1 insertion mutation remodels the immune microenvironment and promotes the progression of childhood acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most common malignant hematological diseases in children. An immunosuppressive microenvironment, particularly regulatory T cell (Treg) infiltration, has been documented to be highly associated with childhood ALL. This present study, based on genetic factors, was aimed at investigating the mutations potentially involved in the immunosuppressive microenvironment in childhood ALL. After whole-exome sequencing was used on DNA extracted from the T cells of ALL bone marrow samples, we found the FOXC1 H446HG induced a increased Treg while decreased cytotoxic T lymphocyte (CTL) in bone marrow. The mutation of FOXC1 in T cell promoted the proliferation of leukemia cells in vitro and in vivo. CpG islands formed by insertion mutation led to an abnormal increase in exon methylation and were associated with the suppression of FOXC1. Decreased FOXC1 attenuated the transcription of HDAC1, thus resulting in the activation of KLF10 through increasing H3K27 acetylation in the promoter region. In conclusion, the de novo insertion mutation in FOXC1 induced suppression of FOXC1, thereby promoting a Treg/CTL shift in the ALL immune microenvironment. The FOXC1 H446HG mutation might be a potential therapeutic target for ALL in the future.

How essential are in-person clinic visits during maintenance treatment of children with acute lymphoblastic leukemia?

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Treatment consists of an initial intensive phase of chemotherapy, followed by a prolonged period of maintenance chemotherapy intended to reduce the risk of relapse. During the COVID-19 pandemic, the need arose to identify and reduce non-essential hospital visits. We aimed to determine which proportion of in-person clinic visits during ALL maintenance therapy was associated with a change of management based on the results of the physical examination. Medical records of children receiving maintenance chemotherapy for B-precursor ALL between September 2019 and February 2020 were reviewed. Visits with a new finding on physical examination were divided into those where an in-person assessment was deemed essential versus not essential. Finally, we determined the proportion of essential in-person visits that resulted in a change of management. A total of 240 maintenance visits by 75 children were analyzed. An abnormal finding on physical examination was noted during 20 visits (8.3%). Of those, 14 (5.8%) uncovered a new finding, six (2.5%) were classified as "in-person visit essential," and among those six visits, three (1.2%) resulted in a change of patient management (one for acute otitis media, one for wheezing, and one for limp). Our findings support the evaluation of care delivery models other than in-person visits during ALL maintenance therapy. A prospective study is required to delineate criteria, benefits/risks, and families' perspectives associated with virtual care delivery and the optimal frequency of in-person visits.