Leukemic Population Research Articles

In Vitro Purging of Acute Lymphoblastic Leukemia (B-ALL) Cells with the Use of PTL, DMAPT, or PU-H71

Acute lymphoblastic leukemia (ALL) is a hematopoietic disorder that mainly affects the child population, and it is characterized by the presence of lymphoid progenitor or precursor cells with different genetic alterations. The origin of this disease is controversial, since some authors assumed that leukemic transformation occurs in a lymphoid progenitor, and there is also evidence that suggests the existence of leukemic initiating cells (LIC). PTL, DMAPT, and PU-H71 are agents that have been shown to eliminate bulk and stem cells from myeloid leukemias, but this effect has not been analyzed in lymphoblastic leukemias. In this study, we evaluated the effect of these compounds in different populations from pediatric B-ALL. For this, bone marrow samples from pediatric patients without treatment were obtained and cultured in the presence or absence of PTL, DMAPT, and PU-H71. The viability and apoptosis index were analyzed by flow cytometry in different hematopoietic subpopulations. These observations indicate that PTL and DMAPT are able to reduce B-ALL cells with a minimum effect in normal hematopoietic and non-hematopoietic cells. In contrast, PU-H71 was able to reduce the leukemic population and had a minimal effect in normal cells. These results present evidence that PTL and DMAPT are able to abrogate in vitro different populations of B-ALL and could represent a possibility of treatment, as well as prevent disease progression or relapse.

Tracking Rare Single Donor and Recipient Immune and Leukemia Cells after Allogeneic Hematopoietic Cell Transplantation Using Mitochondrial DNA Mutations.

Combined tracking of clonal evolution and chimeric cell phenotypes could enable detection of the key cellular populations associated with response following therapy, including after allogeneic hematopoietic stem cell transplantation (HSCT). We demonstrate that mitochondrial DNA (mtDNA) mutations coevolve with somatic nuclear DNA mutations at relapse post-HSCT and provide a sensitive means to monitor these cellular populations. Furthermore, detection of mtDNA mutations via single-cell assay for transposase-accessible chromatin with select antigen profiling by sequencing (ASAP-seq) simultaneously determines not only donor and recipient cells but also their phenotype at frequencies of 0.1% to 1%. Finally, integration of mtDNA mutations, surface markers, and chromatin accessibility profiles enables the phenotypic resolution of leukemic populations from normal immune cells, thereby providing fresh insights into residual donor-derived engraftment and short-term clonal evolution following therapy for post-transplant leukemia relapse. As throughput evolves, we envision future development of single-cell sequencing-based post-transplant monitoring as a powerful approach for guiding clinical decision-making. Significance: mtDNA mutations enable single-cell tracking of leukemic clonal evolution and donor-recipient origin following allogeneic HSCT. This provides unprecedented insight into chimeric cellular phenotypes of early immune reconstitution, incipient relapse, and quality of donor engraftment with immediate translational potential for future clinical post-transplant monitoring and decision-making.

Immunophenotypic but Not Genetic Changes Reclassify the Majority of Relapsed/Refractory Pediatric Cases of Early T-Cell Precursor Acute Lymphoblastic Leukemia.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) develops from very early cells with the potential for both T-cell and myeloid differentiation. The ambiguous nature of leukemic blasts in ETP-ALL may lead to immunophenotypic alterations at relapse. Here, we address immunophenotypic alterations and related classification issues, as well as genetic features of relapsed pediatric ETP-ALL. Between 2017 and 2022, 7518 patients were diagnosed with acute leukemia (AL). In addition to conventional immunophenotyping, karyotyping, and FISH studies, we performed next-generation sequencing of the T-cell receptor clonal repertoire and reverse transcription PCR and RNA sequencing for patients with ETP-ALL at both initial diagnosis and relapse. Among a total of 534 patients diagnosed with T-cell ALL (7.1%), 60 had ETP-ALL (11.2%). Ten patients with ETP-ALL experienced relapse or progression on therapy (16.7%), with a median time to event of 5 months (ranging from two weeks to 5 years). Most relapses were classified as AL of ambiguous lineage (n = 5) and acute myeloid leukemia (AML) (n = 4). Major genetic markers of leukemic cells remained unchanged at relapse. Of the patients with relapse, four had polyclonal leukemic populations and a relapse with AML or bilineal mixed-phenotype AL (MPAL). Three patients had clonal TRD rearrangements and relapse with AML, undifferentiated AL, or retention of the ETP-ALL phenotype. ETP-ALL relapse requires careful clinical and laboratory diagnosis. Treatment decisions should rely mainly on initial examination data, taking into account both immunophenotypic and molecular/genetic characteristics.

Abstract 618: Preclinical assessment of APL-030, a selective and orally bioavailable inhibitor of the integrated stress response regulator GCN2 with activity against acute myeloid leukemia

Abstract GCN2 (EIF2AK4) is an evolutionarily conserved kinase and a pivotal regulator of the Integrated Stress Response (ISR), an adaptive cellular program triggered primarily by amino acid scarcity. Active GCN2 phosphorylates the translation initiation factor eIF2α, resulting in the attenuation of global protein synthesis and a largely ATF4-driven program of resource enhancement. GCN2 promotes cancer cell survival under conditions of microenvironmental or drug-induced intracellular nutrient scarcity. Here, we describe the preclinical development of APL-030, a novel and selective ATP-competitive inhibitor of GCN2. APL-030 was confirmed as a potent inhibitor of GCN2 in an on-target biochemical kinase assay with a Ki of 4.4nM. In a cell-based assay APL-030 decreased eIF2α phosphorylation in a dose-dependent manner via direct GCN2 inhibition with an IC50 of 50.8nM. In acute myeloid leukemia (AML) cell lines GCN2 inhibition resulted in a decrease in gene expression of effector genes CHAC1 and DDIT3. Moreover, treatment with APL-030 dose-dependently decreased cell viability in multiple AML cell lines, which was accompanied by an increase in caspase 3/7 activity. Bulk mRNA sequencing of MOLM-13 AML cells showed that APL-030 inhibited the extensive transcriptome changes triggered by glutamine depletion, in line with a high degree of selectivity of APL-030 for GCN2-mediated ISR signaling. In cells grown in rich medium, APL-030 triggered a largely ISR-independent transcriptome response that was dominated by the enrichment of pathways related to cell cycle as well as RNA and protein metabolism. In vivo, APL-030 treatment of an AML xenograft animal model completely inhibited tumor growth and prolonged survival time. Inhibition of AML tumor growth was shown to be a consequence of decreased cell viability and induction of caspase-dependent apoptosis. APL-030 was also tested on primary diagnostic AML cells, which were assessed for cell death by flow cytometry following staining for Annexin-V, 7-AAD, CD45, CD34, and CD38. Treatment with APL-030 caused a statistically significant increase in AML cell death in all six samples studied. Three samples exhibited a measurable CD34+/CD38- compartment, enabling evaluation of this leukemic stem cell-enriched population. APL-030 caused a statistically significant increase in cell death in the leukemic stem cell-enriched population in all three samples studied. APL-030 is a novel GCN2 inhibitor that that has shown encouraging efficacy in preclinical studies using both AML cell lines and patient-derived AML samples. Based on these preclinical results, a phase 1/2 study is planned in hematological tumors. Citation Format: Monica Roman-Trufero, Gavin Whitlock, Matthew Fuchter, Maxmila Jeyakumar, Panagiota Chaida, Sereina Annik Herzog, Armin Zebisch, Richard Butt, Holger W. Auner, Nadine Clemo. Preclinical assessment of APL-030, a selective and orally bioavailable inhibitor of the integrated stress response regulator GCN2 with activity against acute myeloid 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 618.

Pre-Clinical Developmental Hematopoiesis Demonstrates the Heterogeneity of KMT2A-Rearranged Infant Acute Lymphoblastic Leukemic Populations

KMT2A-rearranged (KMT2A-r) infant acute lymphoblastic leukemia (ALL) has a poor prognosis with increased risk of relapse when diagnosed at younger ages. Understanding the origin of these leukemic cells is crucial to comprehend disease development and prognosis differences. The unclear delineation of the identity of early blood populations in humans is a challenge in defining the cell type(s) driving this infant ALL. Recent pre-clinical studies revealed that definitive hematopoietic stem cells (HSCs) have minimal contribution to embryonic hematopoiesis, while independently arising embryonic hematopoietic progenitors (HPCs) that possess more limited self-renewal and/or multilineage differentiation capacities, are both necessary and sufficient to sustain the developing embryo. Here, we aim to leverage pre-clinical knowledge on the markers that distinguish HSCs from HPCs to investigate the potential contribution of HPCs in driving prenatal and infant leukemias. We defined transcriptional signatures from developmental HSC and HPC populations using thirteen single cell RNA sequencing (scRNA-seq) datasets from zebrafish, eight datasets generated by our research group and five from independent published studies. We then employed a neural network approach to define the orthologous cell type signatures most likely correlating with human healthy and blast cells from publicly available data of 18 KMT2A-r infant ALL patients. In accordance with previous literature, HSC and lymphomyeloid progenitor signatures are both highly represented in the leukemic blast populations. Additionally, we found subpopulation differences in gene expression within leukemic blasts cells including lymphoid progenitor, granulocytic, and T cell signatures, possibly suggesting heterogeneity in either their cell type of origin or developmental differentiation potential. Our findings support the ability to use orthologous scRNA-seq to decipher new understanding on the role of HSCs and HPCs in hematological malignances affecting infants and children.

Targeting CDK8/CDK19 to Disrupt Leukemic Stem Cell-like Population in Acute Myeloid Leukemia: Exploring RVU120 As a Promising Frontline Therapy

Leukemic stem cells (LSCs) are implicated in the initiation, maintenance, and relapse of leukemia. The failure of current standard induction chemotherapy, which primarily targets dividing cells, has been linked to the quiescence of LSCs, leading to resistance to treatment. Consequently, there is an urgent need for novel therapies specifically aimed at eradicating LSCs to achieve more effective and durable treatment responses. CDK8 and its paralog CDK19 associate with the mediator complex and regulate gene expression programs linked to cancer and stem cell pluripotency. RVU120, a highly selective and potent CDK8/19 inhibitor, is currently undergoing clinical trials for AML and high-risk myelodysplastic syndrome (HR-MDS) (NCT04021368). Previous studies have demonstrated its potential mechanism of action on LSCs, wherein inhibition of CDK8/CDK19 led to the activation of lineage commitment genes in sensitive AML cells. Through recent gene expression analysis, it was discovered that inhibition of CDK8/CDK19 in transformed CD34+ cord blood cells resulted in the downregulation of stemness and quiescence-associated genes, while simultaneously upregulating genes associated with hematopoietic lineage-committed progenitors. Building on these findings, this study further investigates the potential of CDK8/CDK19 inhibition as a means to target LSCs, providing a rationale for RVU120 as a frontline treatment option in AML. The impact of RVU120 on LSCs has been investigated using an in vitro hierarchical AML-8227 model. This primary model maintains distinct populations in culture conditions, including CD34+CD38- cells with quiescent LSC potential, CD34+CD38+ cells enriched in progenitors, and CD34- more mature blasts (Lechman et al., 2016, Cancer Cell). The CD34+CD38- LSC population was further characterized by flow cytometry showing the expression of LSC surface markers, namely CD45RA, CD123, CD44, and negative expression for CD90, rendering it a suitable model for mechanistic studies. Next, AML-8227 cells were subjected to RVU120 treatment, resulting in a noticeable reduction in cell proliferation and viability. Flow cytometric analysis demonstrated a significant decrease in the percentage and total cell number of LSC-enriched/progenitor populations within the AML hierarchical model upon inhibition of CDK8/CDK19. Furthermore, RVU120 treatment induced the differentiation of cells, as evidenced by a sequential increase in the percentage of early myeloid differentiation marker, CD64, and late myeloid differentiation marker, CD14 (Fig. 1). In order to deconvolute the activity and mechanism of action of RVU120 in different cell populations, single-cell RNAseq analysis on AML-8227 cells has been performed at 24h and 3 days of treatment using technology from 10xGenomics. Additionally, analyzed AML-8227 cells have been enriched in the CD34+ population to evaluate the effect of RVU120 on stem and progenitor fraction at the single-cell level. The bioinformatic analysis of the performed experiment is ongoing and its results will be integrated into the data described above. CDK8 activity can be conveniently tracked by measuring specific phosphorylation of its target S726 on STAT5 protein. Dose and exposure-dependent inhibition of this marker has been established in cells exposed to RVU120. Levels of STAT5 S726 have been measured in different cell populations in patients from the first-in-human study of RVU120 in AML/HR-MDS. Consistent with established roles of CDK8 in LSCs, significantly elevated levels of pSTAT5 were observed in CD34+ stem/progenitor-enriched population when compared to CD34- blast cells measured at baseline before the start of the treatment (p<0.0001; ratio paired t-test; updated data from ASH 2022; Fig. 2). In summary, RVU120 displays cytotoxic and differentiating effects on well-characterized LSC populations in various models. Additionally, the CDK8 activity marker is directly associated with LSC-enriched bone marrow cells in AML patients, highlighting RVU120's potential as a promising therapeutic for targeting AML LSCs. Moreover, single-cell studies offer insights into its inhibitory effects on LSC-enriched populations and capacity for inducing differentiation. Overall, these results support RVU120 as a frontline candidate in AML therapy, countering therapeutic failure caused by persistent LSCs.

Minimal Residual Disease Detection By Single Cell DNA Sequencing Technology: A Feasible Approach for Clinical Application and Identification of the Landscape of MRD Clones

Introduction:Despite recent advances in AML treatment, patients continue to relapse and become chemotherapy resistant, leading to poor long-term overall survival. The identification of this measurable residual disease (MRD), defined as the post-therapy presence of leukemic cells, currently stands as one of the most well-established risk factors. The abnormal phenotypic and molecular characteristics of AML cells offer an opportunity for disease monitoring using techniques like multiparameter flow cytometry (MFC) and qPCR, which are currently considered the gold standard for MRD detection. However, these techniques have limitations. The aim of this study was to validate a different approach using single-cell DNA sequencing (scDNAseq) technology to detect MRD in patients achieving complete response (CR) and to characterize the genomic landscape of MRD clones and the potential identification of clonal evolution. Methods:We selected 24 cryopreserved bone marrow samples from 15 AML patients who participated in the QUIWI-PETHEMA clinical trial (NCT04107727) and achieved CR after induction and consolidation (n=20). 4 diagnosis samples were analyzed to determine genomic differences between the MRD clone and the initial leukemic population. All patients had bulk targeted NGS data available at diagnosis. Selection of CD34+ and/or CD117+ cells was performed using magnetic beads for enrichment of blasts and Mission Bio multiome single cell DNA+protein was performed using an AML-related 469 amplicon panel and 19 surface antibody mix. Multiplex of 3 independent samples was performed in each library preparation. Analysis was done according to manufacturer's instructions. MRD was also analyzed using MFC (n=14) EuroFlow panel with a limit of detection of 0.1% aberrant cells. RNA qRT-PCR was used in cases with NPM1 mutations (n=6). Quantification of MRD by scDNAseq was calculated according to the enrichment performance obtained from the manufacturer and the percentage of mutant cells. Results: The concordance between gold standard techniques for MRD and scDNAseq was 75% (15/20) (Table 1). Concordance between MFC and scDNAseq was 78% (11/14). The 3 discordant cases, positives by scDNAseq, MRD levels ranged between 0.04-0.09%, below the consensus cutoff of 0.1% to define MRD+. These results suggest that scDNAseq may complement MFC in the detection of very low levels of MRD. Concordance with qRT-PCR was 66% (4/6), but we only detected one patient with a persistent NPM1 clone. Taking advantage of the single cell approach, we were able to assess the genomic landscape of MRD clones and the clonal evolution in sequential samples. The number of clones/subclones and the number of variants per clone varied between patients with positive MRD as shown in Table 1. Interestingly, we analyzed 4 samples at diagnosis by scDNAseq and observed 2 cases in which MRD mutation was already present at diagnosis but was not informed as VAF was < 1%. In 4 patients, we detected small clones (around 1%) that remained unchanged in size despite treatment, suggesting that they likely represent clonal hematopoiesis. In 6 cases, consecutive samples were obtained showing clearance of some clones with other ones remaining stable (UPN2), progressive clearance of clones (UPN3, UPN4), acquisition of new clones (UPN9) and clearance of some clones and acquisition of new ones (UPN15) (Figure 1). Integration of scDNA and cell surface protein expression in the same cell allowed us to perform mutation-clone specific immunophenotypic analysis. Some cases showed a clear pattern in which one of the mutant clones had a significantly higher expression of some markers that was correlated with previous flow cytometry data (e.g. UPN1). Conclusions: Our study suggests that the use of scDNA technology is a feasible approach for detection of MRD in AML patients. Moreover, the use of this approach in sequential samples may allow deciphering clonal evolution, the co-occurrence of different mutations including potential clonal hematopoiesis mutations and identifying winner clones potentially responsible for disease persistence and relapse. Finally, the integration of mutations and surface antibody markers in the same cell provides a means for identifying the presence of mutations in different cell populations. Validation of these results in larger series of patients and correlation with clinical outcome are the next steps for validation of this technology.

Real-World Performance of 2022 Revised World Health Organization (WHO) Criteria for Mixed Phenotype Acute Leukemia (MPAL) B/Myeloid Defined Only By Isolated MPO Expression (isoMPO)

Mixed phenotype acute leukemia (MPAL) is biologically and immunophenotypically heterogeneous, and optimal diagnostic criteria remain unclear. The two current pathologic classifications [2022 World Health Organization (WHO) 5 th Ed. and International Consensus Classification (ICC)] group MPAL by cell lineages and, when present, cytogenetic drivers. In both, myeloperoxidase (MPO) is a key determinant of myeloid lineage. However, the diagnosis of MPAL has been inconsistently applied to cases of acute leukemia expressing MPO alone to define myeloid lineage. To address this, the 2022 WHO revised prior cell lineage definitions to emphasize antigen intensity (≥50%) relative to a positive control population, attempting to standardize lineage assignment across flow cytometer settings and pathologist interpreters. Distinction between pediatric B-lymphoblastic leukemia (B-ALL) and MPAL B/Myeloid with isolated MPO is crucial for therapeutic decision-making. For example, on Children's Oncology Group (COG) regimens, patients with MPAL B/Myeloid receive an anthracycline during induction and intensified chemotherapy post-induction, whereas standard-risk B-ALL patients do not. We hypothesized that the 2022 WHO criteria applied to MPO expression in children with acute leukemia would identify leukemic subtypes (B-ALL vs. MPAL B/Myeloid with isolated MPO) with differences in clinical characteristics and/or outcome. We applied 2022 WHO criteria to flow cytometry data from a multicenter pediatric cohort of leukemias originally classified as MPAL B/Myeloid or B-ALL (Oberley AJCP 2017; Raikar Blood 2018; Oberley Leukemia 2020). Maximal antigen intensity on the leukemic population was measured in 0.5 log increments and normalized to maximal intensity of a control (neutrophil) population. MPO expression was scored as none, low (<50% intensity of mature neutrophils, the typical level in B-ALL with isolated MPO expression) or high (≥50% intensity of neutrophils, meeting 2022 WHO criteria for myeloid lineage assignment). Since the current study focused on MPAL B/Myeloid cases with isolated MPO expression, cases of MPAL T/Myeloid and of MPAL B/Myeloid with myeloid lineage assignment based on monocytic markers were excluded. Clinicopathologic data were compared with MPO expression scores by univariate and multivariate analyses. Primary endpoint was categorical difference in presenting features. Secondary endpoints were end of induction minimal residual disease (EOI MRD) ≥0.01%, event-free survival (EFS) and overall survival (OS). All analyses were two-sided; p<0.05 was the threshold for statistical significance. Among all patients at diagnosis (n=176), median age was 6.14 years (range 0.3-21), 68% were <10 years old, 59% were male, 63% were Hispanic, 76% had initial white blood cell (WBC) count <50K/µL, and 81% were CNS1. MPO was absent, low (<50%) or high (≥50%) in 48%, 29% and 23% of the cohort, respectively. Prevalence of favorable cytogenetics ( ETV6:: RUNX1 or double trisomy) was inversely proportional to MPO intensity [no MPO (50%), low MPO (27%), high MPO (17%); p<0.001). MPO expression (low or high) was more prevalent in patients >10 years of age (p<0.001). There was no difference in MPO by presenting WBC count (p=0.40). Among patients treated with ALL induction with known EOI MRD results (n=166), MPO intensity was not significantly associated with EOI MRD positivity, EFS or OS (multivariate p>0.40 for all three endpoints after accounting for presenting features and initial therapy). Our study demonstrates that 2022 WHO criteria for interpreting MPO intensity can be consistently applied to flow cytometry data from a variety of laboratories. Based on our analysis, only 44% of previous MPAL B/Myeloid cases with isolated MPO were now classified as MPAL. However, the clinical significance of this distinction remains unclear. After accounting for differences in presenting features, MPO expression was not additionally predictive of MRD positivity, EFS or OS. Further investigation is required to support the proposed threshold of 50% MPO intensity of a neutrophil control population as clinically relevant to assign myeloid lineage. Prospective standardized application of 2022 WHO cell lineage assignment criteria across centers will facilitate detailed understanding of the biological underpinnings and optimal therapeutic strategies for this heterogeneous disease.

RUNX1::RUNX1T1 Impairs the Differentiation Potential of Primary AML Cells

Acute myeloid leukemias (AMLs) are often driven by genomic translocations, of which t(8;21) is the most frequent. The resulting gene fusion RUNX1::RUNX1T has been shown in cell line and transgenic expression models to impede cell differentiation and promote leukemic self-renewal. However, it has remained unclear if and how RUNX1::RUNX1T1 influences leukemic propagation in patient-derived AML cells. To address the role of RUNX1::RUNX1T1 in the differentiation status and heterogeneity of patient-derived leukemic blasts, we developed an siRNA lipid nanoparticle (LNP) delivery system for the fusion transcript knockdown (Issa et al, Leukemia 2023). For efficient delivery to primary cells, the LNPs were functionalized with a Leu-Asp-Val (LDV) tripeptide binding the Very Late antigen-4 receptor on leukocytes. Primary AML cells were cultured and expanded for several weeks using a serum-free co-culture system with human bone marrow-derived mesenchymal stromal cells (MSCs) (Swart et al, Pharmaceutics 2023). Treatment of t(8;21) patient-derived xenograft (PDX) and primary bone marrow aspirates depleted RUNX1::RUNX1T1 transcript and protein, as determined by RT-qPCR and western blotting. We unraveled the transcriptional repercussions of RUNX1::RUNX1T1 knockdown using bulk RNAseq and ATACseq in a PDX and identified 1068 up- and 1521 downregulated genes upon RUNX1::RUNX1T1 knockdown, as compared to mismatched siRNA-treated cells (padj < 0.001, log2FC > 2). Gene set enrichment analysis revealed loss of self-renewal potential and enhanced myeloid differentiation. Similarly, ATACseq revealed an increased GMP/monocyte-like chromatin accessibility and a decreased HCS/MPP-like one upon RUNX1::RUNX1T1 knockdown (Figure 1). Surprisingly and in contrast to previous cell line results, RUNX1::RUNX1T1 depletion correlated with increased cell cycle and DNA repair signatures. These combined data demonstrate that RUNX1::RUNX1T1 perturbance promotes differentiation of leukemic stem cells towards granulocytic and monocytic lineages. Furthermore, they suggest that the initial stages of differentiation are linked to increased proliferation in line with the increased proliferation capacity of GMPs compared to more immature progenitor states. To investigate the impact of RUNX1::RUNX1T1 on leukemic blast populations at greater granularity, we knocked RUNX1::RUNX1T1 down in 2 primary bone marrow aspirates and 1 PDX and performed single-cell (sc) RNAseq. Notably, principal component analysis revealed that loss of RUNX1::RUNX1T1 shifted all leukemic populations, while hardly affecting normal hematopoietic cells or MSCs (Figure 2). In all samples, pseudotime analysis showed a more differentiated state of cells upon RUNX1::RUNX1T1 depletion. For cell type annotation, we projected our datasets onto a reference normal bone marrow dataset (van Galen et al, Cell 2019). This projection confirmed differentiation toward granulocyte-monocyte progenitors (GMPs), granulocytes and monocytes. Strikingly, the confidence of cell type prediction according to the reference normal bone marrow was significantly and substantially higher upon RUNX1::RUNX1T1 depletion than in cells with unimpaired fusion transcript level, suggesting restoration of normal myeloid differentiation. In addition to that, primary RUNX1::RUNX1T1 AML samples also contained a new, distinct cell subpopulation that emerged upon RUNX1::RUNX1T1 depletion. That subpopulation could not be annotated as resembling any normal bone marrow cell type. However, it expressed eosinophil marker genes such as RNASE3 and EPX. We, therefore, labeled this cell subpopulation as aberrant eosinophilic. This reveals RUNX1::RUNX1T1's role in disrupting differentiation towards multiple lineages. Our work illustrates a strong dependency of the differentiation potential of t(8;21) AML cells on their driver gene RUNX1::RUNX1T1 in an ex vivo setting with PDX or primary cells. We demonstrate that RUNX1::RUNX1T1 hijacks more than one differentiation trajectory of the AML blasts. Our findings also highlight siRNA-loaded LNPs as an innovative therapeutic approach for fusion gene-driven AML by directly targeting the fusion gene as the leukemic driver.

Multiomic Single-Cell Sequencing May Distinguish BCR-ABL-Mutated Acute Myeloid Leukemia (AML) from Blast Crisis Chronic Myelogenous Leukemia (BC-CML)

Introduction The existence of BCR-ABL+ AML as an entity distinct from BC-CML is controversial. The WHO recently recognized BCR-ABL+ AML, but with the caveat that it is poorly characterized (Khoury, 2022). Clinically, BCR-ABL+ AML is associated with high risk of relapse (Neuendorff, 2018). Further, acquired BCR-ABL mutations have been identified as a cause of relapse on FLT3 inhibitors (McMahon, 2019). We hypothesize that BCR-ABL+ AML can be distinguished as a distinct biologic entity from BC-CML by the observation of BCR-ABL as a sub-clonal rather than a founder mutation in leukemogenesis. Methods To test the utility of multi-omic single cell (SC) sequencing in determining tumor phylogeny and identification of BCR-ABL+ AML vs BC-CML, we analyzed bone marrow samples from 4 patients (pts): 1 relapsed AML with clinical development of a new BCR-ABL fusion at the time of relapse, and 3 newly diagnosed BC-CML (2 myeloid, 1 lymphoid). We performed high-throughput SC DNA sequencing with simultaneous cell-surface immunophenotyping and targeted RNA sequencing for BCR-ABL fusion proteins (Mission Bio Tapestri). All SC DNA sequencing included pt-specific primers for BCR-ABL DNA breakpoints, which were previously identified using nanopore-based technology for sequencing long strands of DNA. Results We analyzed 19,197 single cells (median 1995 cells/pt). Multi-omic SC technology differentiated BCR-ABL+ AML from BC-CML by confirming the fusion arose as a branch mutation from a founding leukemic clone and was not present in all leukemic populations. In Patient 1, who was sampled at time of AML relapse, we identified pathogenic FLT3 ITD and NRAS Q61K mutations. FLT3 co-mutated with either the BCR-ABL DNA breakpoint or NRAS in the same single cell and clone, while the BCR-ABL DNA breakpoint and NRAS were not co-mutated. Phylogenetic reconstruction determined FLT3 to be a founding mutation, which later branched into FLT3/BCR-ABL and FLT3/ NRAS co-mutant subclones. Further, the FLT3-ITD/BCR-ABL and FLT3-ITD/ NRAS subclones had distinct immunophenotypes. Blasts harboring the BCR-ABL DNA breakpoint were predominantly identified in the CD117+/CD33+ population (60.2% of cells) compared to CD34+ (16.4% of cells, p <0.001) and blasts with monocytic differentiation (19.1% of cells, p = 0.02). Notably, BCR-ABL+ cells were not identified in T cells. NRAS mutations were predominantly identified in both CD34+ and monocytic blasts. Together, these data support BCR-ABL arising late within the evolutionary history of this pt's AML. By contrast, in Patients 2 and 3 with myeloid BC-CML, the BCR-ABL DNA breakpoint and corresponding BCR-ABL fusion were detectable in all cell populations, including blasts as well as immunophenotypically normal T cells, B cells, and macrophages. In Patient 2, we identified an IDH2 R132C mutation co-mutated with BCR-ABL. Phylogenetic reconstruction determined the IDH2 mutation arose as a branch from a founding BCR-ABL+clone. Unlike the BCR-ABL breakpoint, which was present in all cell populations, the IDH2 mutation was only identified in immunophenotypically-defined CD33+/CD34+/CD117+ myeloid blasts and was absent from bystander T cells and macrophages. Finally, Patient 4had a clinical diagnosis of lymphoid BC-CML. Like Patients 2 and 3, the BCR-ABL DNA breakpoint and corresponding RNA fusion were present in all immunophenotypically-defined blast populations. Unlike Patients 2 and 3, however, BCR-ABL was absent from immunophenotypically normal CD4+ and CD8+ T cells, macrophages, and monocytes. Additional pts are needed to determine whether this pattern is consistent with all lymphoid BC-CML or whether this pt's biology may have been closer to BCR-ABL+ acute lymphoblastic leukemia (ALL). Conclusions Multi-omic SC technology can distinguish BCR-ABL+ AML from myeloid BC-CML and provide direct confirmation that lineage-restriction of BCR-ABL potentially clarifies the diagnosis of discrete Ph+ leukemias. Future studies are needed to understand the role of RT-PCR based BCR-ABL1 testing as a marker of minimal residual disease in pts with BCR-ABL+ subclones, the impact of BCR-ABL as a potential driver of AML resistance, and the utility of BCR-ABL inhibitors in leukemias with late-arising BCR-ABL+ clones. Support: U10CA180821, U10CA180882, U24CA196171; https://acknowledgments.alliancefound.org. Clinicaltrials.gov Identifiers: NCT00048958, NCT00899223, NCT00900224

Age and ligand specificity influence the outcome of pathogen engagement on preleukemic and leukemic B-cell precursor populations

AbstractCommon infections have long been proposed to play a role in the development of pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, epidemiologic studies report contradictory effects of infection exposure on subsequent B-ALL risk, and no specific pathogen has been definitively linked to the disease. A unifying mechanism to explain the divergent outcomes could inform disease prevention strategies. We previously reported that the pattern recognition receptor (PRR) ligand Poly(I:C) exerted effects on B-ALL cells that were distinct from those observed with other nucleic acid–based PRR ligands. Here, using multiple double-stranded RNA (dsRNA) moieties, we show that the overall outcome of exposure to Poly(I:C) reflects the balance of opposing responses induced by its ligation to endosomal and cytoplasmic receptors. This PRR response biology is shared between mouse and human B-ALL and can increase leukemia-initiating cell burden in vivo during the preleukemia phase of B-ALL, primarily through tumor necrosis factor α signaling. The age of the responding immune system further influences the impact of dsRNA exposure on B-ALL cells in both mouse and human settings. Overall, our study demonstrates that potentially proleukemic and antileukemic effects can each be generated by the stimulation of pathogen recognition pathways and indicates a mechanistic explanation for the contrasting epidemiologic associations reported for infection exposure and B-ALL.

Single-cell transcriptomics reveals multiple chemoresistant properties in leukemic stem and progenitor cells in pediatric AML

BackgroundCancer patients can achieve dramatic responses to chemotherapy yet retain resistant tumor cells, which ultimately results in relapse. Although xenograft model studies have identified several cellular and molecular features that are associated with chemoresistance in acute myeloid leukemia (AML), to what extent AML patients exhibit these properties remains largely unknown.ResultsWe apply single-cell RNA sequencing to paired pre- and post-chemotherapy whole bone marrow samples obtained from 13 pediatric AML patients who had achieved disease remission, and distinguish AML clusters from normal cells based on their unique transcriptomic profiles. Approximately 50% of leukemic stem and progenitor populations actively express leukemia stem cell (LSC) and oxidative phosphorylation (OXPHOS) signatures, respectively. These clusters have a higher chance of tolerating therapy and exhibit an enhanced metabolic program in response to treatment. Interestingly, the transmembrane receptor CD69 is highly expressed in chemoresistant hematopoietic stem cell (HSC)-like populations (named the CD69+ HSC-like subpopulation). Furthermore, overexpression of CD69 results in suppression of the mTOR signaling pathway and promotion of cell quiescence and adhesion in vitro. Finally, the presence of CD69+ HSC-like cells is associated with unfavorable genetic mutations, the persistence of residual tumor cells in chemotherapy, and poor outcomes in independent pediatric and adult public AML cohorts.ConclusionsOur analysis reveals leukemia stem cell and OXPHOS as two major chemoresistant features in human AML patients. CD69 may serve as a potential biomarker in defining a subpopulation of chemoresistant leukemia stem cells. These findings have important implications for targeting residual chemo-surviving AML cells.

Abstract 3334: Epigenetic immune cell markers for CLL

Abstract CLL is a rare hematological cancer and is classified as low-malignancy non-Hodgkin lymphoma. While rare, it is the most frequent form of leukemia in Europe and US accounting for approximately a quarter of all leukemias. As it is a slowly growing tumor, its 5-year survival rate is high compared to faster developing B cell tumors and survival has further improved since introduction of Anti-CD20 Antibody therapies. However, overall the 5-year survival of rare cancers (49%) is significantly lower than for their common counterparts (63%), even though hematological cancers improved outcome over the last years. Due to the currently very late and often incidental diagnosis, some cases turn into aggressively progressing cancers prior to treatment and with concomitant poor outcome. Therefore, diagnosis of CLL and other B cell leukemias remains a challenge. To further improve outcome of CLL and other lymphatic leukemias earlier diagnosis is important. To this end, simple home testing could broaden access to diagnostic tools, but no current method would provide adequate remote initial screening of lymphatic leukemia in populations at risk, such as persons older than 65 years. Epigenetic qPCR has been shown to detect and quantify immune cell populations efficiently from liquid and dried blood samples (DBS). This system is based on specifically unmethylated regulatory elements in gene loci associated with certain cell types. These unique methylation patterns allow specific quantification of cells of interest via qPCR. Different epigenetic qPCR systems were developed for total B cells, naïve and memory B cells as well as IgM producing B cells. While these gene loci are fully unmethylated in the respective target cells, they are fully methylated in all relevant control cell types. The cell type specificity of these markers was shown using bisulfite specific sequencing using purified cells from healthy donors. Here, we investigated blood samples from healthy donors (n = 112) as well as patients at the time of their initial CLL diagnosis. The mean total B cell count of healthy donors at 5.7% was clearly lower than the 60.4% obtained for CLL patients. Similarly drastic differences were observed for naïve B (2.3% normal vs. 20.8% CLL) and IgM (3.3% vs. 49.5%) as well as memory B cells (1.6% vs. 32.6%). All differences were statistically significant. To investigate if these mean differences may also lead to differential (and potentially early) diagnosis, we designed receiver operator characteristics and contingency tables, both indicating near perfect segregation between CLL patients and healthy donors in Ig M, memory and all B cells. Our data indicate applicability of epigenetic immune cell counting as easy, potentially DBS- and home-based approach to test increase of immune cells in people potentially at risk of hematological cancers. Citation Format: Janine Jung, Jeannette Werner, Konstantin Schildknecht, Janika Schulze, Steffi Walter, Christoph Sachsenmaier, Deborah Phippard, Barbara Seliger, Claudia Wickenhauser, Sven Olek. Epigenetic immune cell markers for CLL [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 3334.

Recognizing Minor Leukemic Populations with Monocytic Features in Mixed-Phenotype Acute Leukemia by Flow Cell Sorting Followed by Cytogenetic and Molecular Studies: Report of Five Exemplary Cases

Mixed-phenotype acute leukemia (MPAL), a rare and heterogeneous category of acute leukemia, is characterized by cross-lineage antigen expression. Leukemic blasts in MPAL can be represented either by one population with multiple markers of different lineages or by several single-lineage populations. In some cases, a major blast population may coexist with a smaller population that has minor immunophenotypic abnormalities and may be missed even by an experienced pathologist. To avoid misdiagnosis, we suggest sorting doubtful populations and leukemic blasts and searching for similar genetic aberrations. Using this approach, we examined questionable monocytic populations in five patients with dominant leukemic populations of B-lymphoblastic origin. Cell populations were isolated either for fluorescence in situ hybridization or for clonality assessment by multiplex PCR or next-generation sequencing. In all cases, monocytic cells shared the same gene rearrangements with dominant leukemic populations, unequivocally confirming the same leukemic origin. This approach is able to identify implicit cases of MPAL and therefore leads to the necessary clinical management for patients.

Evaluation of Analysis Strategy for Cytoplasmic Lineage-Associated Markers in Mixed-Phenotype Acute Leukemia (MPAL) As Devised By Who Classification 2022

Background: Conventional rating of flow cytometrically determined expression of cytoplasmic lineage-associated markers MPO, CD3 and CD79a has defined positivity by ≥10% leukemic cells being positive compared to internal negative controls. WHO classification 2022 has devised a new approach defining positivity by part of leukemic population exceeding 50% of expression level of normal counterpart populations used as positive controls. Aim: Evaluate interpretation of cytoplasmic expression of MPO, CD3 and CD79a in MPAL and other acute leukemias. Methods: 277 acute leukemia cases were analyzed (MPAL-BM, n=18; MPAL-TM, 20; AML M0, 42; AML M1, 40; AML M2, 39; APL, 39; BCP-ALL, 40; T-ALL, 39). Following WHO 2022 positivity for cytoplasmic MPO, CD3 and CD79a was determined setting a cutoff at 50% of mean fluorescence intensity (MFI) of granulocytes, mature T-cells and mature B-cells, respectively. An alternative refined approach was evaluated calculating the MFI ratio between leukemic populations and positive controls. Results of MFI ratios are given as log differences, i.e. 0 representing identical MFIs, -1 meaning 1 log lower expression in leukemic population compared to controls. Log difference of -1.5 was set as cutoff. Results: Applying WHO 2022 majority of cases were rated positive for expected markers, i.e. AML, MPAL-BM and MPAL-TM for MPO, T-ALL and MPAL-TM for CD3 and BCP-ALL and MPAL-BM for CD79a (figure, A). However, there were cases rated positive in other groups although in general only few leukemic cells were causing positive rating, indicating limited specificity of this approach. In detail, MPO was positive in 69% of M0, 95% of M1, 92% of M2, 92% of APL, 61% of MPAL-BM and 70% of MPAL-TM, but also in other groups (BCP-ALL, 35%; T-ALL, 51%).Applying WHO 2022, diagnosis would have changed to BCP-ALL in 39% of MPAL-BM and to T-ALL in 30% of MPAL-TM cases. CD3 was positive in 92% T-ALL and 95% MPAL-TM cases, but also in other groups (M0, 29%; M1, 25%; M2, 15%, APL, 49%; BCP-ALL, 13%; MPAL-BM, 56%). Applying WHO 2022, diagnosis would have changed to AML in 5% of MPAL-TM cases. CD79a was positive in 100% of BCP-ALL and 94% of MPAL-BM, but also in other groups (M0, 55%; M1, 43%; M2, 51%, APL, 49%; T-ALL, 33%; MPAL-TM, 75%). Applying WHO 2022, diagnosis would have changed to AML in 6% of MPAL-BM cases. In order to refine the WHO 2022 approach and to improve specificity MFI ratio between leukemic cells and positive controls was applied. Similarly to the WHO 2022 approach the MFI ratio approach resulted in expected differences but also significant overlaps (figure, B), although to a lower degree for MPO. In detail, MPO was positive in 31% of M0, 75% of M1, 77% of M2, 85% of APL, 72% of MPAL-BM and 50% of MPAL-TM, but also in other groups (BCP-ALL, 13%; T-ALL, 23%). Applying the MFI ratio approach, diagnosis would have changed to BCP-ALL in 28% of MPAL-BM and to T-ALL in 50% of MPAL-TM cases. CD3 was positive in 92% of T-ALL and 100% of MPAL-TM cases, but also in other groups (M0, 14%; M1, 23%; M2, 18%, APL, 85%; BCP-ALL, 13%; MPAL-BM, 72%). Applying the MFI ratio approach there would have been no changes in MPAL-TM diagnosis. CD79a was positive in 98% of BCP-ALL and 94% of MPAL-BM, but also in other groups (M0, 50%; M1, 65%; M2, 46%, APL, 87%; T-ALL, 56%; MPAL-TM, 85%). Applying the MFI ratio approach, diagnosis would have changed to AML in 6% of MPAL-BM. In 6 and 7 cases analyzed by WHO 2022 and MFI ratio, respectively, changes in diagnosis from MPAL to ALL were not supported by NGS yielding mutations known to be associated with myeloid neoplasms while changes were supported by NGS in a minority of cases. Discussion: The WHO 2022 definition, as compared to conventional analysis, changed diagnosis in a significant number of MPAL cases, in particular due to rating MPO negative. At the same time, application of WHO 2022 approach to cases with AML, BCP-ALL and T-ALL suggests limited specificity with significant rates of positivity outside the lineage determined by conventional approach. The MFI ratio approach also changed diagnosis in a significant number of MPAL cases in comparison to the conventional approach, however, data suggests a better specificity for MPO. Further, MFI ratio considers the whole leukemic population and thus may result in higher reproducibility. Overall this data argues in favor of considering multiple lineage-associated markers instead of single markers. In addition, classification based on genetic markers should be maximized also in MPAL.

DR-01, a Non-Fucosylated Anti-CD94 Antibody, Depletes Leukemic Cells in Ex Vivo and In Vivo Models of Large Granular Lymphocyte Leukemia

Large granular lymphocyte leukemia (LGLL) is a rare leukemia more frequently occurring in the elderly population with three major subtypes: T-LGLL, chronic lymphoproliferative disorder of NK cells (CLPD-NK) and aggressive NK cell leukemia (ANKL). Over 50% of patients develop complications including severe neutropenia, thrombocytopenia and anemia, thus predisposing patients to opportunistic infections. In addition, treatments are limited to standard immunosuppressive therapy such as methotrexate, cyclosporine or cyclophosphamide, with complete response rates of only 50% or less for each agent. Thus, novel and effective therapies for LGLL patients are critically needed. Dren Bio is developing DR-01, a non-ligand blocking, non-fucosylated monoclonal antibody targeting CD94. CD94 is a type II transmembrane receptor identified on mature, cytotoxic lymphocytes such as healthy NK and terminally differentiated CD8 T cells. In the context of disease, CD94 is known to be upregulated on LGLL cells. DR-01 functions through depletion of target cells via antibody-mediated cellular cytotoxicity (ADCC) by means of fratricide, a method in which the same cell type induces ADCC on each other. Through characterization of DR-01 using various ELISA and cell-based assays, DR-01 was determined to bind with nanomolar affinity to CD16a (EC50: 0.04µg/mL) and to CD94 on healthy human donor NK cells, cynomolgus monkey NK cells and LGLL cells (EC50: 0.5-1nM). DR-01 induces potent, dose-dependent depletion of CD94+ cells in ex vivo ADCC assays (IC50: 1ng/mL) of peripheral blood mononuclear cells (PBMCs) from healthy and diseased individuals. Furthermore, DR-01 has been shown to be well tolerated in a 4-week dose-range finding and Good Laboratory Practice (GLP) study in cynomolgus monkeys from 5-100mg/kg on a weekly dosing regimen. A 12-color flow cytometry panel was applied to a cohort of patient LGLL PBMCs to profile CD94 expression and other cytotoxic markers associated with the disease, including CD16, CD57 and functional binding partners of CD94 such as NKG2A and NKG2C. CD94 was identified on 97% (37/38) of patient samples screened, ranging from 400 to 725,000 receptors per cell. All patient leukemic cells expressed variable levels of CD16 and CD57. NKG2A and NKG2C were present in over 5% of total leukemic cells in 37% (14/38) and 21% (8/38) of patient samples, respectively. In addition, DR-01 was able to deplete LGL leukemic cells with high potency and in a dose-dependent manner in a functional ADCC assay (IC50:1-10ng/mL). The mechanism of fratricide was demonstrated through degranulation of the leukemic population via expression of CD107a. Cytokine profiling results are pending. To demonstrate in vivo efficacy of DR-01 in a disease model of LGLL, humanized NSG-Tg(hIL-15) mice engrafted with patient LGLL splenocytes were utilized. Optimal splenocyte engraftment (5-20% human CD45+ cells in blood) was achieved within 3 weeks post-inoculation with 10 million PBMCs per pre-sublethally irradiated mouse. The engrafted leukemic population partially preserved the CD94 expression profile from the patient leukemic cells and was successfully depleted (>90%) in blood, bone marrow, spleen and liver 1-week post-treatment with a single dose of DR-01 at 5mg/kg. Overall, DR-01 is a non-fucosylated anti-CD94 antibody with high affinity and potent leukemic cell depleting ability ex vivo and in vivo. The favorable toxicity profile of DR-01 in cynomolgus monkeys precede clinical studies in LGLL patients. Additional profiling and evaluation of DR-01 in LGLL patient samples ex vivo as part of a pre-phase I study is ongoing. A phase I/II trial has been initiated to assess the safety and efficacy of DR-01 in previously treated LGLL patients.

Dormancy Is a Feature of Therapy Resistant Mouse and Human Leukemia-Initiating Cells in T-ALL

Addressing therapy failure and relapse is critical in improving outcomes for pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients. Relapse is thought to represent a failure to effectively eliminate leukemia-initiating cells (L-ICs), which survive chemotherapy and self-renew, regenerating the full complement of leukemic cell populations. Results from mouse T-ALL models show that the double negative-3 (DN3) thymic progenitors are enriched for L-IC activity; however, the L-IC remains an undefined subset of this population. We used single cell RNA-sequencing (scRNA-seq) to profile leukemic development in vivo, with a focus on uncovering DN3 heterogeneity. Integrated UMAP clustering of scRNA-seq libraries from wild type (WT), Tal1/Lmo2 preleukemic (PL), and Tal1/Lmo2 T-ALL (Leuk) thymi revealed an expansion of DN3 progenitors in PL and Leuk samples with decreases in mature (single positive) thymocytes. Unlike WT thymocytes, where Notch1 target gene expression was restricted to DN3 cells, Notch1-Myc pathway activation was maintained throughout thymocyte development in Tal1/Lmo2 transgenic mice. Re-clustering of DN3 cells revealed the presence of dormant and proliferative DN3 cells in PL and Leuk thymus. Dormant DN3 signatures resembled quiescent, Notch1-active DN3a (pre-β-selection) thymocytes and proliferative DN3 cells were transcriptionally similar to DN3b thymocytes. Dormant mouse DN3 cells were enriched for signatures of quiescent, therapy resistant ALL patient cells and cells obtained from ALL patients at minimal residual disease (Ebinger et al., Cancer Cell 2016), suggesting that dormant leukemic DN3 cells resemble human L-ICs that mediate drug resistance and relapse. To test this, we employed in vivo nucleosome labeling to profile dormant DN3 cells throughout leukemia development and functionally evaluate their response to chemotherapy. To assess cell divisional history, WT, Tal1, Lmo2, or Tal1/Lmo2 mice harboring the tet-inducible H2B-GFP reporter allele were subjected to in vivo doxycycline pulse (labeling), followed by a two-week chase. Analysis of pulse-chased thymocytes revealed low frequencies of label retaining (GFPHI) DN3 cells in WT mice. However, label retention was significantly increased in Lmo2 DN3 thymocytes (2.9-fold), consistent with other Lmo2 models (Tremblay et al., Nat. Commun. 2018). Tal1 expression also promoted DN3 label retention (6.0-fold) and Tal1/Lmo2 co-expression resulted in further increases in DN3 GFPHI cells (25-fold), indicating that Tal1 and Lmo2 cooperate to promote DN3 dormancy. DN3 GFPHI cells were present at similar frequency in mice with overt T-ALL and in line with scRNA-seq data, GFPHI cells were observed nearly exclusively as a subset of the leukemic DN3a population. Treatment with Vincristine, DeXamethasone, and L-asparaginase (VXL) led to a further increase in GFPHI DN3 frequency in Lmo2 and Tal1 preleukemic mice (3.1- and 9.7-fold, respectively), suggesting that cell cycle restricted L-ICs may evade conventional chemotherapy and mediate relapse. We then tested whether dormancy is a feature of the L-IC in T-ALL patients. Analysis of relapsed pediatric T-ALL cells revealed that the CD7+CD1a- L-IC population (Chiu et al., Blood 2010) is cell cycle restricted compared to CD7+CD1a+ leukemic cells and that VXL treatment enriched for CD7+CD1a- cells in xenografted mice, suggesting that cell cycle restriction may be an important feature of both mouse and human T-ALL L-ICs. Analysis of transcriptomes to reveal mediators of dormancy that may be exploited to chemosensitize L-ICs will be discussed.

Post-Remission Outcomes in Patients with Acute Myeloid Leukemia (AML) Are Similar between Hypomethylating Agent/Venetoclax Therapy and Intensive Chemotherapy after Accounting for Measurable Residual Disease (MRD) Status

Background: Intensive chemotherapy regimens (IC; e.g., 7+3 or CPX-351) broadly target leukemic blast populations in acute myeloid leukemia (AML) by interfering with DNA replication, whereas hypomethylating agent/venetoclax therapy (HMA/ven) takes advantage of metabolic vulnerabilities in leukemic stem cells via inhibition of anti-apoptotic pathways. IC regimens are often targeted to 'fit', younger patients, while HMA/ven is typically used in patients who are older or who have significant comorbidities. Both therapeutic paradigms can induce morphologic remission, with resultant measurable residual disease (MRD) status remaining a prognostically significant factor. Here, we aim to determine whether post-remission outcomes in AML patients depend mainly on MRD status or are further modified by chosen induction (IC vs HMA/ven). Methods: Retrospective chart review was performed on adult (age 18 years and older) patients newly diagnosed with AML and treated at Johns Hopkins Hospital, Baltimore, MD between September 2014 and June 2021. Patients were excluded if they had promyelocytic or mixed phenotype leukemia. Complete remission (CR) was defined by <5% medullary blasts with no extramedullary disease and count recovery. Primary covariates of interest included age in years, performance status (PS; ECOG 0-1 for good vs 2-4 for poor), European LeukemiaNet (ELN) 2022 risk group, number of HMA/ven cycles to best response, hematopoietic stem cell transplantation (HCT) status, chemotherapy type (IC vs HMA/ven), and MRD status by multiparametric flow cytometry (MFC). Groups were compared using Wilcoxon rank sum, Kruskal-Wallis, and Fisher's exact tests. Laplace regression models were used to estimate subgroup median overall survivals (OS) in order to compare them using the Wald test. Associations between OS and the above covariates were examined in Cox models; one multivariable model included an interaction term for therapy type and MRD status. Analyses were performed using Stata version 17. Results: Of the 388 patients initially reviewed, 214 (55%) achieved a CR or CR with incomplete count recovery and were included in further analysis. Most (103 of 159) IC patients received 7+3; 20 received CPX-351 and 36 received a high-dose cytarabine-based regimen. Fifty-five patients received HMA/ven. The HMA/ven patients were significantly older than those receiving IC regardless of MRD status (median age 72 vs 58 years, respectively, P<0.001). MRD negative (MRD-) remission was achieved in 134 (84.3%) patients after IC and 44 (80.0%) patients after HMA/ven. Patients who achieved MRD- CRs after IC were more likely to undergo HCT than those with identical MRD status after HMA/ven (77.6% vs 40.9%, P<0.001) and were younger (median age 60 vs 73 years, P<0.001). Univariate analysis showed significantly decreased hazard of death in patients with favorable ELN 2022 risk score, MRD- status in first CR, and HCT (Table 1). Kaplan-Meier curves showed patients with MRD- remissions had superior survival to those with MRD positive (MRD+) remissions (median OS not reached vs. 21.9 months, P<0.05), whereas there was no statistically significant difference in median OS between therapies (36.1 months HMA/ven vs. 61.2 months IC). Patients in the IC, MRD- group and HMA/ven, MRD- group had similar outcomes (median OS not reached for both); both groups had superior survival to MRD+ groups (P<0.05; Figure 1). Multivariable analysis showed decreased hazards of death for ELN favorable risk patients (hazard ratio [HR] 0.49, 95% confidence interval [CI] 0.28-0.85) and those MRD- in first CR (HR 0.47, 95% CI 0.28-0.77) after adjusting for age, PS, therapy type, and cycles. In a subsequent model, an interaction term between therapy type and MRD status was not significant (HR=0.96, suggesting no difference in impact of MRD between therapy types). Conclusions: In this retrospective study evaluating outcomes in patients with AML, chemotherapy type does not appear to differentially impact survival by MRD status in those achieving remission. MRD status as measured by MFC significantly impacts survival when adjusting for relevant baseline covariates. These results suggest that obtaining deep, MRD- remissions matters regardless of whether these are achieved with HMA/venetoclax or intensive therapy. Ultimately, randomized controlled trials are needed to better determine the value of MRD in AML after different induction strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Erythroid and Megakaryocytic Differentiation Program in JAK2-Mutated Acute Myeloid Leukemia with or without Antecedent Myeloproliferative Neoplasm

JAK2 V617F is the most common driver mutation for Ph-negative MPN and related blast transformation (MPN-BP). In contrast, JAK2 V617F is rare (<5%) in acute myeloid leukemia (AML) lacking an antecedent MPN (nonMPN AML). JAK2 V617F skews the differentiation of hematopoietic stem/progenitor cells (HSPC) towards erythroid and megakaryocytic lineages and in conjunction with TP53 deficiency/mutations results in acute erythroid leukemia in mice (Rampal et al., PNAS 2014). These phenotypes, although also reported in patients with MPN-BP (Mesa et al., Blood 2005), remain incompletely understood. In this study, we set out to study these phenotypes at the protein, epigenomic and transcriptomic levels in JAK2-mutated AML comparing MPN-BP to nonMPN AML. The study cohort included 58 MPN-BP (16 PV, 16 ET, 14 PMF and 12 MPN-U) and 27 nonMPN AML patients (11 de novo AML, 12 AML-MRC, 4 t-AML) with JAK2 V617F mutation (Table 1). In addition, 100 patients with JAK2 WT AML were also included as control group. The median age at diagnosis in MPN-BP and nonMPN AML subgroups as well as in JAK2 WT AML was similar (70 years vs 71 years vs 70 years, respectively) and showed a male preponderance. The median interval between MPN diagnosis and blast transformation was 4.3 years (range, 0-20.5 years). Overall survival was inferior in MPN-BP compared to nonMPN AML (median survival: 7.4 vs 19.3 months). MPN-BP showed a higher rate of MF-2 or 3 myelofibrosis (78% vs 33%, p<0.05), osteosclerosis (50% vs 15%, p<0.05) and marrow necrosis (7% vs 0%) on bone marrow biopsies than nonMPN AML. On immunohistochemistry, 28% (10/32) MPN-BP cases (vs 0% nonMPN AML, p<0.05) were classified as acute leukemia with erythroid/megakaryocytic differentiation (M6b and/or M7) based on expression of E-cadherin, CD71, Glycophorin A, and CD61 on leukemic blasts. ERG, FLI1, and LMO2, transcription factors that are often expressed in HSPCs, were also expressed in leukemic blasts of MPN-BP and nonMPN AML. Aberrant TP53 expression by immunohistochemistry was seen in 31% of MPN-BP (5/9 and 5/21 cases with and without erythroid/megakaryocytic differentiation, respectively) and 17% (3/18) of nonMPN AML. The most common mutations on next-generation sequencing included TP53, RUNX1, ASXL1, TET2, DNMT3A, SRSF2, IDH1, IDH2 and EZH2 in each group. However, of these, TP53 mutations were more frequent in MPN-BP (34% vs 11% in nonMPN AML, p<0.05) and TET2 mutations more frequent in nonMPN AML (48% vs 24% in MPN-BP, p<0.05). Interestingly, NPM1 and FLT3, which are among the most mutated genes in AML were not detected in either MPN-BP or nonMPN AML. Based on the over-representation of erythroid/megakaryocytic phenotype in MPN-BP seen on immunohistochemistry, we hypothesized that MPN-BP blasts might exhibit an upregulated epigenetic and transcriptional program involved in erythroid/megakaryocytic differentiation. RNA-seq and ATAC-seq analysis were performed on flow cytometry sorted CD34+ leukemic blast populations from 11 MPN-BP and 6 nonMPN AML, as well as similar sorted populations from normal controls and JAK2 WT AML. Principal component analysis showed overlapping MPN-BP and nonMPN AML samples, both of which were separated from JAK2 WT AML. Differential expression analysis confirmed nearly identical gene expression between blasts from MPN-BP and nonMPN AML. In addition, consistent with findings by immunohistochemistry, the expression of genes specifically involved in erythroid/megakaryocytic differentiation (TFRC, GYPA, HBB, SPTA1, EPOR, ITGA2B, ITGB3, PF4, ALAS2, GP1BA, ENG and MPL) were upregulated in MPN-BP. Hierarchical clustering using Ward's method on both RNA-seq and ATAC-seq normalized counts revealed that the JAK2 WT AML cluster was distinct from the MPN-BP and nonMPN AML cluster (Figure 1). Lineage deconvolution of ATAC-seq using hematopoietic differentiation stages (Glass et al., Blood 2017, Dilip et al., Blood 2021) to evaluate lineage characteristics of the blasts showed hematopoietic stem cell (HSC) predominance in JAK2 WT AML while MPN-BP and nonMPN AML displayed enrichment of multipotent progenitor (MPP), megakaryocyte-erythrocyte progenitor (MEP) and erythroid signatures. Collectively, our data demonstrates enrichment of erythroid and megakaryocytic programs in both JAK2-mutated MPN-BP and JAK2-mutated nonMPN AML, suggesting that chromatin accessibility and transcription may be driven by mutated JAK2 regardless of antecedent MPN. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single and Combinational Multiplex Base-Edited 'Universal' CAR T Cells in a Humanised Model of Primary CD7+CD33+ AML

Introduction Universal allogeneic donor CAR T cells are being developed as part of a 'deep conditioning' strategy against AML to secure disease clearance ahead of allogeneic stem cell transplantation (Allo-SCT). CD33 is widely expressed on AML, sometimes on the entire leukemia-associated populations, but is also present on healthy precursors; CD7 may be expressed on AML subpopulations and is present on normal T and NK cells, resulting in problematic fratricide effects during CAR T manufacture. Healthy donor derived CAR T cell banks generated using cytidine deamination base-editing and lentiviral transduction with CARs against both CD33 and CD7 circumvent such issues and could offer cell based anti-leukemic therapy alongside preparative effects ahead of allo-SCT. Here we investigated single and combinational BE-CAR T cells against primary human AML in vivo. Methods: Healthy donor lymphocytes were activated by anti-CD3/28 stimulation ahead of electroporation to deliver codon optimised base editor mRNA and guide RNAs against CD7, CD52 and TRBC, and lentiviral transduction to express either an anti-CD7 or anti-CD33 CAR. Magnetic bead depletion removed residual TCRab T cells, and disruption of CD7 ensured cells avoided fratricide. In vitro, antigen specific function was verified in cytotoxicity and cytokine release assays, and in vivo potency was confirmed against luciferase modified Molm14 and Kasumi lines in NSG mice. Responses against primary AML were investigated in a patient derived xenograft model (PDX) from a KIT mutated AML patient, established by serial transplantation, with full expression of CD33 (100% of blasts) and partial expression of CD7 (30% of blasts). Cells were injected into irradiated NSG mice (n=10 per group). On day 24 at a mean leukemic engraftment of 15% human CD45 positive cells in peripheral blood, mice were treated with either 10x106 BE-CAR33, BE-CAR7 or a combination of both or with anti-CD19-CAR-T cells (CAR19) as a control group (in total 20x106 T cells per mouse). AML engraftment, T cell persistence and expression of checkpoint markers were monitored by multicolour flow cytometric analysis. Results: On days 14 and 28 after CAR-T cell infusion, AML cells were readily detected in peripheral blood of control CAR19 animals (42.8% and 90% respectively) and the BE-CAR7 group (27.8% and 67.6%), but were notably reduced in the BE-CAR33 alone group (2.3% and 8.3%) and the BE-CAR33 and BE-CAR7 combination group (2.5% and 3.4%) (Figure 1A). Survival was significantly prolonged for BE-CAR33 and BE-CAR33/BE-CAR7 treated mice compared to the other groups (P<0.001) (Figure 1B). CD7+ AML cells were depleted to a mean of 1% and 4.5% in the BE-CAR33 group, to 0.6% and 0.7% in the BE-CAR33/BE-CAR7 and to 2.6% and 2.9% in the BE-CAR7 group, while CD7+ AML cells increased to 12% and 30% in the CAR19 group at day 14 and 28 after CAR-T cell infusion. Total CD2+CAR-T cell detection in peripheral blood ranged from 1.6 to 9.8% at day 14, 1 to 3.7% at day 28, and 0.8 to 2.7% at day 42, indicating durable persistence over the investigation period. Interestingly, PD-L1 on AML cells was present in all groups while checkpoint markers PD1, GAL9, TIM3, and LAG3 were significantly upregulated in BE-CAR33 cell treated mice, but not BE-CAR7 or CAR19 cell treated mice (P<0.001 for all comparisons). Conclusions: Lentiviral transduction and multiplexed base editing to remove TCR, CD52 and the shared AML/T lineage antigen CD7, enabled the generation of universal donor CAR T cells for combinational use. This data demonstrates robust activity of BE-CAR33 alone and in combination with BE-CAR7 against human CD7+CD33+AML in mice and highlights the importance of strategies that ensure complete targeting of leukemic populations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal