Acute Lymphoblastic Leukemia Cells Research Articles

DNT cells mediate resistance to CAR-T cells therapy in a pediatric patient with relapsed and refractory B-ALL.

Chimeric antigen receptor T (CAR-T) cells therapy is a milestone achievement in the immunotherapy of relapsed and refractory (R/R) B cell acute lymphoblastic leukemia (B-ALL). However, some patients treated with CAR-T cells do not achieve complete remission, the mechanisms of which have not been elucidated. In the present study, we report a 9-year-old pediatric patient with refractory B-ALL received a triple infusion of autologous CD19 CAR-T cells therapy after the second relapse. CAR-T cells expanded in the peripheral blood and bone marrow. However, the patient did not achieve complete remission, indicating a lack of response to CAR-T cells therapy. Analysis of etiological factors revealed that the number of CD4 and CD8 double-negative T (DNT) cells was significantly upregulated in the peripheral blood, bone marrow, and autologous CAR-T cells products. In conclusiont,these findings indicate that DNT cells mediated resistance to CAR-T cells therapy in this pediatric patient with R/R B-ALL.

Expression of transcriptional repressors of ZBTB genes in acute lymphoblastic leukemia cell lines treated with prednisolone

Expression of transcriptional repressors of ZBTB genes in acute lymphoblastic leukemia cell lines treated with prednisolone

Rituximab potentially improves clinical outcomes of CAR-T therapy for r/r B-ALL via sensitizing leukemia cells to CAR-T-mediated cytotoxicity and reducing CAR-T exhaustion.

Despite chimeric antigen receptor (CAR) T-cell therapy has achieved great advances in recent year, approximately 50% of relapsed/refractory B cell acute lymphoblastic leukemia (r/r B-ALL) patients treated with CAR-T experience relapse 6 months post CAR-T treatment. CD20 express on 30 to 50% of B-ALL, which makes CD20 Monoclonal Antibody as one of the potential therapy strategies to decrease the tumor burden and improve the efficacy of CAR-T therapy. Adding Rituximab to chemotherapy protocol had been demonstrated to improve the outcome for CD20-positive ALL. However, rare study explored the influence of Rituximab combined with CAR-T therapy. We retrospectively analyzed 20 r/r B-ALL patients who received CAR-T therapy, all of whom had failed multiple lines of therapy. Before CAR-T infusion, we administered Rituximab to 10 patients with high CD20 expression at a dose of 375mg/m2 for 1 day. Meanwhile, we selected 10 patients with the comparable features who underwent CAR-T treatment without Rituximab in the same period as the control group. In vitro, the surface molecule expression and killing of CAR-T post Rituximab-treated B-ALL cells co-incubated with CAR-T cells were detected by flow cytometry. The median follow-up of Rituximab and Control groups were 29.27 and 9.83 months. We found that adding Rituximab may confer a favorable prognosis compared with Control group. The 2-year overall survival (OS) and leukemia-free survival (LFS) rates both were longer in the Rituximab group (90% vs. 26.7%, p=0.0342; 41.7% vs. 25%, p=0.308). In vitro, we observed that Rituximab-treated tumour cells are more sensitive to CAR-T killing and a broad range of cytokines and chemokines were produced when Rituximab-treated Nalm-6 cells co-cultured with 19-22CAR-T cells, such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2). To investigate whether Rituximab has an effect on CAR-T persistence, we stimulated CAR-T cells repeatedly in vitro with Rituximab-treated Nalm-6 to evaluate the changes in CAR-T surface exhaustion molecules at different times. We found that the expression of exhaustion molecules (LAG-3, PD-1, TIM-3) on CAR-T cells were significantly lower in the Rituximab group than in the Control group. Rituximab combined with CAR-T therapy is effective for improving the long-term prognosis of B-ALL patients who have failed multiple lines of therapy. In vitro, we observed that rituximab potentially improves CAR-T efficacy by sensitizing ALL to CART-mediated cytotoxicity and reducing CAR-T exhaustion.

Plasma microbial cell-free DNA following chimeric antigen receptor T cell therapy in pediatric patients with relapsed/refractory leukemia.

Chimeric antigen receptor (CAR) T cell therapy is a promising treatment for pediatric patients with relapsed or refractory B cell acute lymphoblastic leukemia (R/R B ALL). Cytokine release syndrome (CRS) is a common toxicity after CAR T cell therapy and fever is often the first symptom. Differentiating CRS from infection after CAR T cell therapy can be challenging. Plasma microbial cell free DNA (mcfDNA) is a novel diagnostic tool which allows for qualitative and quantitative assessment of over 1000 organisms. This pilot study sought to characterize mcfDNA results in pediatric patients with R/R B ALL in the first 2 months after CAR T cell therapy.

Hyperactive STAT5 hijacks T cell receptor signaling and drives immature T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive immature T cell cancer. Mutations in IL7R have been analyzed genetically, but downstream effector functions such as STAT5A and STAT5B hyperactivation are poorly understood. Here, we studied the most frequent and clinically challenging STAT5BN642H driver in T cell development and immature T cell cancer onset and compared it with STAT5A hyperactive variants in transgenic mice. Enhanced STAT5 activity caused disrupted T cell development and promoted an early T cell progenitor-ALL phenotype, with upregulation of genes involved in T cell receptor (TCR) signaling, even in absence of surface TCR. Importantly, TCR pathway genes were overexpressed in human T-ALL and mature T cell cancers and activation of TCR pathway kinases was STAT5 dependent. We confirmed STAT5 binding to these genes using ChIP-Seq analysis in human T-ALL cells, which were sensitive to pharmacologic inhibition by dual STAT3/5 degraders or ZAP70 tyrosine kinase blockers in vitro and in vivo. We provide genetic and biochemical proof that STAT5A and STAT5B hyperactivation can initiate T-ALL through TCR pathway hijacking and suggest similar mechanisms for other T cell cancers. Thus, STAT5 or TCR component blockade are targeted therapy options, particularly in patients with chemoresistant clones carrying STAT5BN642H.

Oncotherapy resistance explained by Darwinian and Lamarckian models.

Cell and antibody therapies directed against surface molecules on B cells, e.g., CD19-targeting chimeric antigen receptor T cells (CD19 CAR-T), are now standard for patients with chemorefractory B cell acute lymphoblastic leukemias and other B cell malignancies. However, early relapse rates remain high. In this issue of the JCI, Aminov, Giricz, and colleagues revealed that leukemia cells resisting CD19-targeted therapy had reduced CD19 but also low CD22 expression and were sensitive to Bruton's tyrosine kinase and/or MEK inhibition. Overall, their observations support the evolution of resistance following a Lamarckian model: the oncotherapy directly elicits adaptive, resistance-conferring reconfigurations, which are then inherited by daughter cells as epigenetic changes. The findings prompt reflection also on the broader role of epigenetics in decoupling of replication from lineage differentiation activation by the B cell lineage master transcription factor hub. Such oncogenesis and resistance mechanisms, being predictable and epigenetic, offer practical opportunities for intervention, potentially non-cross-resistant and safe vis-à-vis present cytotoxic and CAR-T treatments.

CXCR6 defines therapeutic subtypes of CD4+ cytotoxic T cell lineage for adoptive cell transfer therapy in pediatric B cell acute lymphoblastic leukemia

The potential of cytotoxic CD4+ T cells and tissue resident memory T cells (Trm) in achieving adult leukemia remission have been highlighted [1,2]. We hypothesized that CXCR6 could serve as a marker for cytotoxic CD4+ Trm cells in the bone marrow (BM) of pediatric B-ALL patients. Flow cytometry (FCM) and published single cell RNA sequencing (scRNA-seq) datasets were employed to characterize CXCR6+CD4+ T cells in the BM and peripheral blood (PB) of pediatric B-ALL patients and healthy donors. FCM, scRNA-seq and co-culture were utilized to explore the cytotoxicity of CXCR6+CD4+ T cells in vitro based on in vitro induction of CXCR6+CD4+ T cells using tumor antigens and peripheral blood mononuclear cells (PBMCs). The ssGSEA based on the cell markers identified according to the in vivo scRNA-seq data, the TARGET-ALL-P2 datasets, and integrated machine learning algorithm were employed to figure out the key cells with prognostic values, followed by simulation of adoptive cell transfer therapy (ACT). Integrated machine learning identified the high-risk cells for disease free survival, and overall survival, while simulation of ACT therapy using CXCR6+CD4+T cells indicated that CXCR6+CD4+ T cells could remodel the bone marrow microenvironments towards anti-tumor. Based on the expression of genes involved in formation of resident memory T cells, CXCR6 is not a marker of resident memory CD4+T cells but defines therapeutic subtypes of CD4+ cytotoxic T cell lineage for pediatric B-ALL.

Wiskott Aldrich syndrome protein (WASp)-deficient Th1 cells promote R-loop-driven transcriptional insufficiency and transcription-coupled nucleotide excision repair factor (TC-NER)-driven genome-instability in the pathogenesis of T cell acute lymphoblastic leukemia

BackgroundT-ALL is an aggressive hematological tumor that develops as the result of a multi-step oncogenic process which causes expansion of hematopoietic progenitors that are primed for T cell development to undergo malignant transformation and growth. Even though first-line therapy has a significant response rate, 40% of adult patients and 20% of pediatric patients will relapse. Therefore, there is an unmet need for treatment for relapsed/refractory T-ALL to develop potential targeted therapies. MethodsPediatric T-ALL patient derived T cells were grown under either nonskewingTh0 or Th1-skewing conditions to further process for ChIP-qPCR, RDIP-qPCR and other RT-PCR assays. Endogenous WASp was knocked out using CRISPR-Cas9 and was confirmed using flow cytometry and western blotting. LC-MS/MS was performed to find out proteomic dataset of WASp-interactors generated from Th1-skewed, human primary Th-cells. DNA-damage was assessed by immunofluorescence confocal-imaging and single-cell gel electrophoresis (comet assay). Overexpression of RNaseH1 was also done to restore normal Th1-transcription in WASp-deficient Th1-skewed cells. ResultsWe discovered that nuclear-WASp is required for suppressing R-loop production (RNA/DNA-hybrids) at Th1-network genes by ribonucleaseH2 (RNH2) and topoisomerase1. Nuclear-WASp is associated with the factors involved in preventing and dissolving R-loops in Th1 cells. In nuclear- WASp-reduced malignant Th1-cells, R-loops accumulate in vivo and are processed into DNA-breaks by transcription-coupled-nucleotide-excision repair (TC-NER). Several epigenetic modifications were also found to be involved at Th1 gene locus which are responsible for active/repressive marks of particular genes. By demonstrating WASp as a physiologic regulator of programmed versus unprogrammed R-loops, we suggest that the transcriptional role of WASp in vivo extends also to prevent transcription-linked DNA damage during malignancy and through modification of epigenetic dysregulations. ConclusionOur findings present a provocative possibility of resetting R-loops as a therapeutic intervention to correct both immune deficiency and malignancy in T-cell acute lymphoblastic leukemia patients and a novel role of WASp in the epigenetic regulation of T helper cell differentiation in T-ALL patients, anticipating WASp's requirement for the suppression of T-ALL progression.

Metabolic Profiling as an Approach to Differentiate T-Cell Acute Lymphoblastic Leukemia Cell Lines Belonging to the Same Genetic Subgroup.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive tumor mainly affecting children and adolescents. It is driven by multiple genetic mutations that together define the leukemic phenotype. Interestingly, based on genetic alterations and/or deregulated expression, at least six genetic subgroups have been recognized. The TAL/LMO subgroup is one of the most represented genetic subgroups, characterizing 30-45% of pediatric T-ALL cases. The study of lipid and metabolic profiles is increasingly recognized as a valuable tool for comprehending the development and progression of tumors. In this study, metabolic and lipidomic analysis via LC/MS have been carried out on four T-ALL cell lines belonging to the TAL/LMO subgroup (Jurkat, Molt-4, Molt-16, and CCRF-CEM) to identify new potential metabolic biomarkers and to provide a subclassification of T-ALL cell lines belonging to the same subgroup. A total of 343 metabolites were annotated, including 126 polar metabolites and 217 lipid molecules. The statistical analysis, for both metabolic and lipid profiles, shows significant differences and similarities among the four cell lines. The Molt-4 cell line is the most distant cell line and CCRF-CEM shows a high activity in specific pathways when compared to the other cell lines, while Molt-16 and Jurkat show a similar metabolic profile. Additionally, this study highlighted the pathways that differ in each cell line and the possible enzymes involved using bioinformatic tools, capable of predicting the pathways involved by studying the differences in the metabolic profiles. This experiment offers an approach to differentiate T-ALL cell lines and could open the way to verify and confirm the obtained results directly in patients.

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.

Chemotherapy-initiated cysteine-rich protein 61 decreases acute B-lymphoblastic leukemia chemosensitivity

PurposeChemoresistance is a major challenge for acute lymphoblastic leukemia (ALL) treatment. Cysteine-rich protein 61 (Cyr61) plays an important role in drug resistance modulation of tumor cells, and Cyr61 levels are increased in the bone marrow of patients with ALL and contribute to ALL cell survival. However, the effect of Cyr61 on B cell acute lymphoblastic leukemia (B-ALL) cell chemosensitivity and the regulatory mechanisms underlying Cyr61 production in bone marrow remain unknown.MethodsNalm-6 and Reh human B-ALL cell lines were used in this study. Cyr61 levels were assessed using quantitative real-time PCR (qRT-PCR), western blot analysis, and enzyme-linked immunosorbent assay. The effect of Cyr61 on B-ALL cell chemosensitivity to daunorubicin (DNR) was evaluated using cell viability and flow cytometry analyses. The regulatory mechanisms of Cyr61 production in bone marrow were examined using qRT-PCR and western blot analysis.ResultsCyr61 knockdown and overexpression increased and decreased the chemosensitivity of B-ALL cells to DNR, respectively. Cyr61 attenuated chemotherapeutic drug-induced apoptosis by upregulating B cell lymphoma-2. Notably, DNR induced DNA damage response and increased Cyr61 secretion in B-ALL cells through the ataxia telangiectasia mutated (ATM)-dependent nuclear factor kappa B pathway.ConclusionDNR induces Cyr61 production in B-ALL cells, and increased Cyr61 levels reduce the chemosensitivity of B-ALL cells. Consequently, targeting Cyr61 or related ATM signaling pathway may present a promising treatment strategy to enhance the chemosensitivity of patients with B-ALL.

Asciminib Maintains Antibody-Dependent Cellular Cytotoxicity against Leukemic Blasts.

B cell acute lymphoblastic leukemia (B-ALL) is characterized by an accumulation of malignant precursor cells. Treatment consists of multiagent chemotherapy followed by allogeneic stem cell transplantation in high-risk patients. In addition, patients bearing the BCR-ABL1 fusion gene receive concomitant tyrosine kinase inhibitor (TKI) therapy. On the other hand, monoclonal antibody therapy is increasingly used in both clinical trials and real-world settings. The introduction of rituximab has improved the outcomes in CD20 positive cases. Other monoclonal antibodies, such as tafasitamab (anti-CD19), obinutuzumab (anti-CD20) and epratuzumab (anti-CD22) have been tested in trials (NCT05366218, NCT04920968, NCT00098839). The efficacy of monoclonal antibodies is based, at least in part, on their ability to induce antibody-dependent cellular cytotoxicity (ADCC). Combination treatments, e.g., chemotherapy and TKI, should therefore be screened for potential interference with ADCC. Here, we report on in vitro data using BCR-ABL1 positive and negative B-ALL cell lines treated with rituximab and TKI. NK cell activation, proliferation, degranulation, cytokine release and tumor cell lysis were analyzed. In contrast to ATP site inhibitors such as dasatinib and ponatinib, the novel first-in-class selective allosteric ABL myristoyl pocket (STAMP) inhibitor asciminib did not significantly impact ADCC in our settings. Our results suggest that asciminib should be considered in clinical trials.

Abstract 624: Ptpn1 deficiency collaborates with a NUP98::HOXD13 fusion gene to generate B cell precursor acute lymphoblastic leukemia

Abstract Background: Acute lymphoid leukemia is characterized by inherited or acquired mutations that effect critical differentiation and proliferation pathways. We have shown that Mcm2 deficient mice developed T cell acute lymphoblastic leukemia, due to copy number variations, most commonly interstitial deletions throughout the genome. When crossed with mice that expressed a NUP98::HOXD13 (NHD13) fusion gene, Mcm2−/−NHD13 mice developed B cell precursor ALL (BCP-ALL). A majority of these BCP-ALL had acquired homozygous deletions of Ptpn1, a protein tyrosine phosphatase, leading to the hypothesis that Ptpn1 deficiency combined with NHD13 expression leads to BCP ALL. Objective: To investigate the role of Ptpn1 deletion in BCP-ALL development using mouse models. Methods: Mice expressing an NHD13 fusion gene were crossed with Ptpn1 knockout mice, generating 6 possible genotypes. Mice were followed for 18 months. Mice with signs of leukemia were characterized by clinical evaluation, CBC, flow cytometry, and IHC. Primary BCP-ALL and derived BCP-ALL cell lines were also evaluated with RNA-seq and molecular pathway analysis. Results: NHD13+Ptpn1-/- mice developed BCP-ALL with 65% penetrance, characterized by hyperleukocytosis, anemia, thrombocytopenia, and invasion of non-hematopoietic tissues. Similar to human BCP-ALL, NHD13+Ptpn1-/- BCP-ALL had clonal IGH as well as clonal Tcrd gene rearrangements. Flow cytometry revealed CD19 and/or B220 expression. NHD13+Ptpn1+/- mice with BCP-ALL frequently lost the wild-type (WT) Ptpn1 allele in leukemic cells, reinforcing the hypothesis that Ptpn1 can function as a classic tumor suppressor gene in this context. Whole exome sequencing revealed acquired mutations in B-cell differentiation genes (Pax5 or Bcor) and activating mutations in tyrosine kinase genes (Jak1/3 and Flt3). Transcription signature analysis showed significant upregulation of Hoxa/b gene clusters, RNase12 and LncRNAs subsets. Conclusion: This study demonstrates that Ptpn1 loss combined with expression of NHD13 fusion gene leads to highly penetrant BCP-ALL in mice, suggesting a role for Ptpn1 in preventing malignant transformation. These findings present a collaborative model for BCP-ALL in which the NHD13 transgene leads to increased stem cell self-renewal, somatic Bcor or Pax5 mutations block normal B cell differentiation, and somatic signaling mutations (Jak1/3,Flt3) lead to hyperproliferation, which is potentiated by Ptpn1 deficiency. Citation Format: Nupur Nigam, Toshihiro Matsukawa, Ryan Bertoli, Michel L. Tremblay, Mianmian Yin, Peter D. Aplan. Ptpn1 deficiency collaborates with a NUP98::HOXD13 fusion gene to generate B cell precursor 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 624.

The Eμ-Ret mouse is a novel model of hyperdiploid B-cell acute lymphoblastic leukemia

The presence of supernumerary chromosomes is the only abnormality shared by all patients diagnosed with high-hyperdiploid B cell acute lymphoblastic leukemia (HD-ALL). Despite being the most frequently diagnosed pediatric leukemia, the lack of clonal molecular lesions and complete absence of appropriate experimental models have impeded the elucidation of HD-ALL leukemogenesis. Here, we report that for 23 leukemia samples isolated from moribund Eμ-Ret mice, all were characterized by non-random chromosomal gains, involving combinations of trisomy 9, 12, 14, 15, and 17. With a median gain of three chromosomes, leukemia emerged after a prolonged latency from a preleukemic B cell precursor cell population displaying more diverse aneuploidy. Transition from preleukemia to overt disease in Eμ-Ret mice is associated with acquisition of heterogeneous genomic abnormalities affecting the expression of genes implicated in pediatric B-ALL. The development of abnormal centrosomes in parallel with aneuploidy renders both preleukemic and leukemic cells sensitive to inhibitors of centrosome clustering, enabling targeted in vivo depletion of leukemia-propagating cells. This study reveals the Eμ-Ret mouse to be a novel tool for investigating HD-ALL leukemogenesis, including supervision and selection of preleukemic aneuploid clones by the immune system and identification of vulnerabilities that could be targeted to prevent relapse.

Detection of terminal deoxynucleotidyl transferase activity based on self-mediated nucleic acid elongation and elemental labeling inductively coupled plasma-mass spectrometry

Terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase, is recognized as a promising biomarker for acute leukemia. Herein, taking the advantage of the self-mediated strand elongation property of TdT, a simple and sensitive method for TdT activity assay was developed based on gold nanoparticles (AuNPs) labeling inductively coupled plasma mass spectrometry (ICP-MS). In the presence of TdT, the primer DNA on magnetic beads is elongated with an adenine-rich single stranded long chain that can label poly-thymine modified AuNPs. After acid elution, the labeled AuNPs were detected by ICP-MS, and the signal intensity of 197Au reflected the TdT activity. Under the optimal conditions, the limit of detection for TdT activity is down to 0.054 U mL−1, along with good selectivity and strong tolerance to other interfering proteins. Furthermore, it achieves a straightforward and accurate detection of TdT activity in acute lymphoblastic leukemia cells without sample pre-processing and tool enzyme addition. Therefore, the proposed method shows great promise as a valuable tool for TdT-related biological research and leukemia therapeutics.

Abstract 6325: Lineage tracing defines responding CAR T cells in patients with B cell malignancies

Abstract Autologous T cells engineered to express a chimeric antigen receptor (CAR) have transformed the standard of care for patients with B cell malignancies, but >50% of patients progress following therapy. Here, we sought to understand key T cell intrinsic factors impacting efficacy: CAR T cell expansion, persistence, and homing to the tumor. Using an endogenous T cell receptor (TCR) sequence as a ‘barcode’, we followed individual T cell clonotypes at the single-cell level from pre-manufacture apheresis and infusion products to tumor-involved lymph nodes and blood at peak and late expansion in 22 adult patients with relapsed or refractory large B cell lymphoma (LBCL) or acute lymphoblastic leukemia (ALL) treated with axicabtagene ciloleucel, an FDA-approved CD19-CAR T cell therapy, or bispecific CD19/CD22-CAR T cells on an investigator-initiated trial (NCT03233854). The resulting CAR T cell atlas comprises matched transcriptome (scRNA-seq) and surface protein expression (CITE-seq) for 846,344 cells from 97 samples, with 251,175 unique TCR clonotypes identified, including 17,479 clonotypes that could be traced across ≥2 timepoints in CAR+ cells, enabling lineage tracing at scale for the first time. We found that T cells present in apheresis that preferentially expand during manufacturing primarily comprise T central memory (Tcm) cells and are enriched for predicted specificity to common viruses, including influenza, but also include a subset of T regulatory (Treg) cells. In infusion products, CAR T conventional (non-Treg) cells with superior abilities to home to the tumor, expand, and persist post-infusion share features of activated T effector memory (Tem) phenotype enriched for expression of BATF3 and high TCF4 transcription factor activity. Baseline apheresis and infusion products were enriched for cells classified as Tregs in patients with poor response. This observation aligned with our recent work linking post-infusion CAR Tregs to progression following CD19-CAR T cell therapy. We therefore performed lineage tracing and methylation analysis of the Treg-specific demethylated region (TSDR) in the FOXP3 gene and found that post-infusion CAR Tregs are primarily derived from pre-existing Tregs. Of interest, flow cytometry analysis of baseline blood from a cohort of 53 patients treated with CD19-CAR for LBCL showed that Tregs are elevated in baseline blood relative to healthy donors. Removing Tregs from healthy donor T cells prior to manufacturing enhanced CAR T cell expansion and anti-tumor activity against JeKo-1 human lymphoma cell line, whereas adding 5% Tregs to mimic patient T cells rendered the resulting CAR T cells less active. These analyses pinpoint the identities of apheresis T cells and infusion CAR T cells with properties impacting efficacy, and also identify pre-existing Tregs as limiting efficacy of CAR T cell immunotherapies. Citation Format: Zinaida Good, Mark P. Hamilton, Jay Y. Spiegel, Moksha H. Desai, Zachary J. Ehlinger, Patrick J. Quinn, Yiyun Chen, Snehit Prabhu, Shin-Heng Chiou, Sreevidya Kurra, Eric Yang, Michael G. Ozawa, Matthew J. Frank, Lori Muffly, Gursharan K. Claire, Sushma Bharadwaj, Saurabh Dahiya, Katherine A. Kong, Mark M. Davis, Sylvia K. Plevritis, Elena Sotillo, Bita Sahaf, David B. Miklos, Crystal L. Mackall. Lineage tracing defines responding CAR T cells in patients with B cell malignancies [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 6325.

Abstract 6587: Discovery of molecular glue of FBXW7:MYC interaction using small molecule microarray

Abstract MYC deregulation is a key driver and feature of many human cancers. Yet, despite its importance in cancer progression, MYC is considered “undruggable” by small molecules due to its disordered conformation. Therefore, interfering with its interactions with protein partners to modulate MYC activity appears viable. FBXW7 is an E3 ligase, which serves as a constituent of the SCF (Skp1-Cul1-F box) ubiquitin ligase complex. It mediates the degradation of both c-Myc and Cyclin E through ubiquitin-dependent proteolysis. FBXW7 mutations are found in various human tumor types, establishing its role as a general human tumor suppressor. Specifically, FBXW7 down-regulation increases MYC level, and its expression correlates to lethal outcomes of breast epithelial cells, poor prognosis in glioblastoma, and T cell acute lymphoblastic leukemia remission. Therefore, our project aims to discover potent small molecule glues that would enhance the interaction between FBXW7 and MYC, leading to MYC degradation through proteolysis and eventually suppression of various cancers. Taking advantage of our high throughput screening technology, Small Molecule Microarrays (SMMs), we successfully identified 29 compounds that potently bind to both c-Myc and FBXW7 after screening our 65,000 ‘drug-like’ small molecule library. We then performed secondary assays using a mCherry-GFP MYC reporter cell line to identify our lead compound, KI-FBX-001, which decreases MYC’s expression:transcription ratio by over 50% compared to control. Additionally, this down-regulation can be rescued by protease inhibitor MG132, indicating that the effect is achieved via proteolysis. In vitro dose-response and time-course treatment revealed an optimal drugging concentration at 15uM in K562 cells, with inhibition reaching its peak at the 4-hour time point at over 60%. Using the Qiagen MYC reporter assay, the in vitro IC50 of KI-FBX-001 is found at 7.42uM. Beyond the protein level, the qPCR result also reveals a decrease in MYC mRNA level at over 70%. Surface Plasmon Resonance (SPR) is used to confirm direct binding between FBXW7 and KI-FBX-001, which showed promising binding with kd=0.17µM. Proximity Ligation Assay further confirms KI-FBX-001’s effect in enhancing FBXW7:MYC interaction in the cell. In summary, our findings proved that KI-FBX-001 is a promising molecular glue in the FBXW7:MYC interaction, and can effectively modulate the MYC level for future therapeutic purposes. Citation Format: Shenghao Guo, Morgan Stilgenbauer, Bocheng Wu, Yatin Mankan, Angela N. Koehler. Discovery of molecular glue of FBXW7:MYC interaction using small molecule microarray [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 6587.

Abstract 6672: Unraveling the microbiome: antibiotic impact on CAR T therapy and the synergistic potential of vancomycin in enhancing antitumor immunity

Abstract The landscape of treating hematologic malignancies has undergone a revolutionary transformation with the advent of Anti-CD19 chimeric antigen receptor T cell therapy (CART-19). Despite its success, persistent challenges like disease relapse and CAR-mediated toxicities remain. A comprehensive multicenter study involving 228 individuals revealed a significant association between antibiotic administration, particularly piperacillin/tazobactam, meropenem, and imipenem/cilastatin (P-I-M), targeting anaerobes within four weeks preceding CART-19 therapy, and adverse outcomes, including worsened survival and increased neurotoxicity. Intriguingly, another antibiotic, Cefepime, also used for neutropenic fever, displayed a positive trend when compared with P-I-M. This highlights the intricate relationship between the modulation of the intestinal microbiome and outcomes in CART-19 therapy for B cell malignancies. Building on these critical observations, our study delved into the impact of vancomycin-induced gut microbiota dysbiosis on chimeric antigen receptor (CAR) T immunotherapy. Utilizing preclinical models of CD19+ A20 lymphoma and CD19+ B16 melanoma, we discovered that mice receiving vancomycin alongside CART-19 therapy exhibited enhanced tumor control and increased cross-presentation of tumor-associated antigens (TAAs) compared to those receiving CART-19 alone. This effect was further validated through fecal microbiota transplants from healthy human donors to pre-conditioned mice, replicating the favorable findings. Importantly, our study extends to clinical correlates, as B cell acute lymphoblastic leukemia patients treated with CART-19 and oral vancomycin displayed elevated CART-19 peak expansion compared to unexposed patients. These compelling results underscore the profound influence of the gut microbiota on CAR T cell therapy and suggest that vancomycin-induced gut microbiota modulation may emerge as a potent strategy to enhance therapeutic outcomes across various tumor types. Citation Format: Andrea Facciabene, Mireia Uribe-Herranz, Vijay Bhoj. Unraveling the microbiome: antibiotic impact on CAR T therapy and the synergistic potential of vancomycin in enhancing antitumor 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 6672.

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.

Abstract 271: The effects of B-cell acute lymphoblastic leukemia cells on the adipose tissue microenvironment

Abstract Obesity increases the risk of developing many cancers, including breast, colon, kidney, and acute lymphoblastic leukemia (ALL). We have previously shown that ALL cells are recruited to migrate into adipose tissue, where they are protected from chemotherapy. In vitro, ALL cells induce significant changes in adipocytes which may contribute to these protective effects. However, how ALL cells affect the complex cell populations found in adipose tissue in vivo remains unknown. We therefore conducted single-cell RNA sequencing (scRNAseq) of the non-adipocyte stromal vascular fraction (SVF) from adipose tissue in obese mice with and without implanted syngeneic BCR-ABL positive ALL. Differential expression analysis was performed on individual SVF cell types to compare ALL vs. control adipose tissue and pathway enrichment analysis was used in identifying altered molecular processes between conditions. SVF from leukemic mice showed substantial differences in gene expression compared to control mice, suggesting that the ALL potentially induces changes in the microenvironment. Some of the largest differences were found in adipose tissue macrophages, and pathway enrichment analysis identified neutrophil recruitment and regulation as one of the top ten pathways upregulated in adipose macrophages from leukemic mice (4.10 fold enrichment, p<1 × 10-17 by Fisher’s exact test). Selected macrophage genes of interest in this pathway included CYBB (Cytochrome B-245 Beta), a neutrophil interaction gene that has been associated with macrophage infiltration in gastric cancer, was expressed 2.28 log2-fold higher in macrophages from leukemic compared to nonleukemic mice (p<1 × 10−99 by Wilcoxon test). ITGAL (Integrin subunit alpha L), which has been shown to be involved in neutrophil recruitment and implicated in cancer cell growth and transformation was expressed 2.85 log2-fold higher in macrophages from leukemic mice (p<1 × 10−99). Interestingly, adipose from leukemic mice showed a population of neutrophils (1.9% of SVF cells), while neutrophils were largely absent in adipose from nonleukemic mice (<0.1% of SVF cells), which is consistent with macrophage involvement in neutrophil recruitment. Our findings highlight the importance of adipose tissue in the leukemia microenvironment and show that ALL cells can influence this microenvironment in complex ways. Further work will confirm and investigate these cell-cell interactions, potentially improving our understanding of how adipose tissue contributes to cancer mortality. Citation Format: Jia Tan, Michael Cohen, Jessica Ding, In Sook Ahn, Xia Yang, Steven D. Mittelman. The effects of B-cell acute lymphoblastic leukemia cells on the adipose tissue microenvironment [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 271.