Acute Myeloid Leukemia Progenitor Cells Research Articles

Abstract 1687: Targeted degradation of MECOM in high-risk acute myeloid leukemia reveals a novel repressive function that is amenable to therapeutic small-molecule rewiring

Abstract Hematopoiesis relies on an intricate balance between stem-cell self-renewal and terminal maturation of blood cells. Disrupting this balance can lead to the development of deadly blood cancers, like acute myeloid leukemia (AML). Increasing evidence suggests that acquiring hematopoietic stem cell (HSC) gene expression programs in AML confers a particularly poor prognosis and increases risk of relapse with conventional therapy. Acquisition of these features in AML is commonly driven by dysregulation of MECOM, a transcription factor and master regulator of HSC self-renewal. To define the role of MECOM in AMLs, we engineered cell-line models of high-risk AML by endogenously tagging MECOM with an FKBP12F36V degron. These models enable targeted degradation of MECOM which we employed in concert with multi-omic readouts to assess changes in nascent transcription and chromatin accessibility to identify direct transcriptional targets of MECOM. We demonstrate that MECOM degradation results in a rapid increase in chromatin accessibility at MECOM-bound sites and increased expression of cognate genes to establish a pro-differentiation gene program. These findings suggest a previously unappreciated mechanism for MECOM in repressing differentiation-promoting cis-regulatory elements (cisREs) and their cognate target genes in AML. We then employed a pooled CRISPR inhibition (CRISPRi) screen to functionally characterize the role that each cisRE plays in promoting high-risk features in AML. Utilizing CRISPRi to repress MECOM-bound cisREs in concert with dTAG-mediated MECOM degradation, we examined if repression of over 500 individual MECOM-bound cisREs is sufficient to maintain AML progenitors in the absence of MECOM. This functional dissection revealed that the individual repression of three cisREs that regulate CEBPA, GFI1B, and RUNX1 is sufficient to maintain AML progenitor cells in the absence of MECOM. This result implicates MECOM as a direct repressor of known regulators of myeloid differentiation. Given these findings, we hypothesized that small molecule-based recruitment of a transcriptional coactivator to MECOM could rewire its repressive function to activate myeloid differentiation and subsequent leukemia cell death. To test this approach, we treated a MECOM-FKBP12F36V AML degron model with the bifunctional small molecule NICE-01 (AP1867-PEG2-JQ1) to recruit an epigenetic activator (BRD4) to MECOM to functionally rewire this transcriptional network. NICE-01 treatment induced significant differentiation of AML progenitor cells relative to JQ1 treatment alone. Our work highlights the utility of targeted protein degradation to mechanistically interrogate the function of a key driver of high-risk AMLs and suggests the potential for small molecule-rewiring of stem cell gene regulatory networks to confer therapeutic benefit. Citation Format: Travis J. Fleming, Michael Gundry, Richard Voit, Mateusz Antoszewski, William J. Gibson, Ananthan Sadagopan, Vijay G. Sankaran. Targeted degradation of MECOM in high-risk acute myeloid leukemia reveals a novel repressive function that is amenable to therapeutic small-molecule rewiring [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 1687.

Synthetic Lethal Interactions with IRAK4 Inhibition in Myeloid Malignancies

Overexpression of immune-related genes is widely reported in acute myeloid leukemia (AML). Research from our lab and others has demonstrated that AML leukemic stem and progenitor cells (LSPC) rely on innate immune signaling through IRAK4 (reviewed in Bennett et al., Blood 2023). Moreover, AML LSPCs express a hypermorphic isoform of IRAK4 (IRAK4-L), due to U2AF1- and SF3B1-dependent mis-splicing. Building on this preclinical evidence, several IRAK4 inhibitors are now undergoing clinical trials in AML patients. In Phase-1 findings with a selective IRAK4 inhibitor (CA-4948; Curis Therapeutics), it was found that MDS and AML patients with splicing factor mutations responded best to monotherapy IRAK4 inhibition, although the overall response rate with monotherapy was modest. These findings suggest that IRAK4 is a relevant target in myeloid malignancies, but the identification of synthetic lethal dependencies may be crucial in revealing effective combination therapies. In this study, we sought to identify synthetic lethal interactions upon IRAK4 inhibition in AML. For this, we first generated isogenic AML cells that are proficient (WT THP1) or deficient for IRAK4 (IRAK4 KO THP1) using site-directed mutagenesis with CRISPR-Cas9. Deletion of IRAK4 resulted in a modest (~50%) reduction in leukemic colony formation and negligible differences in growth potential. We used the isogenic WT and IRAK4 KO AML cells to screen for synthetic lethal interactions by performing a 2400+ drug in vitro screen. Hits were prioritized for potency in IRAK4 KO cells vs WT AML cells, degree of fitness, and overall effect. Among these hits, the Cereblon E3 ligase modulator (CELMoD) CC-885 emerged as the top target. Independent validation confirmed that CC-885 significantly decreased proliferation and viability of isogenic IRAK4 KO MDS (MDSL) and AML (THP1) cells by approximately 3-4-fold compared to WT MDS or AML cells. Colony formation in methylcellulose after treatment with CC-885 was also significantly reduced in IRAK4 KO compared to WT MDS and AML cells. Based on Caspase-3 activation and AnnexinV staining, CC-885 induced significantly more apoptosis of IRAK4 KO cells as compared to WT AML cells. We extended our synthetic lethal analysis to a clinical-stage IRAK4 inhibitor (CA-4948). MDS and AML cells were treated with CA-4948 concurrently with CC-885 or pretreated for 7 days prior to treatment with CC-885. Unexpectedly, we found that only prior exposure of the AML cells to the IRAK4 inhibitors resulted in sensitivity to CC-885. These findings indicate that IRAK4 inhibition reprograms AML cells so that they acquire a synthetic lethal interaction with CC-885. CC-885 is known to target multiple substrates for degradation by the ubiquitin proteasome, including IKZF1, IKZF3, CK1a, and GSPT1. We compared the effect of CC-885 to CC-90009, a CELMoD with GSPT1 specific activity in the isogenic IRAK4 KO and WT AML cell lines. CC-90009 did not result in complete cell death up to concentrations of 10mM in both WT and IRAK4 KO, suggesting that the selective sensitivity of IRAK4 KO AML cells to CC-885 is not due to inhibition of GSPT1. In support of these interpretations, IRAK4 deficient AML cells exhibit increased protein expression of IKZF2 and IKZF3, but not GSPT1 or CK1a. Given the unique responses of IRAK4 KO cells to CELMoDs, we posited that IRAK4 inhibition alters the neosubstrate profile of CELMoDs in AML cells. Total proteome characterization of CC-885 treated WT and IRAK4 KO THP1 cells by mass spectrometry identified a significant decrease in 90 protein substrates in IRAK4 KO cells treated with CC-885 as compared to WT cells treated with CC-885. These findings suggest that IRAK4 inhibition alters the pool of neosubstrates in AML cells for certain CELMoDs. Overall, our study demonstrates that IRAK4 is a therapeutic target in AML, but that combination therapies, such as with certain CELMoDs, will be necessary to achieve better clinical responses.

Identifying Surface Protein Markers for AML Immunotherapy

Background: Acute Myeloid Leukemia (AML) is a hematologic malignancy characterized by clonal expansion and differentiation arrest of myeloid precursor cells in the bone marrow. It results in the accumulation of abnormal and immature myeloblasts, which impairs the production of normal blood cells, including erythrocytes, leukocytes, and platelets. Chimeric Antigen Receptor T-cell therapy (CAR-T therapy) and Antibody-Drug Conjugate (ADC) immunotherapies have shown significant success in the treatment of hematological malignancies. However, their efficacy in AML is limited due to the disease's genetic diversity, lack of uniform antigen targets and immunosuppressive microenvironment. Single-cell RNA sequencing (scRNA-seq) is a powerful technique that can be utilized for surface antigen discovery in various cell types, including immune cells and cancer cells. Methods: To identify potential AML cell-surface markers, we employed a comprehensive approach that involved the utilization of 3' single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing (RNA-seq), and enhanced whole exome sequencing (eWES). A total of 21 samples were analyzed, comprising 18 distinct AML patients and 3 healthy bone marrow aspirates, serving as controls. Results: Using enhanced whole exome sequencing (eWES) on tumor/normal paired samples, we defined the significant driver mutations within our sample cohort, notably DNMT3A (N=4, 21%), FLT3 (N=3, 16%), TET2 (N=3, 16%), KRAS (N=2, 11%), and GATA2 (N=2, 11%). Subsequently, we conducted scRNAseq on 21 samples, yielding a total of 185,106 high-quality cells, which were effectively clustered into 38 unbiased groups. Each sample demonstrated an average capture of 8862 cells, showcasing robust data acquisition. Our scRNAseq analysis revealed a diverse representation of major lymphoid and myeloid lineages, with prominent populations of AML blasts, T/NK cells, and progenitor cells. Notably, tumor clusters exhibited highest copy number variations and formed separate clusters from other non-tumor immune cells. To uncover key functional pathways, we conducted pathway enrichment analysis comparing tumor cells (N=131,196 cells) to non-tumor cells (N=53,910 cells). Our findings demonstrated a significant overrepresentation of cancer hallmarks, such as Allograft rejection (FDR= 0.000002), IL2/STAT5 signaling (FDR= 0.0000005), IFN-gamma response (FDR= 0.00000001) and apoptosis (FDR= 0.00000001) within the AML blasts, suggesting their potential involvement in AML pathogenesis. To identify AML specific markers, we developed analysis pipeline and performed an unbiased systematic search of the scRNAseq dataset for genes with unique expression patterns in tumor cells compared to the healthy bone marrow controls. Our analysis identified 302 differentially expressed genes, with 8 genes displaying relative cell and tissue specificity. Notably, among these genes, we unveiled key potential targets with cell surface specificity, including novel markers alongside previously known ones. Noteworthy candidates, such as CD86, CSF1R, CD33, CLEC12A, and FLT3, were prominently and significantly expressed, showcasing their potential significance as targets for engineered immunotherapy. To validate and consolidate our discoveries, we conducted a cross-referencing exercise by comparing our scRNAseq data against bulk RNA data from primary human AML samples and cell lines. Bulk global proteomics and flow cytometry-based studies are planned to confirm our genetic discoveries. Summary: Our rigorous computational and experimental approach provides valuable insights into tumor-specific cell surface proteins, which hold promise as potential targets for innovative and precision immunotherapeutic interventions in the realm of AML treatment.

A STAT3 Degrader Demonstrates Pre-Clinical Efficacy in Venetoclax Resistant Acute Myeloid Leukemia

Introduction: Acute Myeloid leukemia (AML) is an aggressive hematological malignancy resulting from the transformation of immature myeloid progenitor cells followed by an uncontrolled clonal proliferation and accumulation of the transformed cells. AML is the most common myeloid malignancy in the elderly [1]. Venetoclax (Ven) is a selective inhibitor of the anti-apoptotic BCL2 protein. Ven is FDA approved in combination with hypomethylating agents (HMA's) or low dose cytarabine for the treatment of de-novo AML in patients > 75 years or those ineligible for standard induction therapies. Despite the recent FDA approval of HMA/Ven and other novel therapies, 5-year survival in newly diagnosed AML remain less than 30% and disease relapse is inevitable without a bridge to allogeneic stem cell transplantation [2]. Signal transducer and activator of transcription 3 (STAT3) belonging to the STAT family of transcription factors is well known to be inappropriately activated in several malignancies [3]. On stimulation by cytokines such as IL-6, STAT3 undergoes dimerization and phosphorylation. Phosphorylation at Tyr 705 leads to its translocation to the nucleus whereas phosphorylation at Ser 727 translocates it to the mitochondria [4]. Previous data from our lab demonstrated de-methylation and overexpression of STAT3 in MDS & AML stem cells, that is associated with an adverse prognosis [5]. We have also reported that STAT3 controls several important leukemic drivers such as the anti-apoptotic protein myeloid cell leukemia-1 (MCL1). MCL1 overexpression is the central mechanism of resistance to BCL2 inhibition (Ven) in AML [5]. While MCL1 is a well-known direct transcriptional target of STAT3, the role of STAT3 in venetoclax resistance (Ven-res) is unknown. Methods & Results: To understand the role of STAT3 in Venetoclax resistance (Ven-res), Ven-res AML cell lines (MOLM-13, MV-4-11) as well as a Ven-res large cell lymphoma cell line (SU-DHL-1) were generated, which exhibited increased levels of both total STAT3, phospho-STAT3 and its downstream effector, MCL1, when compared to their parental cell lines (Fig. 1A). Data from > 90 AML patients treated with prior Ven also showed high expression of total STAT3 along with enhanced phosphorylation for p Ser727 and p Tyr-705 STAT3, that strongly correlated with worse overall survival (OS) and reduced remission duration (RemDur) (p<0.05). A highly specific potent heterobifunctional degrader of STAT3 (degrader) resulted in a dose dependent and selective degradation of STAT3 in both parental and Ven-res hematological malignancy cell lines. Treatment with degrader showed a significant (>60%) decrease in p Tyr-705 as wellas p Ser-727 STAT3 levels in MOLM-13 parental as well as MOLM-13 Ven-res cells. STAT3 degradation also led to induction of apoptosis in both parental and Ven-res AML cell lines (p < 0.001). Importantly, colony assay of Ven-res AML patient samples showed effective degradation of STAT3 (>90%) together with the increased erythroid and myeloid differentiation (~ 2 fold) on treatment with degrader. Interestingly, no differentiation was observed in healthy samples, suggesting the specificity of the degrader to AML stem and progenitor cells. In patients with Ven-res AML, erythroid colony counts were seen to increase (~1.5 fold) with a concomitant decrease in myeloid colony counts (>2.5 fold), on treatment with degrader. This observation supports the clinical significance of our studies in AML patients with anemia. Although BH3 profiling of Ven-res MOLM-13 supports an increased dependency on MCL1, we have observed that treatment with degrader led to ~20% reduction in the MCL1 dependent mitochondrial depolarization. Additionally, cell derived xenograft (CDX) models of Ven-res showed significant reduction of p Tyr-705 STAT3(~60%), total STAT3 (>90%) and MCL1 (~70%), on treatment with STAT3 degrader - KT-333 (currently in an early phase clinical trial:NCT05225584), as early as week 2 (Fig 1B). Significant decrease in intracellular STAT3 in the spleen cells was also observed (p < 0.05), with ongoing survival analysis. Conclusion: Our study suggests that targeting STAT3 and the downstream MCL1 represents a novel and effective strategy for Ven-resistant AML patients in clinic, with strong mechanistic rationales that can spur further clinical development of STAT3 degraders especially given the significant side effects of direct MCL1 inhibitors.

Abstract A35: Overcoming Venetoclax Resistance in Acute Myeloid Leukemia (AML) via the Novel Imipridone ONC213

Abstract Older adults make up most of the AML patient population and have an especially dismal prognosis due to limited therapeutic options. The Bcl-2 inhibitor, venetoclax (VEN), was approved for use in combination with low-dose cytarabine or a hypomethylating agent [azacitidine (AZA) or decitabine] in AML patients who are ≥75 years old or ineligible for intensive chemotherapy (IC). Due to superior survival data and tolerability, the combination of VEN and AZA has become a new standard of care for older patients and those unfit for IC. Though this was a promising therapeutic option for these patients, resistance to this therapy results in a 1-year overall survival rate of only 30-40% and the median overall survival following treatment failure is less than three months, highlighting the urgent need of new therapies to address this. Previous studies have found VEN resistance can be overcome by targeting mitochondria and Mcl-1. Our studies on ONC213, a novel imipridone, have found that it has potent antileukemic activity against both AML cell lines and primary patient samples. It reduces Mcl-1 and targets mitochondria of AML cells – and was thus a promising agent to overcome VEN resistance. When used in combination, ONC213 and VEN are highly synergistic in both AML cell lines and primary patient samples, including those intrinsically resistant to VEN. The combination also targets AML progenitor cells determined by colony formation assays and shows in vivo efficacy in a cell line derived xenograft mouse model (median survival of 57 days vs 37 days in control mice, p=0.002). To determine if ONC213 could overcome acquired resistance to VEN and AZA, we developed VEN and AZA double resistant ML-2 and MV4-11 AML cells (designated ML-2/VEN+AZA-R and MV4-11/VEN+AZA-R) by stepwise increasing concentrations of VEN and AZA at 1:3 fixed ratio over an extended period. ML-2/VEN+AZA-R and MV4-11/VEN+AZA-R AML cells demonstrate 437- and 1156-fold, respectively, increase in VEN+AZA (1:3 ratio) IC50 compared to parental cells (5,285.3 nM vs 12.1 nM and 6,704.3 nM vs 5.8 nM, respectively). ONC213 can resensitize resistant cells to VEN and the combination of ONC213 and VEN is highly synergistic in both ML-2/VEN+AZA-R and MV4-11/VEN+AZA-R cells as evident by a combination index (CI) value of < 0.05. ONC213 alone and in combination with VEN suppress oxidative phosphorylation (OXPHOS) and Mcl-1 in AML cells, including those with inherent resistance to VEN or with acquired resistance to VEN+AZA. Overexpression of Mcl-1 can partially rescue cells from combination therapy. These results suggest that ONC213 suppresses OXPHOS and Mcl-1 to overcome VEN resistance. Here we demonstrate that VEN resistance can be addressed through dual targeting of mitochondria and Mcl-1 using the novel imipridone, ONC213. ONC213 resensitizes VEN + AZA resistant cells to VEN and offers a new approach to combat VEN+AZA resistance in AML. Citation Format: Jenn L. Carter, Yongwei Su, Holly Edwards, Lisa Polin, Joshua E. Allen, Varun V. Prabhu, Jay Yang, Maik Huettermann, Yubin Ge. Overcoming Venetoclax Resistance in Acute Myeloid Leukemia (AML) via the Novel Imipridone ONC213 [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A35.

The Composition of Acute Myeloid Leukemia Cell Differentiation States Predicts Response to Immune Checkpoint Blockade

Background: Given the activity of allogeneic T cells in acute myeloid leukemia (AML) patients, immunotherapies stimulating antitumor T cell responses have been explored as a promising therapeutic option. However, these therapies have been associated with modest clinical activity, and further research is needed to identify response and resistance mechanisms. A spectrum of primitive and differentiated cells comprise AML in each individual patient, and this cell composition has been previously shown to predict outcomes. In particular, high proportions of AML stem and progenitor cells have been associated with worse clinical outcomes and lack of response to induction chemotherapy (Ng et al Nature 2016; van Galen et al Cell 2019; Zeng et al Nat Med 2022). The composition of AML cell differentiation states in relation to immunotherapy response or resistance has not been elucidated; herein, we explored whether this composition may be associated with response in AML patients treated with immune checkpoint blockade. Methods: Pre- and posttreatment whole bone marrow (BM) samples were collected from 31 newly diagnosed and relapsed/refractory AML patients treated with azacitidine and pembrolizumab (NCT02845297) and 18 relapsed/refractory AML patients treated with high-dose cytarabine (HiDAC) and pembrolizumab (NCT02768792; Zeidner et al Blood Cancer Discov 2022) on two separate phase II trials. Primary patient samples and associated clinical data were obtained via informed consent in accordance with the Declaration of Helsinki as per research protocols approved by the Institutional Review Boards of the participating institutions. Bulk RNA sequencing was performed on all BM samples. Differential gene expression analysis was performed using the DESeq2 package, and gene set enrichment analysis (GSEA) was performed using the clusterProfiler package. Proportions of AML cell populations present in BM samples were deconvoluted using CIBERSORTx as per Zeng et al Nat Med 2022. The LSC17 score and LinClass7 score were calculated as per Ng et al Nature 2016 and Zeng et al Nat Med 2022, respectively. All analyses and graphs were generated using R (v4.2.1). Results: GSEA using gene sets from a range of primitive and differentiated AML and normal cell types as per van Galen et al Cell 2019 was performed on pre- and posttreatment samples. Patients achieving a complete response (CR) to either immunotherapy regimen were enriched at baseline for the following differentiated AML gene sets: pro monocyte (Promono)-like AML; monocyte-like AML; conventional dendritic cell (cDC)-like AML; and myeloid-like AML (Figure 1A). Patients not responding (NR) to azacitidine/pembrolizumab were enriched for the following stem and progenitor AML gene sets: hematopoietic stem cell (HSC)-like AML; progenitor (Prog)-like AML; and HSC progenitor (HSCProg)-like AML (Figure 1A). To validate these findings, BM samples were deconvoluted into inferred fractions of AML differentiation states using CIBERSORTx and single-cell RNA sequencing expression data from Zeng et al Nat Med 2022. Patients not responding to azacitidine/pembrolizumab had a higher proportion of leukemia stem and progenitor quiescent cells (LSPC Quiescent) at baseline, while patients achieving CR to either regimen had higher baseline proportions of pro monocyte-like AML cells (Figure 1B). Additionally, patients not responding to azacitidine/pembrolizumab had higher baseline LSC17 and LinClass7 scores, two gene expression scores that are reflective of leukemia stemness (Figure 1B). In the posttreatment setting for both regimens, HSC-like, Prog-like, and HSCProg-like gene sets were associated with NR; monocyte-like and myeloid-like gene sets were associated with CR (Figure 1A). Discussion: Patients achieving CR to azacitidine/pembrolizumab and HiDAC/pembrolizumab had higher proportions of differentiated AML cells, while NR patients had a higher proportion of AML stem and progenitor cells. We hypothesize that antitumor T cells may preferentially target differentiated AML cells over AML stem and progenitor cells. Further research is needed to validate these findings in a prospective and mechanistic manner and in other immunotherapy regimens. Analyses of the proportions of AML differentiation states in patients relapsing after allogeneic hematopoietic cell transplantation are ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Novel Bispecific Innate Cell Engager (ICE®) AFM28 Efficiently Directs Allogeneic NK Cells to CD123+ Leukemic Stem- and Progenitor Cells in AML

Introduction: Acute myeloid leukemia (AML) is a common hematologic malignancy characterized by expansion of undifferentiated myeloid cells in the bone marrow (BM) and peripheral blood. Although AML can be treated with curative intent, residual leukemic stem cells (LSCs) are frequently reservoirs for relapse, which is associated with a poor prognosis. Moreover, especially elderly patients are often not eligible for intensive treatment regimen. Therefore, there is a high need for novel treatment strategies, which are capable of eliminating LSCs in order to induce long-term remissions, suppress measurable residual disease (MRD) and prevent relapse. A promising emerging approach is the adoptive transfer of allogeneic natural killer (NK) cells, which was shown to be clinically active and could induce complete remissions in relapsed/refractory (R/R) AML. Innate Cell Engagers (ICE®) may further augment the activity of NK cell therapies, in particular against LSCs and hold the potential to increase frequency and durability of responses. The tetravalent bispecific CD123/CD16A ICE®, AFM28, directs the cytotoxic activity of CD16A-expressing NK cells towards CD123-expressing (CD123+) AML blasts and LSCs. In this study, we evaluated the efficacy of AFM28 in pre-clinical models of AML. Methods: Antibody-dependent cell-mediated cytotoxicity (ADCC) data were obtained by a 4h calcein release assay using AML cell lines and primary human NK cells (E:T ratio 2.5:1) in the presence of titrated antibodies. CD64 and CD123 expression levels on AML cell lines were evaluated by flow cytometry. Intracellular FACS was used to detect STAT5/pSTAT5. Proliferation of TF-1 cells in the presence of IL-3 or GM-CSF was measured via CellTiter Glo Assay (Promega). Using primary BM samples of n=6 AML patients and n=4 healthy donors, ADCC assays and colony-forming unit (CFU) assays were performed. BM-MNC (ADCC assay) or enriched CD34+ cells (CFU assay) and allogeneic NK cells (E:T ratio 1:1) were treated with AFM28 or an Fc-enhanced anti-CD123 antibody comparator at different concentrations of AFM28 (0-500 or 0/10/100/1000 pM, respectively) for 24h. Subsequently, ADCC assays were analyzed by flow cytometry and CFU assays were evaluated after 7-14 days. Systemic anti-tumor activity of AFM28 in vivo was assessed using in-life imaging (BLI) in a T cell-depleted huFcgR-C57BL6 transgenic mouse model against intravenously administered luciferase-transfected murine AML cells expressing human CD123 as tumor antigen. Results: Pre-clinical in vitro models using a panel of AML cell lines showed efficacious ADCC against CD123+ tumor cells by allogeneic NK cells in the presence of AFM28. ADCC induction by AFM28 was independent of leukemic cell mutational profiles and even targeted cells with low levels of CD123 surface expression. Of note, expression of the high-affinity IgG receptor CD64 on AML cell lines completely abolished ADCC induction by an Fc-enhanced anti-CD123 antibody whereas AFM28 also showed high ADCC efficacy against all tested CD64+ cell lines. In addition, AFM28 exerted an NK cell-independent inhibitory effect on the IL-3-induced phosphorylation of STAT5 and abolished proliferation of CD123+ TF-1 cells, indicating an antagonistic effect on IL-3R signaling. In ex vivo models using primary AML patient cells, AFM28 demonstrated strong ADCC mediated by allogeneic NK cells. CFU assays of patient-derived AML BM samples performed subsequent to incubation with allogeneic NK cells with and without AFM28 showed significantly reduced numbers of outgrowing colonies in AFM28 treated samples, indicating that AML LSCs and progenitor cells were eliminated. In the in vivo efficacy study with huFcgR-C57BL6 mice, treatment with AFM28 at 15 mg/kg b.w. led to complete inhibition of tumor growth in 5/5 animals throughout a treatment period of 42 days. In contrast, all untreated control mice (6/6) developed systemic disease. Conclusion: AFM28 induced highly potent and selective lysis of CD123+ leukemic cells, including LSCs and progenitor cells, by allogeneic NK cells in vitro and in vivo. The capacity to eradicate LSCs holds the promise for durable responses and the potential for long-term remissions in patients with R/R AML. AFM28 is currently being prepared for a first-in-human clinical investigation as monotherapy and in combination with allogeneic NK cells.

Autophagy inhibition impairs leukemia stem cell function in FLT3-ITD AML but has antagonistic interactions with tyrosine kinase inhibition.

The FLT3-ITD mutation is associated with poor prognosis in acute myeloid leukemia (AML). FLT3 tyrosine kinase inhibitors (TKIs) demonstrate clinical efficacy but fail to target leukemia stem cells (LSC) and do not generate sustained responses. Autophagy is an important cellular stress response contributing to hematopoietic stem cells (HSC) maintenance and promoting leukemia development. Here we investigated the role of autophagy in regulating FLT3-ITD AML stem cell function and response to TKI treatment. We show that autophagy inhibition reduced quiescence and depleted repopulating potential of FLT3-ITD AML LSC, associated with mitochondrial accumulation and increased oxidative phosphorylation. However, TKI treatment reduced mitochondrial respiration and unexpectedly antagonized the effects of autophagy inhibition on LSC attrition. We further show that TKI-mediated targeting of AML LSC and committed progenitors was p53-dependent, and that autophagy inhibition enhanced p53 activity and increased TKI-mediated targeting of AML progenitors, but decreased p53 activity in LSC and reduced TKI-mediated LSC inhibition. These results provide new insights into the role of autophagy in differentially regulating AML stem and progenitor cells, reveal unexpected antagonistic effects of combined oncogenic tyrosine kinase inhibition and autophagy inhibition in AML LSC, and suggest an alternative approach to target AML LSC quiescence and regenerative potential.

CD93 promotes acute myeloid leukemia development and is a potential therapeutic target

CD93 is a transmembrane receptor belonging to the Group XIV C-Type lectin family. It is expressed in a variety of cellular types such as monocytes, neutrophils, platelets, microglia, and endothelial cells. CD93 has been reported to play important roles in cell proliferation, cell migration, and tumor angiogenesis. Here, we show CD93 is highly expressed in M4 and M5 subtypes of acute myeloid leukemia (AML) patients, and highly expressed in leukemia stem cells, AML progenitor cells, as well as more differentiated AML cells. We found that CD93 promotes AML cell proliferation, while CD93 deficient AML cells commit to differentiation. We further show that CD93 exerts its proliferative function through downstream SHP-2/Syk/CREB cascade in AML cells. Moreover, human AML cells treated with CD93 mAb combined with αMFc–NC–DM1 (an IgG Fc specific antibody conjugated to maytansinoid DM1), showed a striking reduction of proliferation. Our study revealed that CD93 is a critical participator of AML development and provides a potential therapeutic cell surface target. (160 words).

Abstract 3168: Immunopeptidome-defined acute myeloid leukemia progenitor cell-associated antigens are targeted in vivo by AML patients’ T cells

Abstract Despite recent advances and the approval of novel molecular therapies in acute myeloid leukemia (AML), the disease is still characterized by high relapse rates and poor overall survival due to the persistence of therapy-resistant residual leukemic progenitor cells (LPCs). T cell-based immunotherapy has been suggested as a novel therapeutic option to eliminate minimal residual disease and achieve long-lasting remissions. One main prerequisite for immunotherapy development is the selection of immunogenic targets that show natural, high-frequent, and tumor-exclusive presentation on the cell surface of malignant cells. In a previous study we characterized the antigenic landscape of AML by mass spectrometry to identify AML-specific T cell epitopes (Berlin et al. Leukemia 2015). Here, we aimed to analyze the immunopeptidome of primary AML progenitor cells (n = 10, purity >80% CD34+CD38-) to identify LPC-associated antigens that enable the specific targeting of AML LPCs. Additionally, we analyzed an extended set of AML patient samples (n = 47) to screen for naturally presented neoepitopes and to identify broadly applicable AML-associated target antigens that are presented on both AML blasts and LPCs. We identified more than 16,000 HLA class I- and 17,000 HLA class II-presented peptides on LPCs and 72,000 HLA class I and 61,000 HLA class II peptides in the total AML cohort. Comparative profiling of LPCs, AML blasts, and a benign tissue database (n = 332) revealed HLA class I- and HLA class II-presented LPC-exclusive as well as frequently presented AML-associated antigens on both AML blasts and LPCs. Besides these tumor-exclusive self-peptides, we detected naturally presented neoepitopes derived from two frequent mutations (NPM1 and IDH2) in this low-mutational burden malignancy. For immunogenicity analyses we selected 16 HLA class I- and 15 HLA class II-restricted peptides comprising unmutated as well as mutation-derived antigens. In vitro priming experiments showed the induction of peptide-specific, multi-functional, and cytotoxic CD8+ effector cells in samples of healthy volunteers and AML patients. We were able to detect strong preexisting memory T cell responses targeting LPC-associated antigens in AML patients with detection frequencies of up to 20%. Retrospective analyses revealed that patients with preexisting peptide-specific T cell responses showed improved overall survival compared to patients without any memory responses against our targets. Taken together, we identified novel, naturally presented, LPC-exclusive, and AML-associated self-antigens and neoepitopes presented on both AML blasts and LPCs. We could demonstrate the immunogenicity of 14/16 (88%) HLA class I and 13/15 (87%) HLA class II antigens highlighting their potential as promising targets for T cell-based immunotherapy approaches to eliminate minimal residual disease in AML patients. Citation Format: Annika Nelde, Heiko Schuster, Tatjana Bilich, Jens Bauer, Malte Roerden, Sarah Schroeder, Jonas S. Heitmann, Elke Rücker-Braun, Hans-Georg Rammensee, Helmut R. Salih, Juliane S. Walz. Immunopeptidome-defined acute myeloid leukemia progenitor cell-associated antigens are targeted in vivo by AML patients’ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3168.

Chidamide and apatinib are therapeutically synergistic in acute myeloid leukemia stem and progenitor cells

BackgroundLeukemia stem cells (LSCs) are responsible for the initiation and perpetuation of acute myeloid leukemia (AML), and also represent leukemia relapse reservoirs with limited therapeutic approaches. Thus, additional treatment strategies are medical unmet needs to eliminate LSCs.MethodsCell counting kit-8 and Annexin-V-FITC/PI assays were used to examine the interaction of chidamide and apatinib on LSC-like cell lines (CD34+CD38− KG1α and Kasumi-1 cells) and primary CD34+ AML cells. AML patient-derived xenografts were established to investigate the in vivo efficacy of the combined regimen. RNA sequencing, Glutamine uptake assay, oxygen consumption assay, and western blotting were employed to explore the molecule mechanism for the cytotoxicity of chidamide with or without apatinib against LSC-like cell lines and/or primary CD34+ AML cells.ResultsIn this study, chidamide and apatinib were synergisitc to diminish cell viability and induce apoptosis in CD34+CD38− KG1α and Kasumi-1 cells and in CD34+ primary AML cells. Importantly, chidamide combined with apatinib had more powerful in reducing leukemia burden and improving prognosis than single drug alone in an AML PDX model without significant adverse effects. Chidamide cytotoxicity was associated with decreasing glutamine uptake. The therapeutic synergy of chidamide and apatinib correlated with reprogramming of energy metabolic pathways. In addition, inactivating the VEGFR function and reducing the anti-apoptotic ability of the Bcl2 family contributed to the synergism of chidamide and apatinib in CD34+CD38− KG1α cells and CD34+ primary AML cells.ConclusionChidamide in combination with apatinib might be a promising therapeutic strategy to get rid of the population of AML stem and progenitor cells, and thus provide a potentially curative option in the treatment of patients with AML, although further clinical evaluations are required to substantiate the conclusion.

Blocking UBE2N abrogates oncogenic immune signaling in acute myeloid leukemia.

Dysregulation of innate immune signaling pathways is implicated in various hematologic malignancies. However, these pathways have not been systematically examined in acute myeloid leukemia (AML). We report that AML hematopoietic stem and progenitor cells (HSPCs) exhibit a high frequency of dysregulated innate immune-related and inflammatory pathways, referred to as oncogenic immune signaling states. Through gene expression analyses and functional studies in human AML cell lines and patient-derived samples, we found that the ubiquitin-conjugating enzyme UBE2N is required for leukemic cell function in vitro and in vivo by maintaining oncogenic immune signaling states. It is known that the enzyme function of UBE2N can be inhibited by interfering with thioester formation between ubiquitin and the active site. We performed in silico structure-based and cellular-based screens and identified two related small-molecule inhibitors UC-764864/65 that targeted UBE2N at its active site. Using these small-molecule inhibitors as chemical probes, we further revealed the therapeutic efficacy of interfering with UBE2N function. This resulted in the blocking of ubiquitination of innate immune- and inflammatory-related substrates in human AML cell lines. Inhibition of UBE2N function disrupted oncogenic immune signaling by promoting cell death of leukemic HSPCs while sparing normal HSPCs in vitro. Moreover, baseline oncogenic immune signaling states in leukemic cells derived from discrete subsets of patients with AML exhibited a selective dependency on UBE2N function in vitro and in vivo. Our study reveals that interfering with UBE2N abrogates leukemic HSPC function and underscores the dependency of AML cells on UBE2N-dependent oncogenic immune signaling states.

Deregulation of Splicing in Pediatric Acute Myeloid Stem and Progenitor Cells

-Currently, the limited capacity of pediatric acute myeloid leukemia (AML) therapies to prevent recurrence has contributed to high mortality rates. While dormant self-renewing leukemia stem cells (LSCs) contribute to adult AML relapse, their role in pediatric AML therapeutic resistance has not been clearly elucidated and thus was investigated in the context of this study.Through whole transcriptome sequencing (RNA-seq) analyses of FACS-purified human hematopoietic stem cells (HSCs; CD34 +CD38 -Lineage -) and progenitor cells (HPCs; CD34 +CD38 + Lineage -) from pediatric AML (n=10) compared with adult de novo (n=5) and secondary AML (n=6) as well as non-leukemic pediatric bone marrow samples (n=6), we identified widespread splicing alterations in pediatric AML compared to non-leukemic donors, indicative of a disruption in splicing regulation. In this study, we identified 2,000 exon skipping events in pediatric AML HSCs and HPCs. Moreover, we detected increased exon skipping and intron retention in stem cell self-renewal and survival transcripts in pediatric AML stem and progenitor cells. Specifically, the pro-survival isoform of MCL1, MCL1 long, was significantly increased in comparison to its pro-apoptotic counterpart, MCL1 short. In addition, self-renewal, RNA editing and splice isoform altering adenosine deaminase RNA specific 1 (ADAR1) p150 isoform levels were significantly (p=0.05) upregulated in pediatric AML progenitors suggesting that splicing and RNA editing deregulation could fuel pediatric AML stem and progenitor cell propagation.After successful completion of pre-IND development of a pharmacologically stable, potent, and selective small molecule splicing modulator, Rebecsinib (17S-FD-895) (Crews, Balain et al Cell Stem Cell 2016; Chan et al Cell Reports 2020), we developed a dual fluorescence lentiviral splicing reporter that assays the on target anti-leukemic efficacy of Rebcsinib and to assess the therapeutic index between LSCs and normal hematopoietic stem and progenitor cells. In hematopoietic progenitor assays, we observed a dose-dependent reduction in clonogenicity and replating of CD34 + cells isolated from pediatric AML samples following treatment with Rebecsinib. While pediatric AML samples were more sensitive to splicing modulation than adult de novo or adult secondary AML samples, normal cord blood progenitor samples were unaffected by splicing modulator treatment. In addition, we identified dose-dependent alterations in lentiviral splicing reporter activity in pediatric leukemia cells engrafted in a humanized AML mouse xenograft model following intravenous treatment with one dose of 10mg/kg and 20mg/kg of Rebecsinib. Finally, we observed a reduction in ADAR1 p150 transcripts by RNA-seq analysis of hematopoietic tissues in serially transplanted patient derived AML xenografts after Rebecsinib treatment suggesting that inhibition of ADAR1 splicing prevents LSC self-renewal.Cumulatively, these data demonstrate that stem and progenitor cell specific deregulation of pre-mRNA splicing and ADAR1 activation represent a therapeutic vulnerability to splicing modulation, which provides a strong rationale for developing Rebecsinib for preventing pediatric AML recurrence. DisclosuresCloos: Astellas: Speakers Bureau; DC-One: Other, Research Funding; Genentech: Research Funding; Helsinn: Other; Janssen: Research Funding; Merus: Other, Research Funding; Navigate: Patents & Royalties; Novartis: Consultancy, Other, Research Funding; Takeda: Research Funding. Crews: Ionis Pharmaceuticals: Research Funding. Burkart: Algenesis: Other: Co-founder. Jamieson: Forty Seven Inc.: Patents & Royalties.

Single-Cell Transcriptional Profiling of Acute Myeloid Leukemia Identifies Prognostic Transcriptional Profiles at Diagnosis

Acute myeloid leukemia (AML) is a lethal cancer with a survival of less than 50%. Standard cytotoxic therapies frequently induce complete remission, but patients frequently relapse and die of their disease. Leukemia stem cells (LSCs) are the leukemia cells with self-renewal potential and ability to recapitulate the disease. In hematopoietic stem cells, the mechanisms of proliferation are distinct from self-renewal (Li et al. Nature 2013). Consequently, targeting proliferation may explain the failure of traditional chemotherapy to target LSCs and eradicate AML. Our goal is define the molecular mechanisms that allow AML to relapse. We previously showed that activated NRAS (NRASG12V) facilitates self-renewal in the LSC-enriched subpopulation of a mouse model of AML (Mll-AF9 / NRASG12V, Sachs et al. Blood 2014, Kim et al. Blood 2009). We hypothesize that understanding the NRAS-activated pathways required for self-renewal at the single-cell level can be used to identify clinically relevant features of human AML.We used single-cell RNA sequencing to identify the self-renewing cells among the LSC-enriched subgroup in this model (Mac1LowKit+Sca1+, “MKS”). We identified three discrete transcriptional profiles among the LSC-enriched subpopulation and found that that two of these profiles (Profile 1 and Profile 2) are NRASG12V-dependent. These two profiles can be differentiated by CD36 and CD69 expression. We sorted the MKS LSCs based on CD36 and CD69 expression. Sorted LSC subsets were transplanted into recipient mice to compare their ability to transfer leukemia as a measure of their self-renewal capacity. We found that MKS-CD36-CD69+ cells (consistent with Profile 1) rapidly transferred leukemia with high penetrance in 20 of 22 mice. In contrast, MKS-CD36+CD69- cells (Profile 2) failed transfer leukemia in most mice; only 2 of 25 of these mice developed AML (p < 0.004).In our previous work, we demonstrated that the NRASG12V-activated self-renewal gene expression profile that we identified in our murine model was expressed in human AML, suggesting that the gene expression behavior of LSCs from this model may recapitulate the gene expression behavior of human LSCs (Sachs et al. Blood 2014). We used our murine single-cell self-renewal transcriptional profile to define a 96-gene panel consisting of 88 genes from this profile and 8 housekeeping genes. We sorted primary, diagnostic human AML cells for leukemia stem and progenitor cells (CD34+CD38-) and performed single-cell qPCR on these cells using our 96-gene panel. We found that a subset of these human LSCs preferentially expresses Profile 1, the self-renewal gene expression profile that we identified in our murine model, and another subset preferentially expresses Profile 2 (the profile associated with no leukemia-reconstituting capacity).Next, we investigated whether CD36 and CD69 delineate clinically relevant transcriptional subgroups in human AML. We used previously published, clinically annotated gene expression datasets of human AML that include associated survival outcomes data (TCGA, GSE12417 (Metzeler et al. Blood 2008), and GSE6891 (Verhaak et al. Haematologica 2009)). Gene Cluster Expression Summary Score (GCESS, Scott et al. under review) analysis identified gene clusters associated with CD69 and CD36 in our murine single-cell dataset. We found that the CD36 and CD69-associated clusters are conserved in the human transcriptional datasets. More importantly, patients that express either the CD69 or the CD36-associated gene expression profile suffer from shorter survival times in these three, independent human transcriptional datasets.In these experiments, we use a murine model of AML to define the LSC self-renewal gene expression profile at the single-cell level and functionally validate this profile in vivo . Analogous to the murine model, a subset of human AML stem and progenitor cells expresses this LSC self-renewal gene expression profile at the single-cell level. Finally, we found that the gene expression profiles we identified in our murine single-cell data are associated with poor outcomes in human AML indicating that our single-cell gene expression analyses can be used to identify prognostic patient subgroups. These findings suggest that these gene expression profiles can be used to identify functional transcriptional modules associated with poor outcome in human AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Blocking CD70/CD27 Signaling in Combination with Hypomethylating Agents Eradicates Human CD34+ AML Stem and Progenitor Cells in Vitro and In Vivo

Leukemia stem cells (LSCs) are the origin of acute myeloid leukemia (AML) and are resistant to standard therapeutic regimens resulting in frequent relapse of the disease and poor prognosis especially for AML patients not fit for intensive chemotherapy. Consequently, LSCs represent a major obstacle for AML therapy. We recently identified the interaction of the TNF ligand CD70 and its receptor CD27 on LSCs as a promising therapeutic strategy to target LSCs.In this study, we demonstrate for the first time that treatment with hypomethylating agents (HMA, azacitidine or decitabine) significantly up-regulates CD70 mRNA and protein expression on primary CD34+ stem/progenitor cells from newly diagnosed AML patients independent of the cytogenetic/molecular risk category in vitro (mRNA: 4.3±1.6 fold increase, FACS: 4.0±0.9 fold increase vs. vehicle treatment, n=15). Similarly, CD34+ stem/progenitor cells from AML patients after 5 days decitabine administration (20mg/kg by continuous intravenous infusion over 1 hour repeated daily) exhibited a significant increase in CD70 expression as analyzed by flow-cytometry (6.8±1.1 fold increase vs. diagnostic sample, n=6). In contrast, CD70 expression of CD34+ stem/progenitor cells from healthy donor bone marrow (n=3) was not affected by the treatment.Consequently, co-treatment of CD34+ stem/progenitor cells from AML patients of each risk group (n=3) with the HMA decitabine and a blocking αCD70 monoclonal antibody strongly reduced colony-forming and re-plating capacity in vitro compared to single agent treatment. To confirm and extend our findings towards disease-initiating CD34+ AML LSCs in vivo, we performed patient-derived AML xenografts (PDX) from one patient with intermediate and one patient with adverse cytogenetic risk profile. Combining decitabine treatment with CD70 blockade significantly reduced AML engraftment in blood, spleen and BM compared to controls. Moreover, co-treatment significantly reduced frequency and absolute numbers of CD34+CD38- and CD34+CD38-CD45RA- AML LSCs in the BM as analyzed phenotypically by FACS and eliminated human LSCs as analyzed in limiting dilution PDX experiments compared to single treatments. Importantly, CD34+ hematopoietic stem/progenitor cells from healthy human bone marrow (n=3) were only marginally affected by the co-treatment.In summary, combining HMAs with blocking CD70/CD27 signaling could represent a novel strategy to eradicate human LSCs. DisclosuresVan Rompaey:argenX: Employment. Leupin:argenX: Employment. De Haard:argenX: Employment.

Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

SummaryThe aberrant expression of dopamine receptors (DRDs) in acute myeloid leukemia (AML) cells has encouraged the repurposing of DRD antagonists such as thioridazine (TDZ) as anti-leukemic agents. Here, we access patient cells from a Phase I dose escalation trial to resolve the cellular and molecular bases of response to TDZ, and we extend these findings to an additional independent cohort of AML patient samples tested preclinically. We reveal that in DRD2+ AML patients, DRD signaling in leukemic progenitors provides leukemia-exclusive networks of sensitivity that spare healthy hematopoiesis. AML progenitor cell suppression can be increased by the isolation of the positive enantiomer from the racemic TDZ mixture (TDZ+), and this is accompanied by reduced cardiac liability. Our study indicates that the development of DRD-directed therapies provides a targeting strategy for a subset of AML patients and potentially other cancers that acquire DRD expression upon transformation from healthy tissue.

TFEB links MYC signaling to epigenetic control of myeloid differentiation and acute myeloid leukemia.

MYC oncoproteins regulate transcription of genes directing cell proliferation, metabolism and tumorigenesis. A variety of alterations drive MYC expression in acute myeloid leukemia (AML) and enforced MYC expression in hematopoietic progenitors is sufficient to induce AML. Here we report that AML and myeloid progenitor cell growth and survival rely on MYC-directed suppression of Transcription Factor EB (TFEB), a master regulator of the autophagy-lysosome pathway. Notably, although originally identified as an oncogene, TFEB functions as a tumor suppressor in AML, where it provokes AML cell differentiation and death. These responses reflect TFEB control of myeloid epigenetic programs, by inducing expression of isocitrate dehydrogenase-1 (IDH1) and IDH2, resulting in global hydroxylation of 5-methycytosine. Finally, activating the TFEB-IDH1/IDH2-TET2 axis is revealed as a targetable vulnerability in AML. Thus, epigenetic control by a MYC-TFEB circuit dictates myeloid cell fate and is essential for maintenance of AML.

Evaluation of the human type 3 adenoviral dodecahedron as a vector to target acute myeloid leukemia

Intensive systemic chemotherapy is the gold standard of acute myeloid leukemia (AML) treatment and is associated with considerable off-target toxicities. Safer and targeted delivery systems are thus urgently needed. In this study, we evaluated a virus-like particle derived from the human type 3 adenovirus, called the adenoviral dodecahedron (Dd) to target AML cells. The vectorization of leukemic cells was proved very effective at nanomolar concentrations in a time- and dose-dependent manner, without vector toxicity. The internalization involved clathrin-mediated energy-dependent endocytosis and strongly correlated with the expression of αVβ3 integrin. The treatment of healthy donor peripheral blood mononuclear cells showed a preferential targeting of monocytes compared to lymphocytes and granulocytes. Similarly, monocytes but also AML blasts were the best-vectorized populations in patients while acute lymphoid leukemia blasts were less efficiently targeted. Importantly, AML leukemic stem cells (LSCs) could be addressed. Finally, Dd reached peripheral monocytes and bone marrow hematopoietic stem and progenitor cells following intravenous injection in mice, without excessive spreading in other organs. These findings reveal Dd as a promising myeloid vector especially for therapeutic purposes in AML blasts, LSCs, and progenitor cells.

Dual Inhibition of MDM2 and XPO1 Synergizes to Induce Apoptosis in Acute Myeloid Leukemia Progenitor Cells with Wild-Type TP53 through Nuclear Accumulation of p53 and Suppression of c-Myc

Dual Inhibition of MDM2 and XPO1 Synergizes to Induce Apoptosis in Acute Myeloid Leukemia Progenitor Cells with Wild-Type TP53 through Nuclear Accumulation of p53 and Suppression of c-Myc

Abstract 3003: Inhibiting the mitochondrial enzyme phosphatidylserine decarboxylase (PISD) reduces stemness and increases differentiation in acute myeloid leukemia (AML)

Abstract Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by the accumulation of malignant myeloid cells that have arrested maturation. Most therapeutic regimens approved or under development are cytotoxics. An alternate, but less explored therapeutic approach, is to induce terminal differentiation of AML cells. Upon differentiation, AML cells cease to proliferate or die. Phosphatidylserine decarboxylase (PISD) is a mitochondrial enzyme that converts phosphatidylserine (PS) to phosphatidylethanolamine (PE). Here, we explored the effects of inhibiting PISD on AML growth, stemness and differentiation. Knockout of PISD by CRISPR reduced the growth and clonogenic growth of OCI-AML2 cells. The reported chemical PISD inhibitor, 7-chloro-N-(4-ethoxyphenyl)-4-quinolinamine (aka: MMV007285), reduced growth and viability of OCI-AML2 cells (IC50 = 4.741 μM) and TEX cells (IC50 = 4.868 μM). Using the 8227 primary AML cell culture model, we showed that inhibiting PISD induced cell death in the functionally defined stem cell fraction (CD34+CD38-). MMV007285 also preferentially inhibited the clonogenic growth of primary AML cells (n = 7) over normal hematopoietic cells (n= 3). Moreover, MMV007285 induced AML cell differentiation as evidenced by increased CD11b expression and staining for non-specific esterase. Using high-performance thin layer chromatography (HPTLC), we found that inhibition of PISD with MMV007285 increased intracellular PS. To determine whether increased PS was functionally important, OCI-AML2 cells were treated with PS, resulting in reduced growth and clonogenic growth. Furthermore, PS supplementation targeted AML progenitor cells as it decreased engraftment of TEX cells in mice. Mechanistically, inhibiting PISD induced differentiation and decreased stemness in AML by activating Toll-like receptor (TLR) signaling. Specifically, inhibiting PISD upregulated TLR4 and 8 expression and increased expression of cytokines downstream of TLR activation. We also showed that TLR activation was functionally important to induce AML differentiation. Finally, we evaluated the effects of PISD inhibition in AML mouse models. MMV007285 (300 mg/kg/5 of 7 days orally for 10 days) decreased the growth of OCI-AML2 cells in SCID mice. Moreover, MMV007285 (150 mg/kg/5 of 7 days orally for 5 weeks) impeded the leukemic engraftment of primary AML cell in NOD/SCID mice without toxicity. Using secondary transplants, we showed that MMV007285 also targeted the leukemic stem cells. Taken together, inhibition of PISD altered phospholipid metabolism, inhibited growth and stemness, and increased differentiation in AML cells. Our findings reveal a previously undescribed link between mitochondrial phospholipid metabolism and AML stemness and differentiation, highlighting a potential new therapeutic strategy for AML. Citation Format: Mingjing Xu, Ayesh Seneviratne, Val A. Fajardo, Geethu E. Thomas, G. Wei Xu, Rose Hurren, S. Kim, Neil MacLean, Xiaoming Wang, Marcela Gronda, Danny Jeyaraju, Yulia Jitkova, David Sharon, Ahmed Aman, Rima Al-awar, Steven Chan, Mark D. Minden, Paul LeBlanc, Aaron D. Schimmer. Inhibiting the mitochondrial enzyme phosphatidylserine decarboxylase (PISD) reduces stemness and increases differentiation in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3003.