Leukemic Stem Cell Population Research Articles

Abstract 5184: Biomarker results from PEVENAZA, a randomized phase 2 study of venetoclax and azacitidine +/- pevonedistat in newly diagnosed AML patients unfit for intensive chemotherapy: Increased efficacy in a subset of patients with IDH1/2 mutations and other observations

Abstract Background: Many newly diagnosed patients with AML are ineligible for intensive chemotherapy due to pre-existing comorbidities and older age. VIALE-A, a phase-3 trial with venetoclax (ven) and azacitidine (aza) established this combination as a new standard of care (SOC) for this patient population. The addition of pevonedistat (pevo) to this combination was evaluated in the same patient population in the current trial (NCT04266795). Here we present results of biomarker analyses from baseline and on-treatment bone marrow samples. Methods: Molecular mutational analysis was performed using a next generation sequencing (NGS) panel on baseline bone marrow samples. Combining this data with cytogenetics allowed centralized assignment of risk category based on ELN2017 guidelines. Longitudinal bone marrow aspirate samples were also examined by multi-color flow cytometry for expression of select surface antigen expression and to identify AML leukemic stem cells (LSCs) from the blast population. Results: Eleven genes were selected for analyses based on prevalence and scientific interest, including DNMT3A, ASXL1, TET2, FLT3, and IDH1/2. In contrast to other genes and ELN risk categories, we observed a statistically significant increase (p-value 0.013) in CR+CRi rates for IDH1/2 mutant patients when treated with pevo + ven + aza (95%, n=21) compared to ven+aza control arm (62%, n=21). In addition, we evaluated the prevalence of CD33, CD123 and CLEC12a, targets of current AML developmental therapeutics, in both the blast and LSC populations at baseline. The median (n=120 pts) % blast cells expressing CD33, CD123, or CLEC12a were 89.8%, 63.2%, and 66.0%, respectively. The median (n=78 pts) % LSCs expressing CD33, CD123, or CLEC12a were 48.2%, 90.9%, and 32.0%, respectively. Importantly, we did not see any significant difference in median fluorescence intensity (MFI) or % expression in any of the three proteins after treatment with ven and aza (+/- pevo). Finally, no strong associations were observed between expression levels and either mutations or ELN risk categories. Conclusions: Analyses of biomarker samples as part of the PEVENAZA trial resulted in several insights for not only the pevonedistat program but also for other AML therapeutics in development. The observation that newly diagnosed patients with IDH1/2 mutations may benefit from the addition of pevo to ven+aza is certainly hypothesis-generating but would require further follow-up with higher patient numbers. In addition, baseline CD33, CD123, and CLEC12a expression levels were in line with published studies. Here we have also shown they remain similar after SOC treatment (ven+aza) supporting the development of agents that target these proteins in both first and second line settings for AML. Citation Format: Radha Ramesh, Xiang Fang, Shuli Li, Sharon Friedlander, Farhad Sedarati, Lionel Adès, Nicholas Short, Cristina Papayannidis, Tammie Yeh. Biomarker results from PEVENAZA, a randomized phase 2 study of venetoclax and azacitidine +/- pevonedistat in newly diagnosed AML patients unfit for intensive chemotherapy: Increased efficacy in a subset of patients with IDH1/2 mutations and other observations [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 5184.

An immunophenotype-coupled transcriptomic atlas of human hematopoietic progenitors.

Analysis of the human hematopoietic progenitor compartment is being transformed by single-cell multimodal approaches. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) enables coupled surface protein and transcriptome profiling, thereby revealing genomic programs underlying progenitor states. To perform CITE-seq systematically on primary human bone marrow cells, we used titrations with 266 CITE-seq antibodies (antibody-derived tags) and machine learning to optimize a panel of 132 antibodies. Multimodal analysis resolved >80 stem, progenitor, immune, stromal and transitional cells defined by distinctive surface markers and transcriptomes. This dataset enables flow cytometry solutions for in silico-predicted cell states and identifies dozens of cell surface markers consistently detected across donors spanning race and sex. Finally, aligning annotations from this atlas, we nominate normal marrow equivalents for acute myeloid leukemia stem cell populations that differ in clinical response. This atlas serves as an advanced digital resource for hematopoietic progenitor analyses in human health and disease.

Mathematical modelling of clonal reduction therapeutic strategies in acute myeloid leukemia

Over the years, the overall survival of older patients diagnosed with acute myeloid leukemia (AML) has not significantly increased. Although standard cytotoxic therapies that rapidly eliminate dividing myeloblasts are used to induce remission, relapse can occur due to surviving therapy-resistant leukemic stem cells (LSCs). Hence, anti-LSC strategies have become a key target to cure AML. We have recently shown that previously approved cardiac glycosides and glucocorticoids target LSC-enriched CD34+ cells in the primary human AML 8227 model with more efficacy than normal hematopoietic stem cells (HSCs). To translate these in vitro findings into humans, we developed a mathematical model of stem cell dynamics that describes the stochastic evolution of LSCs in AML post-standard-of-care. To this, we integrated population pharmacokinetic-pharmacodynamic (PKPD) models to investigate the clonal reduction potential of several promising candidate drugs in comparison to cytarabine, which is commonly used in high doses for consolidation therapy in AML patients. Our results suggest that cardiac glycosides (proscillaridin A, digoxin and ouabain) and glucocorticoids (budesonide and mometasone) reduce the expansion of LSCs through a decrease in their viability. While our model predicts that effective doses of cardiac glycosides are potentially too toxic to use in patients, simulations show the possibility of mometasone to prevent relapse through the glucocorticoid’s ability to drastically reduce LSC population size. This work therefore highlights the prospect of these treatments for anti-LSC strategies and underlines the use of quantitative approaches to preclinical drug translation in AML.

Leukemic stem cells activate lineage inappropriate signalling pathways to promote their growth

Acute Myeloid Leukemia (AML) is caused by multiple mutations which dysregulate growth and differentiation of myeloid cells. Cells adopt different gene regulatory networks specific to individual mutations, maintaining a rapidly proliferating blast cell population with fatal consequences for the patient if not treated. The most common treatment option is still chemotherapy which targets such cells. However, patients harbour a population of quiescent leukemic stem cells (LSCs) which can emerge from quiescence to trigger relapse after therapy. The processes that allow such cells to re-grow remain unknown. Here, we examine the well characterised t(8;21) AML sub-type as a model to address this question. Using four primary AML samples and a novel t(8;21) patient-derived xenograft model, we show that t(8;21) LSCs aberrantly activate the VEGF and IL-5 signalling pathways. Both pathways operate within a regulatory circuit consisting of the driver oncoprotein RUNX1::ETO and an AP-1/GATA2 axis allowing LSCs to re-enter the cell cycle while preserving self-renewal capacity.

NKX101, an Allogeneic Off-the-Shelf CAR NK Therapy Targeting NKG2D-Ls, Has Potent In Vitro Cytotoxicity Against Patient-Derived AML Leukemic Stem Cell (LSC) and Non-Leukemic Stem Cell (Non-LSC) Blasts

Introduction Acute myeloid leukemia (AML) is a malignancy of immature myeloid cells characterized by rapid proliferation of abnormal myeloblasts. Treatment options for patients with AML are limited, especially for relapsed/refractory (r/r) disease. Leukemic cells with stem cell features, known as leukemic stem cells (LSCs), have been implicated as the origin of relapse in minimal residual disease positive (MRD +) AML (Khaldoyanidi et al. 2022, Kamel et al. 2022). Additionally, absence of NKG2D-ligand (NKG2D-L) expression has been reported to be associated with stemness and the LSC population in AML (Paczulla et. al, 2019). NKX101 is an allogeneic, off-the-shelf, healthy donor-derived chimeric antigen receptor (CAR) NK cell therapy candidate engineered to express an NKG2D-L targeting CAR as well membrane bound IL-15. In a clinical trial, NKX101 has shown promise for the treatment of r/r AML, including generating complete responses (CR) with MRD negativity (MRD -). In this study, we utilize a flow cytometry-based approach to (i) evaluate NKG2D-L cell surface expression on normal hematopoietic cells vs. primary AML blasts, (ii) assess the expression pattern of NKG2D-Ls on AML blast subsets, and (iii) determine the potency of NKX101 cytotoxic killing of both LSC and non-LSC AML blasts. Methods NKX101 cells were generated from peripheral blood leukopaks from healthy donors. Cryopreserved bone marrow mononuclear cells (BMMCs) were obtained from AML patients in accordance with approved IRB protocols (n=20). BMMCs from healthy donors were used as controls (n=10). AML somatic mutation status was determined using an Illumina TruSight® Myeloid targeted sequencing panel. NKX101 cytotoxicity against patient derived AML blasts was assessed using a flow cytometry-based assay that simultaneously measured specific cytotoxicity AML blast/LSC markers. NKG2D-L protein expression on LSC and non-LSC subsets was determined using flow cytometry with fluorescently labeled monoclonal antibodies specific to MICA, MICB, and ULBP1-6. Results Utilizing patient-derived primary samples, we demonstrate that NKG2D-Ls are expressed on AML blast cells and expressed at higher levels on AML blast cells compared to healthy normal hematopoietic cells (p = 0.002). Furthermore, we show that NKG2D-Ls are expressed at similar levels on both LSC and non-LSC AML blast populations. Target cell specific flow cytometry-based cytotoxicity assays revealed that NKX101 cells potently kill patient-derived AML blasts in a dose-dependent manner. Moreover, NKX101 cells had similar cytotoxic potency on both LSC and non-LSC AML blasts subsets. Conclusions In summary, we show that NKG2D-Ls are expressed on the surface of AML blasts at levels above those observed in normal hematopoietic cells. NKG2D-Ls are expressed on both non-LSC AML blasts and on the LSC population implicated as the origin of relapse in AML. Additionally, the ability of NKX101 to potently kill AML LSC blasts suggests that NKG2D-L targeting may be a viable mechanism for eliminating LSC in the blood and bone marrow of patients with r/r AML. Taken together, these data support NKG2D-Ls as promising therapeutic targets for AML and support further investigation of NKX101 CAR NK cell therapy for the treatment of r/r AML.

Ferritinophagy Is a Druggable Vulnerability of Quiescent Leukemic Stem Cells

Acute myeloid leukemia (AML) poses significant challenges in terms of prognosis and treatment options. Leukemic stem cells (LSCs) contribute to therapy resistance, relapse and adverse outcomes. This study aimed to explore one of the major features of LSCs (e.g. quiescence) and its underlying molecular mechanisms, as well as identify potential targets for therapeutic intervention. Using in vivo cell labeling in patient-derived xenograft (PDX) assays, we identified a quiescent cell subpopulation with high LSC potential. Transcriptomics experiments conducted separately on quiescent and cycling cell subsets revealed gene signatures characteristic of quiescent (Q_AML_UP) and cycling cells (C_AML_UP). Using single-cell RNA sequencing, we identified clusters of quiescent leukemic cell that were highly enriched in gene signatures related to LSCs (Figure 1A). Through a machine learning approach, we identified a gene set highly specific of quiescent cells (named the QS35 gene signature) that emerged as a significant prognostic factor for poor outcomes in independent cohorts of AML patients (Figure 1B). Next, we aimed to identify new vulnerabilities of LSCs by investigating the quiescent subset of leukemic cells. Transcriptomics and proteomics approaches on quiescent vs cycling cells revealed enhanced autophagy within the quiescent subset. Additionally, the transferrin receptor TFRC and ferritin, two key regulators of iron metabolism, exhibited lower expression in the quiescent subpopulations. As intracellular iron availability is tightly regulated by ferritinophagy, a specific form of autophagy leading to the degradation of ferritin, we investigated the coordinated regulation of autophagy and iron metabolism in the quiescent LSC subset. Intriguingly, the intracellular iron pool was lower in quiescent LSCs, particularly in the CD34+CD38- compartment compared to more mature leukemic cells. Moreover, autophagy inhibition selectively induced cytotoxicity in the CD34+CD38- compartment and reduced the frequency of long-term culture-initiating cells (LTC-IC), and these effects were rescued by exogenous iron supply, underscoring the critical role of autophagy in iron bioavailability within the quiescent LSC subpopulation. To target ferritinophagy in this compartment, we depleted NCOA4, the main carrier in ferritinophagy responsible for delivering ferritin-bound iron to the autophagosome, using shRNAs in primary AML samples. Interestingly, NCOA4 was found more expressed in quiescent compared to cycling AML cells. In ex vivo assays, NCOA4 downregulation significantly reduced the frequency of LTC-IC and inhibited the clonogenic capacities of leukemic cells. Furthermore, NCOA4 invalidation abrogated leukemia initiation and self-renewal in serial transplantation experiments in vivo, particularly within the quiescent LSC population (Figure 2A). Additionally, treatment with a novel small molecule inhibitor named compound 9a that disrupts the NCOA4-ferritin interaction, depleted intracellular iron in primary AML cells, reduced their clonogenic potential and LTC-IC frequency ex vivo, and induced cell death specifically within the quiescent CD34+CD38- subset. Importantly, the viability of LSCs treated with compound 9a was rescued by the addition of iron, demonstrating that the cytotoxic effects of this compound were dependent on iron depletion. Notably, compound 9a exhibited minimal toxicity to normal hematopoietic cells ex vivo. In PDX models, in vivo short-term treatment with compound 9a decreased tumor burden and induced cell death within the CD34+CD38- LSC population (Figure 2B). Mechanistically, genetic of chemical inhibition of NCOA4 led to the accumulation of dysfunctional mitochondria, and to the generation of reactive oxygen species especially within the LSCs population. Furthermore, inhibition of NCOA4 by compound 9a decreased mitophagy, strongly suggesting that NCOA4 coordinates ferritinophagy and mitophagy pathways to increase intracellular iron, thereby specifically promoting the survival of LSCs. These findings highlight the critical vulnerability of quiescent LSCs to ferritinophagy. Thus, NCOA4 inhibition represents an innovative therapeutic approach for AML patients.

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.

NUP98-Rearranged Acute Myeloid Leukemia Stem Cells Superimpose Type I Interferon Signaling with Canonical Self-Renewal Programs

The clinical outcomes of pediatric acute myeloid leukemia (AML) have shown improvement with combination therapies; however, relapse remains a significant concern due to the resistance of leukemia cells to conventional treatments. In some cases, epigenetic heterogeneity within leukemias can contribute to therapy resistance. For example, leukemia stem cells (LSCs) have been shown maintain AML, both before and after therapy. This study aims to identify LSCs within a mouse model of high-risk, NUP98-rearranged pediatric AML, and to identify mechanisms that sustain this population. To identify and isolate a LSC subpopulation, we characterized murine NUP98-HOXD13/Flt3-ITD-driven AML in mice by performing cellular indexing of transcriptomes and epitopes (CITE-seq) analysis at two stages: pre-AML and fully transformed AML. Uniform Manifold Approximation and Projection (UMAP) revealed a highly reproducible pattern of heterogeneity within each AML specimen. Each AML had a small percentage of cells that resembled normal and pre-leukemic multipotent progenitors (MPPs), as well as additional populations with B-cell or myeloid gene expression. Pseudotime analysis indicated a trajectory from MPPs to mature cell-like lineages, implicating the MPP-like population as a potential LSC. This population expressed type I interferon (IFN-1) target genes more highly than non-LSC AML cells and pre-AML cells. This finding is significant because we previously identified IFN-1 as an effector of Flt3-ITD/NUP98-HOXD13 synergy during AML initiation, and in human NUP98-rearranged AML (PMID: 36395068). Differential gene expression analysis also identified several established self-renewal genes, including Hlf, Myct1, Mpl, Slc7a8, and Gata2, as highly expressed in putative LSCs relative to non-LSCs (figure B). This suggests that AML LSCs superimpose inflammatory signaling programs, particularly IFN-1, and core stem cell self-renewal programs to maintain a malignant state. The data raise the question of how IFN-1 shapes LSC identity. We next sought to isolate and validate the putative LSC population. CITE-seq feature barcode data indicated high expression of CD117 and CD48, medium expression of B220, and low expression of Gr1 and CD11B in putative LSCs. We isolated LSCs based on these markers (Gr1-CD11b-B220 MidCD117 highCD48+). Several non-LSC populations were isolated for comparison (e.g., Gr1+CD11B+ and Gr1-CD11B-B220 highCD117 Mid populations. Cytospin observation revealed no morphological differences among the three populations, but cell culture assays showed that only the Gr1-CD11b-B220 MidCD117 highCD48+ cells exhibited proliferative capacity after multiple passages. Limit dilution transplant assays showed that the Gr1-CD11b-B220 MidCD117 highCD48+ population was greatly enriched for LSC function relative to whole bone marrow (figure A). Altogether, we have shown that we can prospectively isolate a LSC population from NUP98-HOXD13/Flt3-ITD AML. The LSC population is highly reproducible across specimens and it demonstrates high degrees of IFN-1 activity. Further work will establish the role of IFN-1/self-renewal gene interactions in LSC maintenance.

Results from a Phase (Ph) 1 Clinical Study of the All-Oral Regimen of CC-486 and Venetoclax for Relapsed and Refractory Acute Myeloid Leukemia

Background: Venetoclax (Ven) with azacitidine (Aza) is the standard of care for newly diagnosed AML patients who are unfit for intensive induction chemotherapy. It is well known that this treatment also has activity in the relapsed and refractory (R/R) setting. However, this regimen requires 7 consecutive daily subcutaneous or intravenous doses of Aza each month, which continues indefinitely. The relative inconvenience of this, and its negative impact on quality of life, may lead to its early discontinuation, which may decrease the efficacy or durability of this regimen. A more convenient all-oral regimen may decrease visits to an infusion center, improve quality of life, and potentially increase the efficacy of the regimen due to improved compliance. CC-486 is the oral formulation of Aza that is currently approved for post-induction chemotherapy maintenance in AML. Methods: This is a single center open label, Ph I study investigating CC-486 and Ven in R/R AML patients. In the dose escalation phase, subjects received CC-486 at one of two cohorts (200 mg PO days 1-14 and 300 mg PO days 1-14). Ven was given at the 400 mg/day PO regimen, for 28 days, after an initial intra-patient dose escalation per the standard of care. Using a 3+3 study design for these two cohorts, we aimed to determine the maximum tolerated dose (MTD) of CC-486 in combination with Ven. An expansion cohort is planned for 10 additional patients treated at the MTD. To assess whether CC-486 exerts similar effects in the leukemia stem cell population (LSCs) compared to conventional IV Aza, we retrospectively analyzed patient samples from cohort 1 with a patient sample treated with Ven/IV Aza outside of the study. We performed mass spectrometry-based metabolomics analyses in LSCs isolated from patient samples as well as single-cell RNA sequencing. Results: A total of 9 patients were accrued for this Ph I study (Figure 1A). The average age at the time of enrolment was 66, and median prior treatment regimens was 1 (range 1-3). Five patients had received Ven/Aza in prior treatments. There were no dose-limiting toxicity (DLT) events and the MTD was determined to be 300mg. The most common hematologic toxicities were Gr 3-4 neutropenia (n = 8, 89%), Gr 3-4 anemia (n = 7, 78%), and Gr 3-4 thrombocytopenia (n = 5, 56%). The most common non-hematologic toxicities were Gr 1-2 nausea (n = 5, 56%), fatigue (n = 2, 22%), and diarrhea (n = 2, 22%). Febrile neutropenia was also seen in two subjects (Gr 1-2 n = 1, 11%, Gr 3-4 n= 1, 11%). Best responses were complete remission (CR) (n = 2, 22%) partial remission (PR) (n = 1, 11%), and stable disease (SD) (n= 3, 33%). Three patients proceeded to an allogeneic stem cell transplant. Overall survival was 288 days, 95% CI: (39 days, 424 days). To determine whether CC-486/Ven exerts similar effects on the LSC population compared to conventional IV Aza/Ven, LSCs were isolated from the bone marrow of a responder and non-responder, and these were compared to LSCs from a patient who underwent treatment and responded to conventional IV Aza/Ven. Given subjects who achieved a CR in the study cleared their blasts by day 4 rendering no leukemia cells for analyses, samples used were from Subject 1 (stable disease with unchanged blasts), and Subject 2 (stable disease with significant blast reduction). Metabolomics assays performed in LSCs at day 4 and day 28 suggested mitochondrial dysfunction, decreased electron transport chain complex II activity and decreased glutathione levels in Subject 2 and conventional IV Aza/Ven but not on Subject 1. These findings are consistent with Pollyea et al. 2018 suggesting CC-486/Ven targets LSCs by perturbing complex II activity and OXPHOS in LSCs. Further, scRNA seq performed in Subject 1 and 2 showed gene expression patterns similar to those seen in patients who respond to Aza/Ven vs not, including a significant shift of BCL-2 to MCL-1 expression in the cell population harboring LSCs (Figure 1B). Conclusion: CC-486 and Ven is an all-oral regimen currently being investigated for the treatment of R/R AML. Our Ph I dose escalation demonstrated a favorable safety profile for this regimen with a MTD of 300mg CC-486. Responses were seen in both dose cohorts including 2 CRs and 1 PR, and patients have been successfully bridged to hematopoietic stem cell transplant. Analyses performed in patient samples from this study suggested a similar effect in LSCs in CC-486/Ven compared to conventional IV Aza/Ven.

Mitochondrial STAT3 Plays a Critical Role in Survival of AML Cells

Introduction: Signal transducer and activator of transcription 3 (STAT3) has been shown to be critical to the survival of acute myeloid leukemia (AML) cells. It has been previously shown to mediate oxidative phosphorylation (OXPHOS) in acute myeloid leukemia bulk and stem cells via its transcriptional regulation of the amino acid transporter SLC1A5 and thereby glutamine uptake to AML cells (Amaya et al. Blood 2022). Further, STAT3 has been shown to regulate the transcription of important mitochondrial genes such as MCL-1 (Shastri et al. JCI 2018). Although STAT3 has been previously shown to also translocate to the mitochondria of myeloid malignant cells, its role in the mitochondria is less well defined in this cell population. In this study, we sought to understand the role of mitochondrial STAT3 in AML bulk leukemia and leukemia stem cell population. Methods: Human primary AML samples and MOLM-13 cells were utilized for experiments. To assess STAT3 localization to the mitochondria, mitochondrial fractions were isolated followed by Western Blot analysis. Flow cytometry-based imaging (ImageStream) was also utilized to quantify localization of STAT3 to the mitochondria. Mitochondrial mass was assessed using flow cytometry based MitoTracker assays. Metabolomic assays were performed by mass spectrometry at the University of Colorado metabolomics core. Viability assays were performed with flow cytometry using Annexin V and Dapi stains. Patient derived xenograft models were performed as previously described (Amaya et al. Blood 2022) in NSG-S mouse with Busulfan conditioning. Results: In this study, we show that primary human AML cells have constitutive STAT3 activation via phosphorylation of S727, which has been shown to signal mitochondrial localization. Further, we show that STAT3 is localized in the mitochondria of leukemia cells in primary patient samples and the cell line MOLM-13. Using genetic (siRNA) and pharmacologic (Stattic) inhibition of STAT3, we show a decrease in pSTAT3 at S727, with subsequent decreased translocation of STAT3 to the mitochondria. This leads to mitochondrial dysfunction including decreased TCA cycle metabolites, as well as decreased intracellular ATP upon STAT3 inhibition. STAT3 inhibition also leads to decreased mitochondrial mass suggesting autophagy upon mitochondrial STAT3 (mitoSTAT3) inhibition. To assess whether mitoSTAT3 is functionally important, we performed viability assays in MOLM-13, as well as leukemia stem cells isolated from primary AML cells. Inhibition of mitochondrial STAT3 leads to decreased viability in MOLM-13 and primary AML cells, and reduced engraftment in PDX mouse models using primary AML samples (Figure 1A), suggesting a decrease in leukemia stem cell potential. Interestingly, STAT3 inhibition with Stattic is able to overcome venetoclax resistance for primary AML patient samples in vitro (Figure 1B). Conclusions: In this study, we show mitoSTAT3 plays a key role in mitochondrial function of AML cells. mitoSTAT3 inhibition leads to mitochondrial dysfunction, a decrease in mitochondrial mass and eventually cell death. We show that Stattic is an effective inhibitor of mitoSTAT3 in AML, leading to cell death and decreased leukemia stem cell function. Further studies are needed to assess the mechanistic function of STAT3 in the mitochondria.

Tetraspanin CD81 Supports Cancer Stem Cell Function and Represents a Therapeutic Vulnerability in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease associated with a poor outcome, necessitating aggressive therapies to achieve remission. A major contributing factor to treatment resistance and disease relapse is the presence of leukemic stem cells (LSC). Although challenging, targeting LSC constitutes an imperative approach to improve patient outcomes and limit side effects. In a previous study, we have uncovered a prognostic association between CD81 cell surface overexpression and worse outcome for AML patients. In the present work, we validated that CD81 overexpression of either protein or mRNA in AML blasts represents a specific prognostic marker for non-CBF AML with an extended cohort (n=150). We then confirmed our data using a publicly available dataset (BeatAML cohort). This association was also verified within NPM1 mutated patients, which was not associated to co- occurring FLT3-ITD mutations. When further investigating the underlying mechanisms related to CD81 overexpression and its impact on AML progression, we found out that CD81 expression was increased at relapse using paired diagnostic-relapsed samples (19% vs. 67%, P = 0.002). We demonstrated presence of CD81 within the LSC population, defined as CD34+/CD38-/CD90-/CD123+ blasts. Moreover, we showed that the CD81+ LSC fraction was higher at relapse compared to diagnosis (63% vs. 33%, P = 0.0001). In our cohort, non-CBF AML patients with higher level of CD81+LSC had worse outcome (RFS: P = 0.001, HR [95%CI] = 2.01 [1.16-3.48]; OS: P = 0.003; HR [95% CI] = 2.38 [1.32-4.29]. Next, we aimed to explore the potential impact of CD81 expression on LSC function, particularly focusing on its ability to serially engraft in immunocompromised mice (NSG). We observed a positive association between CD81 expression and an increased probability of positive engraftment, and a correlation with time-to-engraftment i.e., faster engraftment of xenografts from AML with high CD81 expression. Ex-vivo drug testing revealed primary AML cells as being more resistant to chemotherapy when engraftment was achieved. To investigate the mechanisms implied, we engineered either overexpression or knocked-down expression of CD81 in three different AML cell lines using cDNA transfection or shRNA, respectively. By means of these controlled CD81 expression models, we were able to demonstrate that CD81 overexpression increased AML cell adhesion, migration and invasion. Imaging by confocal microscopy exposed the development of membrane protrusions for CD81+ models. In vivo, higher CD81 expression increased blast homing, tumor burden and xenoengraftment efficiency. Moreover, we demonstrated that higher CD81 expression was linked to chemoresistance to anthracyclines and cytarabine, the two cornerstone drugs commonly used in AML. Finally, preclinical use of CD81 blocking antibody combined with chemotherapy in xenografts (AML cell line and patient-derived xenografts) resulted in longer relapse-free survival and reduced tumor aggressiveness. These results highlight the functional significance of CD81 overexpression in AML and its potential role as a biomarker for assessing LSC activity. Further investigations may improve this new avenue for targeted therapeutic intervention aimed at disrupting LSC function and improving treatment outcomes in AML patients.

Multiomic Single-Cell Analysis Identifies Von Willebrand Factor and TIM3-Expressing BCR-ABL1+ Chronic Myeloid Leukemia Stem Cells

Characterization of the leukemic stem cell (LSC) population in chronic myeloid leukemia (CML) may identify therapeutic targets for sustained elimination of leukemic cells. High-throughput single-cell RNAseq (scRNAseq) has paved the way for detailed transcriptional assessment of CML LSC. Building on this technique, multiomic CITE-seq approaches carry additional benefit in that the paired protein expression information may enable identification of associated cell surface marker profiles, subsequent FACS-based isolation and functional characterization. However, detection of BCR-ABL1 transcripts, which is arguably the only unequivocal marker to distinguish CML LSC from healthy hematopoietic stem cells (HSC), remains challenging using current high-throughput 3' end capture-based scRNAseq methods. For the present study, we performed single-cell CITE-seq analysis of the expression of 597 genes and 51 proteins in >70,000 stem and progenitor cells from 16 chronic phase CML patients and five healthy donors. Furthermore, we developed and employed a strategy allowing parallel detection of BCR-ABL1 transcripts at the single-cell level. In bone marrow samples from CML patients, we observed pronounced expansion of erythroid and myeloid progenitors within the CD14 -CD34 + compartment, and substantial heterogeneity within the traditionally defined CD34 +CD38 -/low LSC compartment. In-depth analysis of the latter identified a group of immature BCR-ABL1 +cells displaying a CD45RA -cKIT -CD26 + TKI resistance phenotype sitting atop a hierarchy of myeloid progenitors. Within this group of potential LSC, expression of both previously reported ( e.g. CD25 and CD26) and unreported gene and protein markers was found to distinguish these cells from their healthy counterparts. Unlike HSC, the immature LSC showed high surface expression of TIM3 and high transcription of the von Willebrand factor gene ( VWF). While overexpression of VWF within the stem cell population may be linked to the aberrant myeloid-biased hematopoiesis characterizing the disease, the cell surface upregulation of TIM3 suggests that its targeting may have merit in CML. In conclusion, we here performed detailed multiomic characterization of CML stem and progenitor cells, paired with BCR-ABL1 detection at the single-cell level. Our findings revealed LSC-specific expression patterns that may have implications for the phenotypic definition of CML LSC. Additionally, the results identify TIM3 as a conceivable target for sustained elimination of immature LSC in CML.

The Role of over-Expressed β Globin in Driving Relapsed B - Cell Acute Lymphoblastic Leukemia (B-ALL)

Background: The prognosis for patients with relapsed acute lymphoblastic leukemia (ALL) remains suboptimal despite intensification of chemotherapy, immunotherapy and stem cell transplant. We and others have identified genetic and epigenetic alterations enriched at relapse that drive drug resistance and relapse. In our recent work mapping the epigenetic landscape of B-ALL, we discovered novel super-enhancers (SEs), or regions with increased H3K27 acetylation, enriched at relapse in a majority of patients, implicating a role in clonal evolution. One of these SEs was upstream of the β globin locus and lead to aberrant expression of hemoglobin beta subunit ( HBB) mRNA. HBB upregulation at relapse was also found in a large meta-analysis of gene expression data from pediatric diagnosis/relapse pairs. Of note, b globin is normally found in erythrocytes that interacts with other subunits to form functional hemoglobin. We hypothesized that HBB upregulation in blasts plays a role in relapse and aimed to determine whether it leads to drug tolerance, an early recovery phenotype and/or increased clonal potential. Methods: B-lineage REH, RS4;11 and 697 ALL cell lines were transduced with lentiviral vectors overexpressing either wild-type (WT) HBB or an empty vector (EV) control. Real time PCR was performed to confirm RNA expression and Western Blot analysis was performed to detect protein expression. We generated additional cell lines with a mutation in the translation start codon and another leading to a premature stop codon to unequivocally interrupt protein expression. The generated cell lines were then plated and evaluated for differences in apoptosis via Annexin V assay and proliferation, in response to chemotherapeutic agents commonly used in the treatment of ALL. Clonogenic growth in vitro was also assessed using MethoCult TM media. To test the possibility that HBB acted in generating a persistor cell phenotype, cell lines were placed in mercaptopurine and prednisone for 10-14 days at two different concentrations and counted throughout drug exposure and for an additional 10-14 days following removal of drug. Results: We did not observe significant differences in chemosensitivity to various chemotherapies used in the treatment of B-ALL. However, we did observe less cells undergoing apoptosis in HBB expressing cells compared to control following prolonged treatment with 6-mercaptopurine, suggesting an increase in a small population of drug tolerant cells. However, this did not result in a difference in recovery time following removal of the drug. We also demonstrated that β globin overexpression led to increased clonal growth in methylcellulose compared to control (Fig. 1A, REH) and this observation was replicated in a second cell line (Fig. 1B, RS4;11). While we confirmed mRNA expression, we were not able to confirm protein expression by Western blot or by immunoprecipitation. We therefore hypothesized that b globin mRNA, rather than protein, mediates the observed increase in clonal growth. Interestingly, we observed the same phenotype of increased clonal growth (Fig. 1) in mutated cell lines, suggesting a catalytic role for β globin RNA in expanding a leukemia stem cell population. Conclusions: The increased clonal potential upon overexpression of HBB could be indicative of the ability to reconstitute a tumor from a small subpopulation following treatment. Additionally, our data suggests that protein is not required for increased clonal growth leading us to hypothesize that catalytic RNA is responsible. We are also currently analyzing RNAseq data from the cell lines and will compare differential gene expression between the empty vector and HBB expressing cells to discover downstream pathways impacted by HBB. We will be testing this hypothesis further by performing limiting dilution analysis in an immunocompromised mouse model (with David Teachey, Children's Hospital of Philadelphia).

A Rapid Test to Evaluate the Tumor Differentiation State of Acute Myeloid Leukemias

Introduction: The combination of the hypomethylating agent Azacitidine with Venetoclax, a BCL2 inhibitor (Aza+Ven), is the frontline therapy for patients with acute myeloid leukemia (AML) who are elderly or unfit for intensive chemotherapy. However, identifying responsiveness to Aza+Ven a priori remains a clinical challenge.Aza+Ven effectively targets the primitive leukemia stem cell population (Pollyea, 2018), whereas resistance is observed in differentiated tumors (Pei, 2020). Further insights into the hierarchical organization of hematopoiesis have led to a definition of malignant AML cell types that follow the progression from hematopoietic stem cell to myeloid differentiated cells based on gene expression signatures, and these have been used to classify AML tumors (van Galen, 2019). Classification schemes using gene expression to capture variations in leukemic composition represent key biomarkers of response to targeted agents (Zeng, 2022; Bottomly, 2022). However, the prompt availability of nuanced cell state signatures to guide clinical decision making is limited by the laborious nature of performing and analyzing RNA-sequencing studies. Rapid AML stratification based on these classifications permits the possibility of identifying patients most likely to benefit from a drug or combination. Methods: To classify cell states based on transcriptomic signatures, we developed two assay formats, involving custom Nanostring and TaqMan Array card panels, for evaluation of tumor cell states for AML patients. Both formats contained gene sets representative of the six hematopoietic lineage states (van Galen, 2019) and both provide outputs that are compatible with the timeline for clinical decision-making. Assays were run according to the manufacturers' specifications, and enrichment of each of the six gene signatures was assessed using the Singscore stable method (Foroutan, 2018). Banked AML patient samples with matched RNA-seq and ex vivo Aza+Ven drug sensitivity data were used to validate the assay formats. Data obtained from the rapid assays were validated against cell states determined by deconvolution of bulk RNA sequence and compared with respect to clinical immunophenotype data where available. Results : Both the Nanostring and quantitative PCR-based assay formats detected gene expression representative of the appropriate cell state as previously determined by bulk RNA sequencing (Fig. 1A). Correlation of Aza+Ven ex vivo sensitivity with tumor cell state signatures revealed patterns where primitive but not differentiated cell states associated with sensitivity. In contrast, RNA isolated from healthy donor samples showed expression of all six cell state signatures. Across a cohort of 50 primary AML patient specimens, enrichment scores for each gene set correlated well with those derived for the same patients using deconvolution of bulk RNA-seq (Pearson r values: 0.785 to 0.944). Cell state characterization by rapid gene expression assays also aligned well with clinical immunophenotype, a routinely available diagnostic characterization of tumor blasts. Surface expression of primitive (CD117) and mature (CD11b) markers associated with Aza+Ven ex vivo sensitivity and resistance overall, respectively (p=0.03 & 0.04); however, ex vivo sensitivity could be significantly further stratified among samples expressing these markers when considering the gene expression-based enrichment scores for progenitor- and monocyte-like gene signatures (low vs high p-values: 0.02-0.05) (Fig. 1B). Analysis of matched samples from individual patients before and after treatment also showed cell state signature changes that reflected their temporal disease progression and correlated with differences in ex vivo drug sensitivity and clinical immunophenotype. Conclusion:Incorporation of cell state into prospective evaluations of AML patients provides an early indication of responsiveness to Aza+Ven. With rapid turnaround, assays such as these may augment treatment stratification and improve patient outcomes. These assays offer additional versatility to detect changes in AML cells after forced differentiation or acquired drug resistance.

Stem-Cell Enriched Cellular Hierarchy of TP53 Mutant Acute Myeloid Leukemia Is Vulnerable to Targeted Protein Degradation of c-MYC

Deregulation of MYC genes occurs in up to 70% of all human cancers and is associated with hallmarks of cancer including mitochondrial and ribosomal biogenesis, cell cycle progression, and metabolic abnormalities. TP53 regulates MYC while MYC suppresses TP53, suggesting counteracting negative feedback loops. Therefore, MYC or its function can be activated when TP53 is not functional. TP53 mutations occur in 30% of relapsed/refractory acute myeloid leukemias (AMLs) patients' survival is dismal, and there are no effective therapies for these patients. Compared to TP53 wild-type (TP53wt), TP53 mutant (TP53mut) AMLs have lower percentages and numbers of leukemia blasts with increased immature CD34+ cells and resistance to chemo- or molecularly targeted therapies. However, the exact cellular hierarchy of TP53mut AML has not been elucidated. We observed significantly increased MYC mRNA levels in (TP53mut), as compared to TP53wt AML, and also increased levels in TP53mut versus TP53wt AML leukemia stem cell (LSC) fractions. This finding was confirmed in a dataset from the Munich Leukemia Laboratory (N = 732). We found significantly upregulated MYC pathways in TP53mut compared to TP53wt AML LSC. We confirmed the increased MYC mRNA levels at the protein level in TP53mut AML by single-cell mass cytometry (CyTOF). To dissect the cellular hierarchy in TP53mut AML, we performed single-cell RNA sequencing of 32 BM samples from healthy donors (N = 3), newly diagnosed, high-risk TP53wt (N = 7) and TP53mut (N = 22) AML patients, with 6,685, 10,687 and 10,687 cells from normal, TP53wt and TP53mut AML bone marrow (BM) cells, respectively. We found highly enriched HSC-like cells and reduced progenitor- and GMP-like cells in TP53mut AML compared to normal BM (NBM) and TP53wt AML samples. We overlayed MYC expression levels on the mapping of cellular components and found higher MYC levels in HSC-like cells in TP53mut AML compared to HSC and HSC-like cells in NBM and TP53wt AML samples, suggesting enrichment of immature HSC-like cells and increased activity of MYC in TP53mut AML LSCs ( Fig. A). To target c-MYC and MYC signaling, we utilized GT19715, the first-in-class cereblon modulator (CELMoD) for c-MYC protein (Nishida, ASH 2022). CyTOF confirmed the presence of much increased c-MYC protein levels in primary CD34+ AML than in CD34+ NBM hematopoietic stem cells (HSCs). Notably, CD34+ AML cells showed much greater sensitivity to GT19715 compared to CD34+ NBM cells. Data suggest on-target activity of GT19715 against c-MYC in LSCs with a therapeutic window between LSCs vs. NBM HSCs. Intriguingly, c-MYC protein levels are higher in CD34+CD38+ than in CD34+CD38- AML cells, suggesting that c-MYC drives the proliferation of AML progenitor cells differentiating from quiescent LSCs. Consequently, CD34+CD38+ AML progenitor cells exhibited greater sensitivity to GT19715 compared to CD34+CD38- LSCs. Using paired, TP53 null HL-60 GT19715-sensitive and -resistant cells generated through chronic exposure to GT19715, we interrogated the impact of GT19715 on metabolic changes. GT19715 induced pronounced reductions in basal and maximal oxygen consumption rates (OCRs) in GT19715-sensitive HL-60 cells. GT19715 reduced both basal, glutamine- and glucose-dependent extracellular acidification rates (ECARs) in GT19715-sensitive cells while no inhibition in OCRs or ECARs was observed in GT19715-resistant cells. GT19715 severely inhibited ECARs even after adding glucose to GT19715-sensitive cells, suggesting irreversible inhibition of glycolysis as one of the mechanisms of action of GT19715. GT19715 profoundly reduced AML blasts in TP53mut AML samples (N = 3) ( Fig. B), and in a very aggressive patient-derived xenograft (PDX) TP53mut AML model established from a patient with TP53 p.Y220C and p.P151A mutations along with MECOM rearrangement and K/NRAS mutations. In humanized Crbn I391V mice, where the Crbn-mediated protein degradation is operational, GT19715 only reduced WBC counts along with minimal body weight loss. GT19715 but did not reduce total mouse BM CD45+ cells, suggesting favorable toxicity profiles of GT19715. In conclusion, TP53mut AML comprised highly enriched LSC populations compared to TP53wt AML and targeting of c-MYC protein is highly effective in TP53mut AML in vitro and in vivo with a therapeutic window between AML LSC and normal hematopoietic cells.

Low doses of IFN-γ maintain self-renewal of leukemia stem cells in acute myeloid leukemia.

Conventional therapies for acute myeloid leukemia (AML) often fail to eliminate the disease-initiating leukemia stem cell (LSC) population, leading to disease relapse. Interferon-γ (IFN-γ) is a known inflammatory cytokine that promotes antitumor responses. Here, we found that low serum IFN-γ levels correlated with a higher percentage of LSCs and greater relapse incidence in AML patients. Furthermore, IFNGR1 was overexpressed in relapsed patients with AML and associated with a poor prognosis. We showed that high doses (5-10 μg/day) of IFN-γ exerted an anti-AML effect, while low doses (0.01-0.05 μg/day) of IFN-γ accelerated AML development and supported LSC self-renewal in patient-derived AML-LSCs and in an LSC-enriched MLL-AF9-driven mouse model. Importantly, targeting the IFN-γ receptor IFNGR1 by using lentiviral shRNAs or neutralizing antibodies induced AML differentiation and delayed leukemogenesis in vitro and in mice. Overall, we uncovered essential roles for IFN-γ and IFNGR1 in AML stemness and showed that targeting IFNGR1 is a strategy to decrease stemness and increase differentiation in relapsed AML patients.

A Deeply Quiescent Subset of CML LSC depend on FAO yet Avoid Deleterious ROS by Suppressing Mitochondrial Complex I.

Disease relapse and therapy resistance remain serious impediments to treating cancer. Leukemia stem cells (LSC) are therapy resistant and the cause of relapse. A state of deep quiescence appears to enable cancer stem cells (CSC) to acquire new somatic mutations essential for disease progression and therapy resistance. Both normal hematopoietic stem cells (HSC) and LSC share many common features, thereby complicating the safe elimination of LSC. A recent study demonstrated that long lived normal oocytes exist without mitochondrial complex I (MC-1), expressing it in a developmentally regulated fashion, thereby mitigating their vulnerability to ROS. Quiescent CSC rely on mitochondrial FAO, without complex I expression, thereby avoiding the generation of damaging ROS, similar to long lived normal human stem cells. A deeper understanding of the biology of therapy resistance is important for the development of optimal strategies to attain complete leukemia cures. Here, using scRNA-sequencing and ATAC-seq on primary chronic myelogenous leukemia (CML) patient samples, combined with bioinformatics analyses, we further examine the heterogeneity of a previously characterized in vitro imatinib-selected CD34-CD38- CML LSC population. We utilized a series of functional analyses, including single-cell metabolomic and Seahorse analyses, to validate the existence of the deepest quiescent leukemia initiators (LI) subset. Current study revealed heterogeneity of therapy resistant LSC in CML patients and their existence of two functionally distinct states. The most deeply quiescent LI suppress the expression of MC-1, yet are highly dependent on fatty acid oxidation (FAO) for their metabolic requirements and ATAC-seq demonstrated increased chromatin accessibility in this population, all consistent with an extremely primitive, quiescent stemness transcriptional signature. Importantly, the specific CREB binding protein (CBP)/β-catenin antagonist ICG-001 initiates the differentiation of LSC, including LI, decreases chromatin accessibility with differentiation and increasing expression of MC-1, CD34, CD38 and BCR-ABL1, thereby resensitizing them to imatinib. We investigated the biological aspects related to LSC heterogeneity in CML patients and demonstrated the ability of specific small molecule CBP/β-catenin antagonists to safely eliminate deeply quiescent therapy resistant CSC. These observations may represent an attractive generalizable therapeutic strategy that could help develop better protocols to eradicate the quiescent LSC population.

Beyond αβ T cells: NK, iNKT, and γδT cell biology in leukemic patients and potential for off-the-shelf adoptive cell therapies for AML.

Acute myeloid leukemia (AML) remains an elusive disease to treat, let alone cure, even after highly intensive therapies such as stem cell transplants. Adoptive cell therapeutic strategies based on conventional alpha beta (αβ)T cells are an active area of research in myeloid neoplasms given their remarkable success in other hematologic malignancies, particularly B-cell-derived acute lymphoid leukemia, myeloma, and lymphomas. Several limitations have hindered clinical application of adoptive cell therapies in AML including lack of leukemia-specific antigens, on-target-off-leukemic toxicity, immunosuppressive microenvironments, and leukemic stem cell populations elusive to immune recognition and destruction. While there are promising T cell-based therapies including chimeric antigen receptor (CAR)-T designs under development, other cytotoxic lymphocyte cell subsets have unique phenotypes and capabilities that might be of additional benefit in AML treatment. Of particular interest are the natural killer (NK) and unconventional T cells known as invariant natural killer T (iNKT) and gamma delta (γδ) T cells. NK, iNKT, and γδT cells exhibit intrinsic anti-malignant properties, potential for alloreactivity, and human leukocyte-antigen (HLA)-independent function. Here we review the biology of each of these unconventional cytotoxic lymphocyte cell types and compare and contrast their strengths and limitations as the basis for adoptive cell therapies for AML.

Understanding the Wnt Signaling Pathway in Acute Myeloid Leukemia Stem Cells: A Feasible Key against Relapses.

Wnt signaling is a highly conserved pathway in evolution which controls important processes such as cell proliferation, differentiation and migration, both in the embryo and in the adult. Dysregulation of this pathway can favor the development of different types of cancer, such as acute myeloid leukemia and other hematological malignancies. Overactivation of this pathway may promote the transformation of pre-leukemic stem cells into acute myeloid leukemia stem cells, as well as the maintenance of their quiescent state, which confers them with self-renewal and chemoresistance capacity, favoring relapse of the disease. Although this pathway participates in the regulation of normal hematopoiesis, its requirements seem to be greater in the leukemic stem cell population. In this review, we explore the possible therapeutic targeting of Wnt to eradicate the LSCs of AML.

Abstract 525: Targeting interleukin-1 receptor-associated kinase 1/4 in acute myeloid leukemia stem cells

Abstract Therapies for acute myeloid leukemia (AML) patients are suboptimal due mainly to relapse of disease after treatment. Leukemia stem cell (LSC) populations maintain the disease and serve as a reservoir for relapse. Therefore, treatments that can eradicate LSCs will improve the outcomes of patients with AML. Our preliminary data have shown that the interleukin-1 (IL-1) pathway is upregulated following treatment and disease progression in AML and plays an important role in LSCs in vitro and in vivo. To target IL-1 signaling, we disclose the structure-activity relationship guided development of UR241-2, a novel small molecule compound of interleukin-1 receptor-associated kinase 1/4 (IRAK1/4), which are critical downstream factors in IL-1/Toll-Like Receptor (TLR) signaling. UR241-2, at single dose (5 nM) screening exhibited high target selectivity; other than IRAK1/4 only 9 kinases in a panel of 682 kinases were inhibited by >25% activity. The NanoBRET assay using HEK293 cells transfected with targeted kinase vectors showed notably selective target engagement of UR241-2 against IRAK4 (IC50 = 6.07 nM). By using the in vitro NF-kB reporter assay and immunoblotting analysis, we identified that UR241-2 robustly inhibits IL-1/TLR signaling in AML cells including the activation of NF-κB and the phosphorylation of P65 and P38, following IL-1 stimulation. Next, we treated murine MLL-AF9 AML cells and human MDSL cells with UR241-2 and two IRAK4 inhibitors (PF-06650833 and CA-4948) that are currently in clinical or preclinical studies. We found that UR241-2 significantly inhibits colony-forming ability in AML and myelodysplastic syndromes, with similar or better efficacy compared to PF-06650833 or CA-4948. We also determined that UR241-2 suppresses LSC clonogenicity in primary human AML cells at diagnosis and in relapse while minimally impacting normal hematopoietic stem and progenitor cell function. Last, we investigated the impacts of UR241-2 on LSC engrafted ability in vivo. We treated murine MLL-AF9 cells with UR241-2 and PF-06650833 ex vivo for three days and transplanted them into recipient mice. After four weeks, we observed that UR241-2 significantly suppresses the engraftment levels of AML cells in bone marrow. It also decreases the WBC in peripheral blood and increases the RBC, HGB and PLT counts compared to the vehicle control group. Taken together, our findings demonstrated that targeting IRAK1/4 using the novel compound UR241-2 is able to impair LSC clonogenicity and engraftment capability in AML following treatment and disease progression. This provides a potential therapeutic strategy to eradicate the LSC population and improve the outcome of AML patients. Citation Format: Tzu-Chieh Ho, Rakesh K. Singh, Hiroki Kawano, Mark W. LaMere, Jian Wang, Paula M. Vertino, Nikolay V. Dokholyan, Laura M. Calvi, Craig T. Jordan, Michael W. Becker. Targeting interleukin-1 receptor-associated kinase 1/4 in acute myeloid leukemia stem cells [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 525.