Primary Human Acute Myeloid Leukemia Samples Research Articles

Identification of Nonfunctional Alternatively Spliced Isoforms of STING in Human Acute Myeloid Leukemia.

We find that AML acquires resistance to innate immune activation via the STING pathway through aberrant splicing of the STING transcript including two novel forms described herein that act as dominant negatives. These data broaden understanding of how cancers evolve STING resistance, and suggest that the AML tumor microenvironment, not the cancer cell, should be the target of therapeutic interventions to activate STING.

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.

Identification of Acute Myeloid Leukemia Cell Surface Therapeutic Targets Using Single Cell RNA Sequencing Supported By Surface Proteomics

Background: Acute myeloid leukemia (AML) comprises diverse genomic subgroups and remains hard to treat in most patients. Despite breakthroughs in the therapeutic arsenal in recent years, clinical usage of therapeutic antibodies or chimeric antigen receptor T (CAR-T) cells has been lagging in contrast to other hematological malignancies. In fact, CD33 represents the only antibody-based strategy approved for this disease to date, highlighting the need to identify new promising targets. AML cells span a wide range of aberrant myeloid differentiation programs, complexifying the identification, by bulk genomics, of targets expressed in the most immature leukemic cells. Aims and Methods: To identify the expression landscape of surface proteins in immature leukemic cells, we performed single-cell RNA sequencing (scRNA-seq, 10x 3' Reagent Kits) of primary human AML cells from 20 specimens of the Leucegene cohort enriched in intermediate and adverse genetic backgrounds ( KMT2A-rearranged n=5, chromosome 5 and/or 7 deletions (abn5/7, n=5) complex karyotype (n=4), NPM1/DNMT3A/FLT3-ITD triple-mutant (n=3) and others (n=3)). A Random Forest classifier was developed to unbiasedly classify AML cells into distinct differentiation stages using normal bone marrow-derived scRNA-seq data from the Human Cell Atlas (HCA) consortium. Genes were scored based on their probability of coding for proteins expressed at the cell surface using the SPAT algorithm developed by our group (https://doi.org/10.1101/2023.07.07.547075), retaining high score ones. To validate surface expression, we concomitantly analyzed the surface proteome (hereafter named surfaceome) of 100 primary human AML samples from the Leucegene cohort, including all 20 samples profiled by scRNA-seq. Results: After quality control, we profiled and characterized 103 690 high quality cells (mean of 5185 cells/sample). We trained a Random Forest classifier to annotate cells in a two step process, first identifying plasma cells based on a restricted list of genes abundantly expressed in these cells and subsequently assigning the remaining cells to one of 33 cell types. We performed a five-fold cross validation of the model and subsequently determined the accuracy of our classifier to be 92% on the test subset of the HCA data. Applied to our AML cell collection, a total of 35 053 cells (34%) were unbiasedly classified as Hematopoietic Stem Cell (HSC)-like, corresponding to the most phenotypically immature leukemic cells in each patient sample (ranging from 4 to 74 %). Accordingly, HSC-like AML cells preferentially express genes associated with normal HSCs, such as CD34, FAM30A, and SPINK2, and globally lack expression of mature lineages defining genes, further validating our classifier. The proportion of HSC-like cells varied among AML subgroups, and was lowest in KMT2A-r AML (median 19%) and highest in abn5/7 samples (46%). Integration of our AML atlas using Harmony algorithm preserved differentiation hierarchies across samples, with most cell types, including HSC-like cells, occupying a defined area in the low dimensional embedding. To identify new surface antigens specifically expressed in immature leukemic cells, we compared the high (≥8) SPAT score gene expression profile of AML HSC-like cells with that of normal HSC cells (HCA), and identified 60 genes significantly overexpressed in AML immature cells. Of those, 39 genes were also detected at the protein level by the surfaceome analysis, supporting their predicted expression at the cell surface in AML samples. 59% of these 39 genes (n=23) were detected in over 80% of the specimens analyzed by the surfaceome, and thus are nearly universally expressed in our AML cohort. To identify targets of therapies that could be repurposed, we next evaluated the relevance of our findings by querying the Thera-SAbDab database. Most interestingly, 8 of the 39 AML specific HSC markers are targeted by therapeutic antibodies FDA-approved or in clinical trials for the treatment of AML (n=4, IL3RA, FLT3, CD37 and TNFRSF10B) or other indications (n = 4). Conclusion Our genetically diverse AML single-cell atlas, supported by mass spectrometry, enables the identification of both subset-specific and pan-AML surface protein genes. These represent potential targets for antibody based strategy development or therapy repurposing in AML.

Abstract PR01: Novel anti-leukemia activity of the IRE1-XBP1 signaling pathway in hematopoietic stem and progenitor cells

Abstract Most patients with acute myeloid leukemia (AML) initially achieve clinical remission after chemotherapy but eventually die from disease relapse as they succumb to chemotherapy-resistant AML cells, including the rare fraction of leukemia stem cells (LSC) that persist and often reinitiate a more aggressive disease. While eliminating LSCs has become a desirable strategy to achieve durable AML remission, much remains unknown about LSC vulnerabilities and the specific cellular programs that enable their disease activity. Spurred by increasing evidence that disruptions in proteome homeostasis or “proteostasis” impair stem cell integrity, we investigated how acute myeloid leukemogenesis is impacted by the inositol-requiring enzyme-1 alpha (IRE1a), the most conserved enzyme of the endoplasmic reticulum unfolded protein response that resolves proteotoxic stress in eukaryotic cells. Activated IRE1a catalyzes an unconventional splicing of the XBP1U mRNA to generate an XBP1S transcript that encodes the transcription factor XBP1 (X-box binding protein-1), the main effector of IRE1a-transduced signals. Using transcriptomics and genomics to profile the activation and function of IRE1a in primary human AML samples and murine AML models, we identified the IRE1a-XBP1 axis as a cell-intrinsic brake against pro-myeloid leukemogenic programs in hematopoietic stem and progenitor cells (HSPC). Notably, compared to blast cells from the same AML patient, LSCs expressed significantly reduced levels of the genes encoding IRE1a (ERN1) and its spliced product XBP1S suggesting that downregulation of IRE1a-XBP1 activity promotes LSC activity and acute myeloid leukemogenesis. Consistent with this hypothesis, genetic induction of XBP1S-encoded XBP1 in patient-derived AML cells suppressed their LSC transcriptional program, impaired their clonogenic capacity, and limited their ability to cause AML in immunodeficient animals. By contrast, genetically engineered IRE1a or XBP1 insufficiency enriched the LSC signature in HSPCs and cooperated with the myeloproliferative oncogene FLT3-ITD to initiate a lethal AML disease in mice. Integrated RNA-Seq and ChIP-Seq of XBP1 target genes in HSPCs revealed a mechanism in which IRE1a-induced XBP1 functions as a potent transcriptional repressor of signature pro-LSC gene programs, exemplified by the Wnt/beta-catenin pathway. Accordingly, we defined an independently prognostic “18-gene” signature of XBP1-repressed beta-catenin target genes that when highly expressed correlated with poor survival outcomes in multiple cohorts of AML patients. Taken together, our findings uncover a novel anti-leukemia function for IRE1a-XBP1 signaling that restrains acute myeloid leukemogenesis in HSPCs and suggest that this pathway is likely subverted in LSCs to facilitate AML initiation and progression. Our results further suggest that IRE1a activation is a vulnerability in AML stem cells that can be harnessed therapeutically to achieve durable disease remission. Citation Format: Brendan M Barton, Francheska Son, Akanksha Verma, Laurie Glimcher, Stanley Adoro. Novel anti-leukemia activity of the IRE1-XBP1 signaling pathway in hematopoietic stem and progenitor cells [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 PR01.

Identification of Novel Cell Surface Therapeutic Targets for KMT2A-Rearranged Acute Myeloid Leukemia

INTRODUCTION. Acute myeloid leukemia (AML) is a highly heterogeneous disease that represents 80% of adult leukemias and is characterized by chromosomal rearrangements such as translocations and inversions, as well as genetic mutations. These genomic alterations affect the normal maturation process of myeloid precursor cells and lead to their clonal expansion in the bone marrow and peripheral blood. Genetic aberrations define AML subgroups, which are associated with risk stratification (favorable, intermediate or adverse risk) and prognosis. One AML subgroup, KMT2A-rearranged AML (KMT2Ar), is driven by chromosomal translocations involving the lysine methyltransferase 2A gene (KMT2A) located on chromosome band 11q23.3, leading to gene fusions with over 94 distinct partners. KMT2A rearrangements are present in 5 to 10% of adult AML. Patients with t(9;11)(p21.3;q23.3) translocation generating the MLLT3::KMT2A fusion are classified in the intermediate cytogenetic risk group according to the 2022 ELN genetic risk classification, but KMT2A fusions with other translocation partners are included in the adverse risk group with current therapies. Ongoing clinical trials for KMT2Ar AML focus mainly on chemical inhibitors (DOT1L and Menin inhibitors) that target and disrupt KMT2A fusion protein complexes, with encouraging results. Precision medicine, such as antibody-based immunotherapy, also represents a promising approach for the treatment of KMT2Ar AML, but is currently underexploited due to the limited availability of targetable surface antigens. To overcome this issue, we analyzed the cell surface proteome (surfaceome) of primary human KMT2Ar AML samples to identify novel cell surface antigens with high therapeutic potential. METHODS AND RESULTS. We used a proteomics-based approach to analyze the surfaceome of 100 primary human AML specimens from 13 different AML subgroups, including 13 KMT2Ar AML samples. Briefly, we combined cell surface biotinylation with streptavidin affinity purification to enrich cell extracts with cell surface proteins, which were then analyzed by LC-MS/MS to obtain an overview of primary AML sample surfaceomes. Differential surfaceome analyses were performed to identify surface proteins selectively expressed by KMT2Ar AML specimens, and proteins with low expression in normal hematopoietic cells and normal tissues, as determined by expression analyses using publicly available datasets, were further selected. Using this approach, we identified several potential KMT2Ar AML surface antigens, including CD93, reported to be expressed by KMT2Ar AML stem cells. We then analyzed surface expression of these antigens by flow cytometry using a collection of 112 primary human AML specimens including 10 KMT2Ar AML samples. Results indicate that in the majority of KMT2Ar AML samples analyzed, more than 90% of blasts express selected antigens, suggesting that targeting these antigens with immunotherapeutic approaches could potentially lead to the elimination of most AML cells. Furthermore, flow cytometry data showed specificity of antigen expression for KMT2Ar AML samples, opening new therapeutic opportunities for this AML subgroup. Finally, single cell RNA sequencing of 8 primary KMT2Ar AML specimens was performed to further analyze the expression profile of these antigens in patient samples. AML specimens harboring the MLLT3::KMT2A fusion expressed these antigens in the vast majority of blast cells, confirming the homogeneous expression profile of identified antigens and the high interest to target these antigens for KMT2Ar AML. CONCLUSIONS Using a cell surface proteomics-based approach for the analysis of KMT2Ar AML surfaceome, we identified several antigens for this AML subgroup with high therapeutic potential. These antigens are very specific for KMT2Ar AML and are expressed homogeneously in primary human KMT2Ar AML samples, suggesting that targeting these antigens represents a promising therapeutic strategy for these patients. Several immunotherapeutic approaches to target these antigens are currently being developed and tested by our group, with the hope of identifying novel therapeutic strategies for the treatment of KMT2Ar AML.

TP53 Altered Acute Myeloid Leukemia Stem Cells Hyperactivate Nuclear Factor-Kappa B

In acute myeloid leukemia (AML), standard therapies induce complete remission in 50-70% of patients, but the overall two-year survival is less than 20-30% because of high relapse rates. AML with mutations or deletions in TP53 (TP53 altered, TP53Alt) is a poor-risk AML subtype that is largely insensitive to chemotherapy, modern targeted agents, and hematopoietic stem cell transplantation. TP53Alts are seen in approximately 10-20% of AML and confer a particularly poor prognosis decreasing the 1-2-year survival rates to 0-10%. Leukemia stem cells (LSCs), the cells that recapitulate and propagate leukemia, are central drivers of leukemia progression and relapse. Therapeutically targeting LSCs is important to curing AML, but LSCs are poorly proliferative in contrast to bulk AML cells and are less sensitive to standard chemotherapy. Despite a broad literature defining the immunophenotype (the cell surface marker expression profile) and mechanisms of self-renewal of LSCs, the TP53 status has not been reported in most studies of human LSCs. Our goal is to define the immunophenotype and molecular states of primary human TP53Alt LSCs. First, we asked whether the LSC immunophenotype of TP53Alt AML is consistent with that of the previously reported LSC immunophenotype in AML. We sorted primary human TP53Alt AML samples and plated cells from sorted compartments in serial colony forming assays (which serve as an in vitro surrogate for self-renewal). We found that the Lineage-CD34+CD38-compartment is enriched in serial colony forming capacity in four primary human TP53Alt samples tested. In another TP53Alt sample, we found that the Lineage-CD34+CD38any compartment was enriched for serial colony formation. In vivo engraftment studies recapitulated these findings in one of the TP53Alt AML sample we tested. In the two CD34-TP53Alt AML samples studied the lineage-CD123+ compartment was enriched in serial colony forming capacity. Together, these findings suggest that the standard AML LSC markers also identifies the LSC compartment of TP53Alt AML. Next, we used mass cytometry (CyTOF) to define the proliferative and signaling states of human TP53Alt AML samples. Previous work has demonstrated that the LSC compartment of AML is poorly proliferative, relative to the bulk leukemia population. Consistent with these findings, we found low levels of Ki67 (a proliferation marker) expression, in TP53Alt LSCs, relative to bulk leukemia cells, in every primary TP53Alt AML that we profiled (n=7). Notably, we found that TP53Alt LSCs express high levels of pNFkB, p4EBP1, and pSTAT5, suggesting that these signals may mediate self-renewal in TP53Alt AML. Indeed, we found that CD69, an NFkB target gene, is elevated in TP53Alt LSCs and minimally expressed in bulk leukemia cells. Furthermore, we found that Ki67 levels are low in the CD69High subset, suggesting that this is a poorly proliferative subset (consistent with having self-renewal capacity). Finally, we sorted primary samples according to CD69 status and found that CD69marks a subset with enhanced serial colony forming capacity. Together, these findings demonstrate that traditional LSC markers can be used to study TP53Alt LSCs and that NFkB activation is correlated with self-renewal function in these LSCs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Relocalization of PU.1 Underlies the On- and Off-Tumor Effects of SWI/SNF Blockade in AML

SWI/SNF ATP-dependent chromatin remodeling activity has been identified as a strong dependency of acute myeloid leukemia (AML). In AML cell lines, genetic knockdown or chemical inhibition of the SWI/SNF ATPases SMARCA4 (BRG1) or SMARCA2 (BRM) by the selective small-molecule inhibitor BRM014 reduces proliferation and induces leukemic differentiation. These effects arise because SWI/SNF inhibition reduces DNA accessibility at binding sites of PU.1, a factor essential for driving high levels of MYC in leukemic cells. However, given that both SWI/SNF and PU.1 are essential in hematopoietic development, important questions regarding the use of SWI/SNF inhibitors against AML remain. First, the chromatin features that influence SWI/SNF-dependent PU.1 regulation within healthy hematologic and leukemic cells have not been established. Furthermore, the ability of SWI/SNF inhibitors to target leukemic cells within the bone marrow has remained uncertain, and the effects of these drugs in an immunocompetent setting have not been reported, despite the essential functions of SWI/SNF and PU.1 in normal hematopoiesis. Finally, the sensitivity of primary human AML samples and normal hematopoietic cells has also not been established. As a result, many questions regarding the nature of the therapeutic window for SWI/SNF inhibition against AML have remained unresolved. Here we evaluate SWI/SNF inhibition using BRM014 in clinically relevant models and examine the consequences of the deregulation of SWI/SNF-dependent PU.1 sites. We demonstrate that inhibition of SWI/SNF shifts PU.1 occupancy from enhancers to promoters in both leukemic and healthy hematopoietic cells. In AML, the shift of PU.1 away from enhancers to the promoters of differentiation-related genes results in loss of MYC expression and differentiation of leukemic cells. In non-leukemic mice, SWI/SNF inhibition suppresses PU.1-dependent B cell development and bone marrow-derived monocyte populations, reflecting off-tumor effects caused by SWI/SNF-dependent PU.1 relocalization. These effects influence peripheral immune cells, as well lineage-committed hematopoietic progenitors within the bone marrow. Moreover, we observe profound therapeutic response to SWI/SNF inhibition in an immunocompetent AML mouse model (Figure 1). In vivo, BRM014 induces differentiation of peripheral leukemic blasts and reduces the leukemic stem cell burden in bone marrow but also induces lymphocytopenia due to its off-tumor effects on PU.1. Nevertheless, the robust regression of leukemic burden seen in vivo over a short two-week treatment period provides a compelling justification for continued study of SWI/SNF inhibitors in the treatment of AML. Further, response of primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) and AML specimens highlight that normal hematopoiesis is affected at concentrations similar to those needed for therapeutic response in AML (Figure 2). Hence, hematopoietic and immune-related side effects, particularly at higher doses and over the duration of treatment, will be important considerations for clinical use of SWI/SNF inhibitors against AML and other tumors. Overall, our results reveal a variable therapeutic window for SWI/SNF blockade in AML and highlight important off-tumor effects of such therapies in immunocompetent settings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The tumor-specific antigen landscape of acute myeloid leukemia

In theory, tumor-specific antigens (TSAs) represent ideal targets for cancer immunotherapy, but none have been identified in acute myeloid leukemia (AML) so far. We have recently designed a proteogenomic approach allowing the identification of MHC class I-associated peptides deriving from any reading frame of all genomic regions (not only from exons). Using this approach, we could identify 82 TSAs from 18 primary human AML samples. All these TSAs derived from unmutated transcripts which were aberrantly expressed by allegedly non-coding regions of the genome (that would have been missed by conventional exome-based approaches). In agreement with previous reports showing that high intron retention is a key feature in AML, we found that introns were the main source of TSAs (54.8%). The TSAs were presented by 24 different HLA allotypes, together covering 98,04% of the world population. The examination of the expression of the RNAs coding for the TSAs showed that most of them (68%) were expressed by at least 50% of AML samples in the “The Cancer Genome Atlas” (TCGA) cohort while all of them were either expressed at very low levels or not expressed at all in normal tissues (from the “Genotype-Tissue Expression” (GTEx) project). We have yet to discover the specific epigenetic changes leading to TSA expression in cancer cells. Meanwhile, we propose that vaccination against ab- errantly expressed TSAs could be used to treat a vast majority of patients, with minimal risks of collateral damage to healthy normal tissues.

Targeting the lysine-specific demethylase 1 rewires kinase networks and primes leukemia cells for kinase inhibitor treatment.

Most tumor types either fail to respond or become resistant to kinase inhibitors, often because of compensatory prosurvival pathways in the cancer cell's broader signaling circuitry. Here, we found that intrinsic resistance to kinase inhibitors in cultured primary acute myeloid leukemia (AML) cells may be overcome by reshaping kinase networks into topologies that confer drug sensitivity. We identified several antagonists of chromatin-modifying enzymes that sensitized AML cell lines to kinase inhibitors. Of these, we confirmed that inhibitors of the lysine-specific demethylase (LSD1; also known as KDM1A) rewired kinase signaling in AML cells in a way that increased the activity of the kinase MEK and that broadly suppressed the activity of other kinases and feedback loops. As a result, AML cell lines and about half of primary human AML samples were primed for sensitivity to the MEK inhibitor trametinib. Primary human cells with KRAS mutations and those with high MEK pathway activity were the best responders to sequential treatment with LSD1 inhibitors then trametinib, whereas those with NRAS mutations and high mTOR activity were poor responders. Overall, our study reveals the MEK pathway as a mechanism of resistance to LSD1 inhibitors in AML and shows a way to modulate kinase network circuitry to potentially overcome therapeutic resistance to kinase inhibitors.

Proteasome Inhibition Attenuates Self-Renewal in Human Acute Myeloid Leukemia By Targeting NF-Kappa B in Leukemia Stem Cells

In acute myeloid leukemia (AML), relapse following standard chemotherapy is common, leading to 2-year survival rates of less than 30%. Relapse is caused by leukemia stem cells (LSCs), a rare population of mostly quiescent cells that are chemo-refractory and can recapitulate the disease. Our overall goal is to define mechanisms of self-renewal that could be targeted to cure AML and prevent relapse. Functionally, self-renewal is defined by the ability to propagate leukemia in vivo.We use transcriptional and protein profiling to define the functional states of LSCs. We previously demonstrated that NRAS G12V facilitates self-renewal in a mouse model of AML (Mll-AF9/NRAS G12V). Using single-cell RNA sequencing and in vivo leukemia assays, we showed that the stem cell compartment in this model has two distinct subpopulations which differ in their self-renewal and proliferative abilities. The subset of LSCs marked by CD36 LowCD69 High (CD69 High) expression can self-renew and are poorly proliferative. The CD36 HighCD69 Low (CD36 High) subset is unable to self-renew and is highly proliferative. These data demonstrated that self-renewal and rapid proliferation are mutually exclusive functions among LSCs. We demonstrated that the gene expression profiles associated with these functionally distinct LSC subsets are also similarly differentially expressed in primary human LSCs, at the single-cell level (Sachs Cancer Research 2020). Given the functional differences between the CD36 LowCD69 High and CD36 HighCD69 Low subsets in murine AML, we tested whether CD69 and CD36 likewise discriminate self-renewal and proliferation in human AML. We sorted primary human AML samples according to CD69 and CD36 expression and found that Lin -CD69 High cells formed more colonies than Lin -CD36 High cells in every sample tested (n=6). Notably, in three of the six samples, the Lin -CD36 High population was unable to form any colonies (Panel A). These data suggest that CD69 may identify a self-renewing subset of human AML as it does in murine AML. Next, we asked whether Lin -CD69 High and Lin -CD36 High subsets harbor unique signaling protein activation profiles that could potentially be targeted therapeutically. We used CyTOF (mass cytometry) to compare the levels of 12 intracellular signaling proteins between these subpopulations in 14 human AML samples with intermediate and poor risk genetics. CyTOF quantitatively measures a panel of proteins at the single-cell level and allows us to profile activated signaling pathways within well-defined immunophenotypic subpopulations. We found upregulation of both total and phosphorylated (t- and p-) NF-kappa B, pERK, p4EBP1, pMAPKAPKII, and its upstream activator, pP38 in the Lin -CD69 High subset relative to the Lin -CD36 High. Notably, CD69 is a NF-kappa B target gene. Indeed, our single-cell transcriptional data showed that NF-kappa B target genes were upregulated in the self-renewing, CD69 High subset of murine and human LSCs (Sachs Cancer Research 2020). NF-kappa B is a well-known mediator of AML-self-renewal. Next, we asked whether targeting NF-kappa B with proteasome inhibitors might reduce self-renewal capacity in human AML. We plated six primary human samples in colony-forming assays with pan-proteasome inhibitor, carfilzomib, immunoproteasome inhibitor, PR957, or vehicle and found that both inhibitors reduced colony formation in every sample tested. We harvested colonies from each of the inhibitor and vehicle groups and plated them in secondary colony-forming assays. Colonies harvested from vehicle-treated samples formed colonies but those harvested from either carfilzomib or PR957-treated samples were unable to form any secondary colonies (Panel B). Using CyTOF, we validated that both inhibitors reduce NF-kappa B levels in these samples.These data demonstrate that CD69 marks a self-renewing subset of human AML, as it does in our murine AML model, and suggest that the transcriptional mechanisms of self-renewal that we defined in our murine model at the single-cell level may be shared with human AML. Additionally, these data demonstrate enhanced NF-kappa B levels in this self-renewing subset and that targeting the NF-kappa B pathway with proteasome inhibitors attenuates self-renewal in primary human AML samples (Panel C). These findings suggest that proteasome inhibition may be an effective approach to treating and preventing relapse in AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

DJ4 Targets Rho-associated Protein Kinase Pathway and Attenuates Disease Progression in Pre-clinical Murine Models of Acute Myeloid Leukemia

Introduction: The poor prognosis of acute myeloid leukemia (AML) and the highly heterogenous nature of the diseasemotivates targeted gene therapeutic investigations. Rho-associated protein kinases (ROCKs) are crucial for variousactin cytoskeletal changes, which have established malignant consequences in various cancers, yet are still not beingsuccessfully utilized clinically towards cancer treatment. ROCK 1 and 2 overexpression has been linked to AML celllines and overall survival of AML patients. This work reports the considerable therapeutic efficacy of the ROCKinhibitor DJ4 in both in vitro and in vivo preclinical models of AML to highlight the potential of this class of inhibitors.Experimental Design: The cytotoxic and pro-apoptotic activities of DJ4 for primary human AML samples and humanAML cell lines was determined by cell viability (MTS), colony forming assays, and Annexin V assays. Immunoblotanalysis was used to detect phosphorylation of downstream substrates of ROCK. To assess the preclinical therapeuticefficacy, the luciferase-expressing human AML cell line OCIAML-3-YFP-Luc was injected subcutaneously (SC) andintravenously (IV) into NRG-S mice. Mice were treated through an intraperitoneal (IP) injection with either vehiclecontrol or DJ4 (10 mg/kg) for 3 weeks. Modified AML cell lines, OCI-AML3-YFP-Luc and MV4-11-Luc2-EGFP, werealso treated with their respective IC50 dose of DJ4 or with vehicle control for 24 h and then administered via IV intoNRG-S mice and the survival advantage was monitored over time without further treatment. Disease progression wastracked in mice studies by flow cytometry analysis and examining the survival, bioluminescent signal, tumor volume,and tumor weights of the animals over time.Results: DJ4 induced cytotoxic and pro-apoptotic effects, within the micromolar range and in a dose-dependentmanner, in human AML cell lines (IC50: 0.05-1.68 μM) and primary patient cells (IC50: 0.264-13.43 μM) that harboredvarious mutations; however, normal hematopoietic cells are largely spared (Figures A-C). Treatment of DJ4demonstrated ~5-fold selectivity towards AML patient samples relative to the CB-MNCs of healthy donors (IC50= 25μM, Figure B). Representative flow cytometry plots of the Annexin V assay with AML primary cells (Figure C) depictedthe increase in the apoptotic populations as a result of treatment with increasing concentrations of DJ4. ROCKinhibition by DJ4 disrupts the phosphorylation of downstream targets, myosin light chain (MLC2), and myosin bindingsubunit of MLC phosphatase (MYPT) in OCI-AML3 and MV4-11, yielding a potent yet selective treatment response atmicromolar concentrations, 0.02 to 1 μM (Figure D). Analysis of mice treated with DJ4 via IP for 2.5 weeks indicatedthat DJ4 was well-tolerated with comparable CBC with differential and chemistry values and tissue morphology fromcross-sections of the spleen, liver, lung, and kidney relative to the vehicle treated group. Mice that were IV or SCinjected with OCIAML3-YFP-Luc and treated via IP with DJ4 exhibited an increase in overall survival and reductionin disease progression relative to the vehicle treated control mice (Figure E). Towards the end of the study, theOCIAML3-YFP-Luc SC injected mice exhibited at least a 3-fold reduction in the bioluminescent signal, ~4-fold decreasein tumor volume, and the tumor weight was significantly reduced by ~3-fold among the DJ4 treated group relative tothe control (Figure E). Mice that were IV administered DJ4 pretreated AML cells also exhibited a decreasedbioluminescent signal, increased survival of 10 and 20 days, and a significant reduction in the percent of human CD45cells in the bone marrow or spleen, indicating a diminished tumor burden (Figure F).Conclusion:The observed potency of DJ4 towards various AML cell lines and primary samples with a diverse set of mutationssuggests that this is a promising candidate to be incorporated into the standard AML regimen to help many differentsubsets of AML patients. Therapeutics that induce apoptosis have been shown to be promising candidates towardovercoming chemoresistance and to work well with appropriate combinations of standard of care drugs. This workhighlights the potential of targeting the Rho-ROCK pathway to improve the prognosis of AML and the need for thefuture development of chemotherapeutics that are both less toxic and more effective. [Display omitted] DisclosuresClaxton: Astellas: Other: Clinical Trial; Novartis: Research Funding; Astex: Research Funding; Cyclacel: Research Funding; Daiichi Sankyo: Research Funding; Incyte: Research Funding.

Bispecific Anti-CD123 x Anti-CD3 Adaptir™ Molecules APVO436 and APVO437 Have Broad Activity Against Primary Human AML Cells In Vitro

Background: The IL-3 receptor CD123 is an attractive target for antibody-based therapies in acute myeloid leukemia (AML) because of its frequent expression on AML blasts and its reported overexpression on leukemic stem cells (LSCs) as compared to normal hematopoietic tissues. Previously, two CD123 x CD3 bispecific targeting ADAPTIR constructs (APVO436 and APVO437) have been shown to bind CD123 with high affinity, engage T-cells and, in the presence of effector T-cells, cause cytotoxicity in CD123-expressing cell lines as well as in an in vivo xenograft model. Here, we further investigated the determinants of anti-tumor efficacy of these constructs in human AML.Materials and Methods: Frozen aliquots of Ficoll-isolated mononuclear cells from peripheral blood or bone marrow specimens were obtained from adults with AML. CD123 expression on myeloblasts and cells lines and the percentage of endogenous CD3+ T-cells in AML specimens were quantified by flow cytometry. To determine specific cytotoxicity, cell lines or primary AML cells were incubated in culture medium containing various concentrations of ADAPTIR constructs APVO436 and APVO437. Exogenous T-cells isolated from healthy volunteers were labeled with CellVue membrane dye and added at different effector:target (E:T) cell ratios. After 48 hours, cell numbers and drug-induced cytotoxicity, using DAPI to detect non-viable cells, were determined by flow cytometry; target AML cells were identified by forward/side scatter properties and negativity for CellVue dye. Specific cytotoxicity was calculated as: 100 x (%DAPItreated - %DAPIcontrol)/(100-%DAPIcontrol) and are presented as mean±SEM of all samples tested. T-cell activation, proliferation and depletion of CD123+ cell populations in normal and AML specimens were assessed using multi-color flow cytometry.Results: We examined the ability of APVO436 and APVO437 to induce cytotoxicity in primary human AML specimens. Of 20 samples tested, 10 had both >60% viability on thaw and >40% viability after 48 hours in cultures and were included for further analysis. The median percentage of myeloid blasts and CD3+ T-cells in the analyzed specimens was 78.5% (range 52-91.7%) and 2.95% (range 0.3-8.8%). With no exogenous T-cells, APVO436 and APVO437 induced modest cytotoxicity; at 1000 pM, APVO436 and APVO437 induced specific cytotoxicity of 6.8±3.8% and 3.9±3.6%, respectively. There was no statistically significant correlation between the percentage of endogenous T-cells present in the sample and the amount of cytotoxicity in the absence of exogenous T-cells. The addition of healthy donor T-cells increased cytotoxicity in a E:T-dependent manner (p <0.0001 for both APVO436 and APVO437) and increasing drug concentrations also increased cytotoxicity in a statistically-significant fashion (p <0.0001 for both APVO436 and APVO437). There was no consistent linear relationship between MFICD123 of blasts (MFICD123, arbitrary units) and cytotoxicity. Significant cytotoxicity was achieved even at the lowest levels of blast CD123 expression (MFICD123 range among patient samples: 349-3423). Inclusion of all 20 samples in cytotoxicity analysis did not qualitatively change results. APVO436 induced T-cell activation in both normal and AML samples at 24 hours as assessed by quantification of CD25 and CD69 expressing T-cells. T-cell activation was accompanied by proliferation of T-cells in normal and AML specimens at 96 hours in the presence of APVO436. APVO436 induced dose dependent depletion of CD123+ cells which was evident at 24 hours in both normal and AML samples.Conclusion: APVO436 and APVO437 cause target cell cytolysis in a dose-dependent, target-antigen-dependent, and E:T-dependent manner. Broad activity was seen in primary human AML samples and both constructs were effective in inducing cytotoxicity across a range of blast CD123 expression levels, including specimens with very limited CD123 expression. Endogenous T-cell activation and proliferation accompanied by CD123+ cell depletion demonstrate similar activity of anti-CD123 x anti-CD3 ADAPTIRs in both normal and AML subject samples. These data are supportive of further investigation of anti-CD123 x anti-CD3 ADAPTIR molecules as a potential treatment option for AML. DisclosuresGottschalk:Aptevo Therapeutics: Employment, Equity Ownership. Comeau:Aptevo Therapeutics: Employment, Equity Ownership. Hoyos:Aptevo Therapeutics: Employment, Equity Ownership. Walter:ADC Therapeutics: Research Funding; Aptevo Therapeutics: Research Funding.

The Origin and Functional Impact of Mitochondrial DNA Mutations in Acute Myeloid Leukemia

Recent whole-genome sequencing studies have identified somatic mutations in the mitochondrial genome of several human malignancies including acute myeloid leukemia (AML). Although mitochondrial DNA (mtDNA) mutations have previously been shown to promote tumorigenicity in some solid cancers, their role in AML development is unknown. To address this issue, we traced the origin of mtDNA mutations in primary human AML samples and determined their potential impact on mitochondrial respiration and cell fate decisions in hematopoietic stem and progenitor cells (HSPCs).We first characterized the spectrum of mtDNA mutations in AML by performing hybrid capture-based next generation sequencing of the 13 protein-coding mtDNA genes in 133 AML patient samples collected at the time of diagnosis. We also sequenced 150 recurrently mutated nuclear genes in parallel. We aligned mtDNA sequences to the revised Cambridge Reference Sequence and identified variants with > 5% variant allele frequency. Since matched normal samples were not available, we filtered out variants with a population frequency of > 0.1% in GenBank to enrich for somatic variants. Of the remaining variants, we filtered out ones that were synonymous or caused an amino acid change at a non-conserved residue to enrich for pathogenic mutations. Based on these criteria, we identified 87 different missense mutations in 39% (n=53) of the patients across 12 of the 13 genes. We did not find any hot spot mutations. Interestingly, mtDNA mutations were mutually exclusive with IDH1 R132 mutations (p < 0.05, Chi-square test). Patients with mtDNA mutations had inferior leukemia-free survival compared to those without mtDNA mutations (134 vs 477 days; p = 0.002, Log-rank test).To trace the origin of these mutations, we developed allele-specific TaqMan assays for 9 of the primary AML samples with known mtDNA mutations. The TaqMan assays were sufficiently sensitive to quantify heteroplasmy at the single cell level. Patient-specific mtDNA mutations were detected in single cells sorted from the hematopoietic stem cell (HSC) compartment (defined as CD45dim/CD33-/CD34+/CD38-/CD90+/CD45RA-) in 8 of the 9 samples. The heteroplasmy levels were generally lower in HSCs than leukemic blasts. Interestingly, mtDNA mutations were also detected at low levels in the T cell compartment of 3 of the 8 samples. Analysis of a matched diagnostic/relapsed pair showed persistence of the mtDNA mutation in HSCs and leukemic blasts. These findings collectively indicate that mtDNA mutations are acquired in HSCs and stable during disease evolution.To investigate the potential impact of mtDNA mutations on mitochondrial respiration, we generated a pair of transmitochondrial cybrids by repopulating mtDNA-depleted 143B osteosarcoma cells with mitochondria from either KG-1α AML cells or peripheral blood cells of a healthy donor. KG-1α cells harbor a nonsense mtDNA mutation (C13396T) in the MT-ND5 gene which encodes a core subunit of Complex I in the electron transport chain (ETC). The KG-1α cybrid had lower levels of basal and maximal respiration compared with the healthy control cybrid as determined by oxygen consumption rates. To confirm that this defect was due to impairment of Complex I activity, we ectopically expressed the yeast NADH dehydrogenase (NDI1) protein which bypasses endogenous Complex I function. NDI1 expression completely rescued the mitochondrial respiration defect in KG-1α cybrids, whereas expression of the AOX protein which bypasses Complex II and III failed to do so. To determine the potential impact of Complex I dysfunction on cell fate decisions in HSPCs, we transduced human CD34+ cord blood cells with shRNA lentiviral vectors targeting the nuclear genes NDUFV1 and NDUFB11 which encode critical subunits of Complex I. Knockdown of the genes resulted in an accumulation of CD34+ cells in ex vivo culture over time.In summary, our results demonstrate that mtDNA mutations are commonly found in AML patients and have potential impact on clinical outcomes. The acquisition these mutations appears to be an early event in AML leukemogenesis. Finally, we showed that mtDNA mutations can negatively impact ETC function which we speculate, may in turn drive clonal hematopoiesis by promoting self-renewal and blocking differentiation of HSCs. DisclosuresNo relevant conflicts of interest to declare.

Combined inhibition of XIAP and BCL2 drives maximal therapeutic efficacy in genetically diverse aggressive acute myeloid leukemia.

Aggressive therapy-resistant and refractory acute myeloid leukemia (AML) has an extremely poor outcome. By analyzing a large number of genetically complex and diverse, primary high-risk poor-outcome human AML samples, we identified specific pathways of therapeutic vulnerability. Through drug screens followed by extensive in vivo validation and genomic analyses, we found inhibition of cytosolic and mitochondrial anti-apoptotic proteins XIAP, BCL2 and MCL1, and a key regulator of mitosis, AURKB, as a vulnerability hub based on patient-specific genetic aberrations and transcriptional signatures. Combinatorial therapeutic inhibition of XIAP with an additional patient-specific vulnerability eliminated established AML in vivo in patient-derived xenografts (PDXs) bearing diverse genetic aberrations, with no signs of recurrence during off-treatment follow-up. By integrating genomic profiling and drug-sensitivity testing, this work provides a platform for a precision-medicine approach for treating aggressive AML with high unmet need.

JAK/STAT Inhibition Targets TP53 altered Primary Human Acute Myeloid Leukemia Stem Cells

In acute myeloid leukemia (AML), the impact of genetic drivers on response to therapy and long-term survival has been well characterized. AML with complex cytogenetics and TP53 alterations (TP53Alt) is a poor-risk AML subtype that is largely insensitive to chemotherapy, modern targeted agents, and hematopoietic stem cell transplant leading to survival rates 0-10% at 1 year. In contrast, AML with favorable risk molecular features is highly sensitive to chemotherapy and confers survival rates of 50-70%. AML with intermediate risk molecular features can be responsive to chemotherapy and can be cured with hematopoietic stem cell transplant leading to overall survival rates of 30-60%. Leukemia stem cells (LSCs), the cells that recapitulate and propagate leukemia, are central to leukemia progression and relapse. Given the differences in chemo-sensitivity and clinical behavior of genetic subgroups of AML, we asked whether LSCs from poor risk AMLs exhibit distinct signaling activation profiles. We assembled a panel of 23 primary human AML samples with intermediate- and poor- risk genetics and used CyTOF (mass cytometry) to quantitatively measure the levels of immunophenotypic proteins and intracellular signaling molecules in each sample, at the single-cell level. We gated on CD34+CD123+CD3-CD19- cells (LSCs) and measured the level of intracellular signaling molecules within the LSCs of each sample. Notably, the intracellular signaling activation state of LSCs from each AML subtype was distinct; NFkB, pERK, p4EBP1, and pSTAT3 were uniquely upregulated in complex cytogenetics and TP53Alt LSCs, relative to LSCs from intermediate risk AML, suggesting that these signaling pathways may be important for LSC function in this AML subtype. Given that TP53Alt independently confer treatment resistance in AML, we focused on this genetic subgroup. We compared the gene expression profiles of TP53Alt and TP53-wild-type AML samples from the BEAT AML dataset (Tyner et al. Nature 2018) and found that the gene expression profiles of TP53Alt samples are enriched for gene sets representing JAK/STAT signaling, consistent with our CyTOF data, which identified activation of STAT3 in TP53Alt LSCs. A recent drug screen in AML demonstrated that a JAK1/2 kinase inhibitor, AZD1480, can reduce the in vitro viability of TP53-deleted AML cell lines (Nechiporuk et al. Ca Discovery 2019), but these effects were not tested in primary AML samples or on LSCs. Since LSCs confer treatment resistance, we investigated the effect of the AZD1480 on the LSC population in TP53Alt primary human AML samples. AZD1480 treatment abolished all colony formation in primary human TP53Alt AML samples (n=7, 6 replicates per sample, p&amp;lt;0.01). Treatment of these samples in liquid cultures led to a 50% reduction in LSC frequency. We used CyTOF to profile the intracellular signaling states of in vitro treated samples and found that AZD1480 attenuated pSTAT3, pSTAT5, p4EBP1, and NFkB in the LSCs of these samples. The mTOR/4EBp1 and NF༆B pathways have been implicated as drivers of self-renewal and LSC function in AML. Our data suggest that JAK/STAT inhibition may target these pathways in TP53Alt LSCs. These data demonstrate the unique signaling states of TP53Alt LSCs, relative to other LSCs, and show that inhibition of the JAK/STAT pathway specifically targets LSCs within human TP53Alt AML. Figure Disclosures No relevant conflicts of interest to declare.

Flotetuzumab and Other Cellular Immunotherapies Upregulate MHC Class II Expression on Acute Myeloid Leukemia Cells in Vitro and In Vivo

Background: Allogeneic hematopoietic cell transplantation (allo-HCT) is the only curative therapy for patients with high-risk and refractory acute myeloid leukemia (AML). Unfortunately, up to 50 percent of patients relapse after allo-HCT.Recent research has shown that 30-50 percent of AML samples from patients relapsing after allo-HCT have downregulation of MHC class II (MHC-II) expression, which may promote immune effector evasion and disease relapse. These studies also report that interferon gamma (IFNγ) can restore MHC-II expression. IFNγ has never been systemically administered after allo-HCT and would likely cause significant and potentially life-threatening toxicities. Reinduction of MHC-II expression may lead to re-engagement of immune effectors, restoration of the graft-versus-malignancy effect, and disease control. We hypothesized that T cell immunotherapies targeting AML cells will lead to T cell activation, localized IFNγ release, and upregulation of MHC-II on AML cells. Methods: For in vitro experiments, THP1 cells (THP1s), which have intermediate MHC-II expression, or primary human AML samples with low MHC-II expression from a patient relapsing after allo-HCT (AML-low cells) were co-cultured with or without T-cell immunotherapy and with or without human MHC-mismatched CD3+ T cells. The following T-cell immunotherapies were tested: flotetuzumab (FLZ), an investigational CD123 x CD3 bispecific DART® molecule; a CD33 x CD3 bispecific molecule (Creative Biolabs, Shirley, NY); and CD123-directed chimeric antigen receptor (CAR) T cells. THP1 IFNγ receptor-1 (IFNγR1) knockout cell lines were generated using CRISPR-Cas9. MHC-II expression was measured by flow cytometry and IFNγ concentrations via Luminex immunoflourescence assay. In order to rescue THP1s from FLZ-induced death and allow for longitudinal evaluation, a transwell plate system was used, placing THP1s, human CD3+ T cells, and FLZ in the top well and THP1s in the bottom well. This allowed for diffusion of IFNγ but not human T cells to the bottom wells, permitting MHC-II upregulation while limiting death. The upper and lower wells were coincubated together for 24 hours prior to harvesting of the THP1s in the lower well for longitudinal studies and mixed-lymphocyte reactions. For in vivo experiments, NOD-scid IL2Rgammanull mice expressing human IL-3, GM-CSF, and SCF (NSG-S) were irradiated with 250 rads and injected with 10e6 primary AML-low cells per mouse. After 5.5 weeks, mice were divided into the following groups: 1) untreated control; 2) FLZ only (2mg/kg); 3) human mismatched T cells only (10e7 T cells per mouse); 4) FLZ and T cells. Results: In vitro co-culture of THP1 or AML-low cells with FLZ and T cells led to significantly increased MHC-II expression at 48 hours when compared with the control, FLZ only, and T cell only groups (Figure 1A-B). Co-culture of THP1s with the CD123 CAR-T cells led to similar results. Although co-incubation with a CD33 x CD3 bispecific led to a similar result, the MHC-II upregulation was not nearly as dramatic as that seen with CD123 targeting agents. Using a transwell system to rescue THP1s from FLZ-mediated toxicity, FLZ-induced MHC-II upregulation on THP1s peaked at 48-72 hours (similar kinetics to what is seen with IFNγ alone). These THP1s with upregulated MHC-II activated third-party donor mismatched human CD4+ T cells to a greater extent than untreated THP1s controls. Co-cultures of THP1s with CD4+ T cells and FLZ induced the secretion of very high concentrations of IFNγ, and blockade of IFNγ signaling through knockout of IFNγR1 led to abrogation of the effect (Figure 1C-D). Finally, in an in vivo model, NSG-S mice injected with AML-low samples and treated with FLZ and T cells showed significant upregulation of MHC-II expression on the AML cells. Single cell RNA-sequencing of AML cells purified from these mice is ongoing. Conclusions: Use of FLZ and other T-cell immunotherapies targeting AML antigens led to both direct AML killing as well as significant upregulation of MHC-II expression on AML cells both in vitro and in vivo. The effect appears to be mediated primarily by IFNγ. T-cell immunotherapies represent a promising treatment approach for AML patients relapsing after allo-HCT and may lead to enhanced immune recognition in the 30-50% of patients who relapse after allo-HCT. Based on these results, a clinical trial treating patients relapsing after allo-HCT with FLZ is planned. Disclosures Christopher: Boulder Bioscience: Patents &amp; Royalties: IP around the use of interferon gamma to treat stem cell transplant. Kim:Tmunity: Patents &amp; Royalties: methods for gene editing in hematopoietic stem cells to enhance the therapeutic efficacy of antigen-specific immunotherapy (Licensed by University of Pennsylvania); Neoimmune Tech: Patents &amp; Royalties: use of long-acting IL-7 analogs to enhance CAR T cells (licensed by Washington University). Muth:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Davidson:MacroGenics: Current Employment. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Nicotinamide Phosphoribosyltransferase Inhibitors Induce Apoptosis of AML Stem Cells through Dysregulation of Lipid Metabolism

Current chemotherapeutic regimens for acute myeloid leukemia (AML) often fail to eliminate leukemic stem cells (LSCs) which contribute to disease relapse. A key step towards the development of more effective therapies is the identification of vulnerabilities that are unique to LSCs. Here, we sought to identify LSC-specific metabolic dependencies by performing a flow cytometry-based screen of 110 metabolically-focused drugs against a primary human AML sample. This sample harbored distinct subsets defined by CD34 and CD38 expression, and LSC activity assayed by xenotransplantation was restricted to the CD34+CD38- fraction. Through this screen, we found that inhibitors of nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes the rate-limiting step in the NAD+ salvage pathway, preferentially depleted CD34+CD38- cells, implicating NAMPT inhibitors as potential anti-LSC agents. To evaluate the therapeutic potential of NAMPT inhibitors, we focused on KPT-9274, a small-molecule NAMPT inhibitor currently under clinical development for other cancer types. Treatment with KPT-9274 depleted the CD34+CD38- fraction across multiple primary human AML samples through induction of apoptosis. The preferential sensitivity of CD34+CD38- cells to NAMPT inhibition correlated with a lower basal level of intracellular NAD+ and greater dependency on NAMPT activity for NAD+ generation relative to the other fractions. In contrast, normal CD34+ HSPCs were largely resistant to the cytotoxic effects of KPT-9274 due to their capacity to utilize the Preiss-Handler pathway for NAD+ generation. Consistent with the in vitro findings, KPT-9274 treatment significantly reduced LSC activity as determined by secondary engraftment potential in 2 of 3 patient-derived xenograft (PDX) models of human AML and had minimal impact on normal HSC activity in mice engrafted cord blood cells. To gain mechanistic insights into how NAMPT inhibition induces cell death, we performed transcriptomic analysis of sorted CD34+CD38- cells treated with KPT-9274. This analysis revealed a striking upregulation of genes involved in cholesterol and lipid synthesis including the stearoyl-CoA desaturase (SCD) gene. The upregulated genes were highly enriched for known targets of the sterol regulatory element binding protein (SREBP) transcription factors. Functional studies demonstrated that this transcriptional response was protective against the cytotoxic effect of NAMPT inhibition in AML cells. To uncover the metabolic basis of this protective effect, we performed global metabolomic profiling of AML cells treated with KPT-9274 and observed a decrease in the ratio of monounsaturated fatty acids (MUFAs) to saturated fatty acids (SFAs) upon drug treatment. This drop in MUFA:SFA ratio reflected a reduction in SCD activity which catalyzes the desaturation of SFAs to MUFAs in a NADPH-dependent reaction. Since depletion of intracellular MUFAs could trigger apoptosis, we hypothesized that the SREBP response might protect against cell death through upregulation of SCD activity and consequent increase in MUFA synthesis. In line with this hypothesis, we found that exogenous oleic acid, a MUFA, completely rescued cell death induced by KPT-9274, while treatment with SCD inhibitors sensitized AML cells to the cytotoxic effects of NAMPT inhibition. To explore the translational application of our findings, we tested whether dipyridamole (DP), a clinically approved anti-platelet agent with inhibitory activity against SREBP signaling, can be repurposed to enhance the anti-leukemic effects of KPT-9274. We showed that treatment with DP, at non-toxic concentrations, potentiated the cytotoxicity of KPT-9274 against AML cells in vitro. Importantly, in vivo combination treatment with KPT-9274 and DP effectively targeted LSC activity in a PDX model that was refractory to KPT-9274 as single agent. In summary, our findings demonstrate that LSCs are preferentially dependent on NAMPT activity for survival over non-LSCs and normal HSCs. We further uncovered that NAMPT inhibition results in dysregulation of lipid homeostasis and induces a lipogenic response coordinated by SREBPs that protects AML cells against NAD+ depletion. These findings offer insights into drug combination strategies to enhance the efficacy of NAMPT inhibitors and provide the rationale for testing NAMPT inhibitors in the treatment of AML in clinical trials. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Wang:Trilium therapeutics: Patents &amp; Royalties: There is an existing license agreement between TTI and University Health Network and J.C.Y.W. may be entitled to receive financial benefits further to this license and in accordance with UHN's intellectual property policies. .

CBFB-MYH11 fusion neoantigen enables T cell recognition and killing of acute myeloid leukemia.

Proteins created from recurrent fusion genes like CBFB-MYH11 are prevalent in acute myeloid leukemia (AML), often necessary for leukemogenesis, persistent throughout the disease course, and highly leukemia specific, making them attractive neoantigen targets for immunotherapy. A nonameric peptide derived from a prevalent CBFB-MYH11 fusion protein was found to be immunogenic in HLA-B*40:01+ donors. High-avidity CD8+ T cell clones isolated from healthy donors killed CBFB-MYH11+ HLA-B*40:01+ AML cell lines and primary human AML samples in vitro. CBFB-MYH11-specific T cells also controlled CBFB-MYH11+ HLA-B*40:01+ AML in vivo in a patient-derived murine xenograft model. High-avidity CBFB-MYH11 epitope-specific T cell receptors (TCRs) transduced into CD8+ T cells conferred antileukemic activity in vitro. Our data indicate that the CBFB-MYH11 fusion neoantigen is naturally presented on AML blasts and enables T cell recognition and killing of AML. We provide proof of principle for immunologically targeting AML-initiating fusions and demonstrate that targeting neoantigens has clinical relevance even in low-mutational frequency cancers like fusion-driven AML. This work also represents a first critical step toward the development of TCR T cell immunotherapy targeting fusion gene-driven AML.

Therapeutic Vulnerabilities in Acute Myeloid Leukemia (AML) with TP53 Alterations

Acute myeloid leukemia (AML) is a cancer of the blood and bone marrow with a low two‐year survival rate. Standard chemotherapy achieves complete remission in 60–80% of patients, but 20–30% patients relapse due to leukemia stem cells (LSCs) that can self‐renew and recapitulate disease. AML with TP53 alterations has a lower prognosis with survival rates of 0–10% at one year. In vitro drug screens have identified drugs to target AML, but none have studied the effect of these therapies on LSCs with TP53 alterations. These in vitro drug screens include Crizotinib, Elesclomol, AZD1480, GW2580, Venetoclax, and Entrectinib. Crizotinib, AZD1480, GW2580, Venetoclax, and Entrectinib are signaling pathway inhibitors. Elesclomol induces oxidative stress and apoptosis in cancer cells. The goal of this research is to understand the molecular mechanisms of self‐renewal and therapeutic vulnerabilities in LSCs of AML with TP53 alterations. Specifically, we investigate whether these agents target LSCs from AML with TP53 alterations. We performed in vitro viability and in vitro colony forming assays (CFAs) on primary human AML samples with TP53 alterations, plating them with drugs or vehicle control. Furthermore, we used CYTOF to assess the effects of each drug on signaling within leukemia subpopulations. Crizotinib proved to be effective in reducing in vitro viability, in vitro CFAs, and affecting the expression of immunophenotypic markers, such as CD34 and CD123, which are markers for leukemia stem cell populations. Our data reveals cell‐type specific effects of Crizotinib in human AML with TP53 alterations. Our findings suggest that Crizotinib may be an effective therapy for patients with AML with TP53 alterations.Support or Funding InformationThis research was supported and funded by: NIH/NIGMS MARC U* STAR T34 HHS 00026 National Research Service Award to UMBC, Summer Research at the University of Minnesota Medical School NIH HLBI 2R25HL088728‐11A1, Dr. Colin Campbell, Dr. Craig Henke, American Cancer Society Mentored Research Scholar Grant (MSRG‐16‐195‐01‐DDC), Frederick A. Deluca Foundation, CTSI K to R01 Award, NIH/NCATS, Lois and Richard King Assistant Professorship in Medicine, Division of Hematology, Oncology, Transplantation, Dept. of Medicine, University of Minnesota University of Minnesota Foundation Donors

All-trans retinoic acid enhances, and a pan-RAR antagonist counteracts, the stem cell promoting activity of EVI1 in acute myeloid leukemia

Ecotropic virus integration site 1 (EVI1), whose overexpression characterizes a particularly aggressive subtype of acute myeloid leukemia (AML), enhanced anti-leukemic activities of all-trans retinoic acid (atRA) in cell lines and patient samples. However, the drivers of leukemia formation, therapy resistance, and relapse are leukemic stem cells (LSCs), whose properties were hardly reflected in these experimental setups. The present study was designed to address the effects of, and interactions between, EVI1 and retinoids in AML LSCs. We report that Evi1 reduced the maturation of leukemic cells and promoted the abundance, quiescence, and activity of LSCs in an MLL-AF9-driven mouse model of AML. atRA further augmented these effects in an Evi1 dependent manner. EVI1 also strongly enhanced atRA regulated gene transcription in LSC enriched cells. One of their jointly regulated targets, Notch4, was an important mediator of their effects on leukemic stemness. In vitro exposure of leukemic cells to a pan-RAR antagonist caused effects opposite to those of atRA. In vivo antagonist treatment delayed leukemogenesis and reduced LSC abundance, quiescence, and activity in Evi1high AML. Key results were confirmed in human myeloid cell lines retaining some stem cell characteristics as well as in primary human AML samples. In summary, our study is the first to report the importance of EVI1 for key properties of AML LSCs. Furthermore, it shows that atRA enhances, and a pan-RAR antagonist counteracts, the effects of EVI1 on AML stemness, thus raising the possibility of using RAR antagonists in the therapy of EVI1high AML.