Acute Myeloid Leukemia Progenitor 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.

Combining Allogeneic Gamma Delta T Cells with a Phosphoantigen Prodrug Promotes Apoptosis of Acute Myeloid Leukemia Stem and Progenitor Cells

Despite recent therapeutic advances, patients with relapsed/refractory acute myeloid leukemia (r/r AML) have limited treatment options and poor prognosis. Leukemic stem cells (LSCs) are considered as relapse-initiating cells and the “culprit” of treatment resistance. Allogeneic and autologous gamma delta (γδ) T cell-based therapies have been explored for a long time and proven to be safe in patients through multiple clinical trials. However, quite a few AML blasts obtained from r/r AML patients show resistance to γδ T cell-mediated cytotoxicity. Novel strategies targeted LSCs may improve efficacy of γδ T cell-mediated r/r AML therapy. Given that γδ T cells exert antitumor capacity via multiple pathways, to identify the way matters most in the resistance of γδ T cell-mediated cytotoxicity, we examined the median fluorescence intensity (MFI) of the ligands expressed in tumor cells by flow cytometry, including butyrophilin 3A (BTN3A), butyrophilin 2A1 (BTN2A1), the human MHC class I chain-related genes (MICA and MICB), the unique-long 16 binding proteins (ULBP1, ULBP2/5/6, ULBP3), Fas, the receptor of TNF-related apoptosis-inducing ligand (TRAIL) DR5, CD112 and CD155. The cytotoxicity activity of γδ T cells was measured by flow cytometry-based killing assay or bright-lite luciferase assay system after coculturing with AML cell lines and blasts at an appropriate effector to target (E:T) ratio. Then we employed a Support Vector Machine model to predict the killing efficiency and identified that BTN3A and BTN2A1 play the predominant role in γδ T cell-mediated cytotoxicity based on the Shapley additive explanation. BTN3A and BTN2A1 are the key ligands in the γδ TCR-phosphoantigen (pAg) recognition activity. PAgs, which are produced in tumor cells function as “molecular glues” to promote heteromeric association of BTN3A1 and BTN2A1 (Yuan L et al., BioRxiv 2022). To promote the γδ T cell-mediated cytotoxicity targeting r/r AML, here we synthesized a pAg prodrug, which show great plasma stability and membrane permeability. We anticipate this small molecule would lead pAg accumulation in tumor cells and cause increased γδ T cell activation. To identify the killing efficacy of pAg prodrug-induced γδ T cell activation, we select KG-1α (a human leukemia stem cell-like cell) as target cell. Zoledronate (ZOL), which is employed to stimulate γδ T cells in multiple γδ T cell-based clinical trials, is used in our killing assay as compared group. As expected, combining γδ T cells with pAg prodrug or ZOL both significantly improved the percent of specific killing when the E:T ratio is 1:1. The pAg prodrug showed more potent activity than ZOL group with EC50 value of 0.1 nM vs 17.93μM. And the specific killing percent of pAg prodrug group is 83.26±2.1 % (1 nM), comparing with negative control 15.17±3.4 % (only γδT cells). Futhermore, at relatively low E:T ratio (1:4), γδ T cells with pAg prodrug remain excellent cytotoxicity when targeting MV411 cell and OCI-AML3 cell, which are widely used in r/r AML research. After coculturing with primary leukemia stem and progenitor cells (CD34 +) obtained from r/r AML patients for 6~8 h, the apoptosis percentage of CD34 + leukemia cells was 42.34% comparing with negative control (only γδT cells) 2.53%. Then we evaluated the in vivo efficacy of combining γδT cells and pAg prodrug in NSG mice bearing MV411 cells. Compared with only γδT cells-treated group, combination with pAg prodrug presented better tumor clearing. To elucidate the pathway of pAg prodrug-mediated killing, we tested mitochondria apoptosis related events. After treated by γδ T cells with pAg prodrug, KG-1α cells showed loss of mitochondrial outer membrane potential and increasingly actived caspase 3/7. Meanwhile, pAg prodrug directly activates effector γδ T cells with increased secretion of CD107a and IFN-γ. Considering the possibility of toxicity towards normal hematopoietic stem and progenitor cells, we conducted colony-forming units (CFUs) assay. After treatment with γδT cells and pAg prodrug, the number of CFUs of CD34 + cells from cord blood was not effected compared with the CD34 + cells only group. Overall, we explored the predominant factor in γδ T cell-mediated killing and synthesized a pAg prodrug to improve the killing efficacy. We demonstrated that combining γδ T cells with pAg prodrug has effectively promoted apoptosis of AML stem and progenitor cells sparing normal hematopoietic stem and progenitor cells.

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).

The splicing factor RBM17 drives leukemic stem cell maintenance by evading nonsense-mediated decay of pro-leukemic factors

Chemo-resistance in acute myeloid leukemia (AML) patients is driven by leukemic stem cells (LSCs) resulting in high rates of relapse and low overall survival. Here, we demonstrate that upregulation of the splicing factor, RBM17 preferentially marks and sustains LSCs and directly correlates with shorten patient survival. RBM17 knockdown in primary AML cells leads to myeloid differentiation and impaired colony formation and in vivo engraftment. Integrative multi-omics analyses show that RBM17 repression leads to inclusion of poison exons and production of nonsense-mediated decay (NMD)-sensitive transcripts for pro-leukemic factors and the translation initiation factor, EIF4A2. We show that EIF4A2 is enriched in LSCs and its inhibition impairs primary AML progenitor activity. Proteomic analysis of EIF4A2-depleted AML cells shows recapitulation of the RBM17 knockdown biological effects, including pronounced suppression of proteins involved in ribosome biogenesis. Overall, these results provide a rationale to target RBM17 and/or its downstream NMD-sensitive splicing substrates for AML treatment.

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.

CD33-directed immunotherapy with third-generation chimeric antigen receptor T cells and gemtuzumab ozogamicin in intact and CD33-edited acute myeloid leukemia and hematopoietic stem and progenitor cells.

Immunotherapies, such as chimeric antigen receptor (CAR) modified T cells and antibody-drug conjugates (ADCs), have revolutionized the treatment of cancer, especially of lymphoid malignancies. The application of targeted immunotherapy to patients with acute myeloid leukemia (AML) has been limited in particular by the lack of a tumor-specific target antigen. Gemtuzumab ozogamicin (GO), an ADC targeting CD33, is the only approved immunotherapeutic agent in AML. In our study, we introduce a CD33-directed third-generation CAR T-cell product (3G.CAR33-T) for the treatment of patients with AML. 3G.CAR33-T cells could be expanded up to the end-of-culture, that is, 17 days after transduction, and displayed significant cytokine secretion and robust cytotoxic activity when incubated with CD33-positive cells including cell lines, drug-resistant cells, primary blasts as well as normal hematopoietic stem and progenitor cells (HSPCs). When compared to second-generation CAR33-T cells, 3G.CAR33-T cells exhibited higher viability, increased proliferation and stronger cytotoxicity. Also, GO exerted strong antileukemia activity against CD33-positive AML cells. Upon genomic deletion of CD33 in HSPCs, 3G.CAR33-T cells and GO preferentially killed wildtype leukemia cells, while sparing CD33-deficient HSPCs. Our data provide evidence for the applicability of CD33-targeted immunotherapies in AML and its potential implementation in CD33 genome-edited stem cell transplantation approaches.

Abrogation of a Histone Chaperone Pathway Mitigates Inflammation-Driven AML Progression

Background: Genetic heterogeneity makes clinical interventions challenging for acute myeloid leukemia (AML) patients. Identifying and targeting microenvironment-driven pathways that are active across AML genetic subtypes should allow the development of more broadly effective therapies. Previously, we have shown that AML microenvironment is rich in proinflammatory cytokine interleukin-1β (IL-1β) and significantly promotes the growth of AML progenitors while suppressing healthy progenitors. To elucidate this paradoxical effect, we performed transcriptome (RNA-seq) analysis from IL-1β-stimulated CD34+ AML and normal progenitors and found that ASF1B (anti-silencing function-1B) is one of the most differentially expressed genes. ASF1B is a histone chaperone, which recruits H3-H4 histones onto the replication fork during S-phase. This process is regulated by tousled-like kinase 1 and 2 (TLKs). TLKs and ASF1B are overexpressed in multiple solid tumors and associated with poor prognosis. However, their functional roles in hematopoiesis and inflammation-driven leukemia are unexplored. Here, we reveal a novel molecular mechanism that IL-1β promotes leukemia progression by activating the TLK-ASF1B pathway.Methods and Results: We first confirmed that IL-1β stimulation upregulates ASF1B expression at both mRNA and protein levels in FLT3-ITD, MLL-ENL, and NPM1 positive primary AML samples. ASF1B upregulation is abolished upon treatment with a p38MAPK inhibitor, further suggesting that ASF1B is downstream of IL-1β/p38 signaling. Next, we stably knocked down ASF1B in an AML cell line MOLM-14 using doxycycline-inducible shRNA. ASF1B-depleted AML cells exhibited reduced cell viability, inhibited growth, blocked cell cycle progression, and impaired colony formation ability. We xenografted shASF1B expressing AML cells into NSG mice and induced knockdown in vivo. Flow cytometry analysis of bone marrow cells 3 weeks post-engraftment showed 80 percent reduction in leukemia burden following ASF1B silencing compared to controls.To determine whether upregulation of ASF1B contributes to IL-1β-driven leukemic growth, we overexpressed ASF1B with MLL-ENL oncogene in a murine bone marrow transplantation model. We found that daily IL-1β exposure accelerated leukemia progression compared to vehicle-treated group (median survival = 64 vs. 85 days, p<0.05), and this effect was phenocopied by overexpression of ASF1B (median survival = 62 vs. 85 days, p<0.05). Conversely, heterozygous and complete Asf1b deletion in the MLL-ENL AML model delayed the leukemia progression compared to wildtype mice. Furthermore, Asf1b deletion attenuated IL-1β-mediated AML progression compared to wildtype controls (median survival = 63 vs. 47 days, p <0.01). Immunophenotyping of Asf1b-deficient mice using flow cytometry suggested that ASF1B is dispensable for normal hematopoiesis. Together, these data suggested that targeting of the ASF1B pathway may spare healthy cells. Additionally, we found that TLK2 which regulates ASF1B activation, is also upregulated in AML progenitors compared to healthy cells at the baseline levels and upon IL-1β stimulation. We next knocked down TLKs in human AML cells and observed a similar pattern with growth arrest, higher replication stress and DNA damage response. Finally, we generated Vav-Cre+ Tlk2 mice allowing Tlk2 deletion only in hematopoietic cells and found that Tlk2 deletion prolongs survival of leukemic mice in a dose dependent manner with and without IL-1β stimulation.Conclusions: We demonstrate that increased TLK-ASF1B expression promotes survival of AML cells. We also provide the first in vitro and in vivo evidence that the TLK-ASF1B pathway plays a critical role in potentiating IL-1β-dependent AML growth. Therefore, we establish TLK-ASF1B pathway as a novel route for therapeutic strategy to suppress inflammation-driven growth in various AML genetic subtypes. DisclosuresNo relevant conflicts of interest to declare.

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.

MLN0128, a Potential Target Therapy for Myeloid Proliferation Neoplasia (MPN) with a Benefit of Prolonging Survival

MLN0128 (TAK-228, INK128) is an investigational new drug (IND) that is currently being evaluated in clinical trials and has showed promising efficacies in patients with solid cancer. MLN0128 is one of the second generation of inhibitors for mammalian target of rapamycin (mTORi), which suppresses the dysregulated cell survival signaling activities in cancer cells by specifically inhibiting both mTORC1 and mTORC2 in PI3K/mTOR pathway. MLN0128 was reported inhibiting acute myeloid leukemia (AML) stem cells and progenitors [Zeng et al, Oncotarget, 2016]. However, it is yet to be clarified whether MLN0128 can improve survival time in leukemia patients. Here, we report data from the investigation of the impact of MLN0128 on leukemia survival in mice. We tested MLN0128 in a published mouse model which simultaneously produces experimental mice with genotypes that are associated with pediatric leukemia, including myeloproliferative neoplasm (MPN) and juvenile myelomonocytic leukemia (JMML), and adult chronic myelomonocytic leukemia (CMML) [Vara et al, Blood Adv.2020]. Specifically, mice with deletion of Phosphatase and tensin homolog gene (Pten) and loss-of-heterozygosity of Neurofibromin 1 (Nf1) (Pten Δ/ΔNf1 LOH) develop JMML, mice with Pten deletion and Nf1 heterozygosity (Pten Δ/ΔNf1 Δ/wt) develop pediatric MPN, both of which cause juvenile death; mice with a single copy of Pten deletion and Nf1 LOH (Pten Δ/wtNf1 LOH) develop CMML in adulthood. We previously reported that all of those diseased mice shared a feature with elevated activities in PI3K/MAPK/mTOR pathways. We first collected nucleated cells from bone marrow (BM) of the diseased mice and tested the inhibition of MLN0128 on colony formation of hematopoietic progenitors (CFU-GM) at a concentration range of 1-1000nM. Our data demonstrated that MLN028 significantly inhibited the CFU-GM colony formation of BM cells from mice with all three diseased genotypes, in a dose-dependent fashion. Importantly, the IC50 for CFU-GM was less than 100nM, which is in the concentration range of MLN0128 in the blood from patients who orally received MLN0128 at dosage range of 4-12mg/day [Ghobrial et al AJH 2016]. We then investigated the impact of MLN0128 on life span in diseased mice. We administrated MLN0128 in gavage at dosages of 0.5-1μg/g body weight, starting at an age of postnatal day 17-18 (PND17-18), 3-5 times a week, repeated for 5 weeks followed by a pause in 6 th week every course. Our data showed that MLN0128 significantly reduced the spleen sizes in treated mice with all diseased genotypes comparing to those treated with vehicle. The survival time was significantly prolonged in MLN0128-treated mice with genotypes of MNP (n=7 vs 3, median 150 vs 102 days) and CMML (n=7 vs 6, median 100 vs 56 days), comparing with the mice treated by vehicle. Unfortunately, we did not observe a survival improvement in treated JMML mice, but substantially swollen kidneys were found in the dead JMML mice, which was likely due to tumor-lysis syndrome because JMML mice carried a significantly worse leukemic burden. In conclusion, our data demonstrate that MLN0128 can significantly inhibit colony formation of BM cells from mice with genotypes of JMML, MPN, and CMML; and MLN0128 can significantly expand survival time of mice with genotypes of MPN and CMML, which has never been reported in previous studies that were related to mTORi in pediatric leukemia. Our data suggest that MLN0128 may be a potential target therapy for pediatric MPN and CMML with a benefit of prolonging survival. Further investigation is ongoing to evaluate the potential of MLN0128 in improving survival of JMML mice after minimizing the tumor-lysis syndrome by tipping down MLN0128 dosage. DisclosuresBertaina: Cellevolve Bio: Membership on an entity's Board of Directors or advisory committees; Neovii: Membership on an entity's Board of Directors or advisory committees; AdicetBio: Membership on an entity's Board of Directors or advisory committees.

Isoform Selective HDAC8 Inhibitor (OCH3) Shows Potency in Selectively Targeting Primary AML Cells

The treatment outcomes for patients diagnosed with acute myeloid leukemia (AML) are still dismal. Recent advances in understanding AML indicate that the lack of efficacy is primarily due to non-specificity of currently used chemotherapeutics targeting both leukemic stem/progenitor cells (LSC) and normal hematopoietic stem cells (HSC). Thus, a critical barrier is the identification of innovative therapies that selectively target LSC. Histone deacetylase 8 (HDAC8) has been shown to enhance p53 protein deacetylation, which results in inactivation of p53, promoting LSC survival. We hypothesize that enzymatic/non-enzymatic role of HDAC8 is critical for LSC survival but not for HSCs. Then, we characterized our two tetrahydroisoquinoline (TIQ)-based selective HDAC8 inhibitors (HDAC8i) BIP and OCH3 for growth inhibition, apoptosis, activation of caspase 3, integrity of mitochondrial membrane potential (MMP), and acetylation of histone H4 in human leukemia cell lines. The growth inhibitory effects observed in cell lines were validated using bone marrow (BM) or peripheral blood (PB) cells from AML patients. Colony forming cell (CFC) assays were performed using AML BM/PB cells treated with OCH3 or BIP. OCH3 and BIP were also tested for hematotoxicity using normal CB CD34+ cells. Furthermore, we compared class I HDAC isoform engagement in human normal cord blood (CB) CD34+ cells and in SET-2 leukemia cells using our novel photoreactive probe TH1143. In CD34+ cells, TH1143 had higher level of engagement for HDAC1 and 2, whereas engagement of HDAC3 and 8 was minimal. In SET-2 cells, HDAC3 and HDAC8 displayed relatively higher engagement with TH1143 indicating HDAC engagement is likely cell type specific.The biological efficacies of OCH3 at 50uM and BIP at 25uM were noted to exert >50% growth inhibition in KG1 and in K562 leukemia cells. Both OCH3 and BIP significantly increased the number of apoptotic cells and there was an enhanced active caspase-3 activity. Furthermore, OCH3 and BIP treated cells displayed lower red/green ratio in comparison to control, indicative of poor MMP and depolarization to induce apoptosis (Table 1.a). OCH3 and BIP were further validated by using BM/PB cells from AML patients showing growth inhibition. This was also accompanied by increase in apoptotic cells by OCH3 and BIP. In contrast to BIP, OCH3 spared CB CD34+ cells as demonstrated by notably lower growth inhibition, apoptotic cells vs control when compared with primary AML cells from patients. Both OCH3 and BIP displayed minimal inhibition of CFU growth in CD34+ cells. However, HDAC8i induced significant CFU growth inhibition in primary AML samples suggesting that HDAC8i spares normal CFU progenitors but not leukemia progenitors (Table 1.b). Notably, both BIP and OCH3 lack ability to exert acetylation of histone H4, unlike broad spectrum HDAC inhibitor TSA (MFI with OCH3=0.96±0.03, BIP=0.77±0, TSA =1.63±0.15) which is consistent with isoform selectivity of OCH3 and BIP.The leukemia growth inhibitory effects at LSC level was demonstrated using ex vivo OCH3 treated AML patient derived BM/PB cells transplantation in humanized immunodeficient NSGS mice. After 10 to 12 weeks of transplantation mice receiving untreated AML cells had 7.73±2.18% while with OCH3 treatment mice had 4.84±1.37% human CD34+ leukemia cells, a 38% reduction in CD34+ leukemia cells, despite only a single ex vivo exposure to OCH3. Furthermore, in a second model, NSGS humanized mice were transplanted (IV) with primary leukemia cells from AML patients and after 4 weeks injected (IP) with OCH3 or vehicle control. After 12 weeks of transplantation in this second model human primary AML cell burden was 5.74±1.31% (OCH3) and 18.13±12.76% (vehicle control), while mice transplanted with normal CD34+ cells treated similarly with OCH3 or vehicle control displayed no detectable inhibition of human myeloid cell chimerism (OCH3:12.28 ± 3.31% vs vehicle control: 17.92±11.96%).Taken together, our data indicate that HDAC8 isoform inhibitor, OCH3 displayed significant inhibition of primary AML patient derived leukemia cells growth in vitro and in vivo in contrast to normal CD34+ cells. Selective inhibition of HDAC8 is sufficient to cause growth inhibition in primary AML progenitors including LSCs in vivo while sparing normal HSCs thus offer opportunities for further development of HDAC8i as new experimental therapeutics in AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mutation Signatures of Pediatric Acute Myeloid Leukemia and Normal Blood Progenitors Associated with Differential Patient Outcomes

Acquisition of oncogenic mutations with age is believed to be rate limiting for carcinogenesis. However, the incidence of leukemia in children is higher than in young adults. Here we compare somatic mutations across pediatric acute myeloid leukemia (pAML) patient-matched leukemic blasts and hematopoietic stem and progenitor cells (HSPCs), as well as HSPCs from age-matched healthy donors. HSPCs in the leukemic bone marrow have limited genetic relatedness and share few somatic mutations with the cell-of-origin of the malignant blasts, suggesting polyclonal hematopoiesis in pAML patients. Compared to normal HSPCs, a subset of pAML cases harbored more somatic mutations and a distinct composition of mutational process signatures. We hypothesize these cases might have arisen from a more committed progenitor. This subset had better outcomes than pAML cases with mutation burden comparable to age-matched healthy HSPCs. Our study provides insights into the etiology and patient stratification of pAML.