Normal Hematopoietic Progenitor Research Articles

Maintenance of hematopoietic stem cells by non-tyrosine-phosphorylated STAT5 and JAK inhibition

AbstractAdult hematopoietic stem cells (HSCs) are responsible for the lifelong production of blood and immune cells, a process regulated by extracellular cues, including cytokines. Many cytokines signal through the conserved Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway in which tyrosine-phosphorylated STATs (pSTATs) function as transcription factors. STAT5 is a pivotal downstream mediator of several cytokines known to regulate hematopoiesis, but its function in the HSC compartment remains poorly understood. In this study, we show that STAT5-deficient HSCs exhibit an unusual phenotype, including reduced multilineage repopulation and self-renewal, combined with reduced exit from quiescence and increased differentiation. This was driven not only by the loss of canonical pSTAT5 signaling, but also by the loss of distinct transcriptional functions mediated by STAT5 that lack canonical tyrosine phosphorylation (uSTAT5). Consistent with this concept, expression of an unphosphorylatable STAT5 mutant constrained wild-type HSC differentiation, promoted their maintenance, and upregulated transcriptional programs associated with quiescence and stemness. The JAK1/2 inhibitor, ruxolitinib, which increased the uSTAT5:pSTAT5 ratio, had similar effects on murine HSC function; it constrained HSC differentiation and proliferation, promoted HSC maintenance, and upregulated transcriptional programs associated with stemness. Ruxolitinib also enhanced serial replating of normal human hematopoietic stem and progenitor cells (HSPCs), CALR-mutant murine HSCs, and HSPCs obtained from patients with myelofibrosis. Our results therefore reveal a previously unrecognized interplay between pSTAT5 and uSTAT5 in the control of HSC function and highlight JAK inhibition as a potential strategy for enhancing HSC function during ex vivo culture. Increased levels of uSTAT5 may also contribute to the failure of JAK inhibitors to eradicate myeloproliferative neoplasms.

RNA sequestration in P-bodies sustains myeloid leukaemia.

Post-transcriptional mechanisms are fundamental safeguards of progenitor cell identity and are often dysregulated in cancer. Here, we identified regulators of P-bodies as crucial vulnerabilities in acute myeloid leukaemia (AML) through genome-wide CRISPR screens in normal and malignant haematopoietic progenitors. We found that leukaemia cells harbour aberrantly elevated numbers of P-bodies and show that P-body assembly is crucial for initiation and maintenance of AML. Notably, P-body loss had little effect upon homoeostatic haematopoiesis but impacted regenerative haematopoiesis. Molecular characterization of P-bodies purified from human AML cells unveiled their critical role in sequestering messenger RNAs encoding potent tumour suppressors from the translational machinery. P-body dissolution promoted translation of these mRNAs, which in turn rewired gene expression and chromatin architecture in leukaemia cells. Collectively, our findings highlight the contrasting and unique roles of RNA sequestration in P-bodies during tissue homoeostasis and oncogenesis. These insights open potential avenues for understanding myeloid leukaemia and future therapeutic interventions.

Peds1 deficiency in zebrafish results in myeloid cell apoptosis and exacerbated inflammation

Plasmalogens are glycerophospholipids with a vinyl ether bond that confers unique properties. Recent identification of the gene encoding PEDS1, the desaturase generating the vinyl ether bond, enables evaluation of the role of plasmalogens in health and disease. Here, we report that Peds1-deficient zebrafish larvae display delayed development, increased basal inflammation, normal hematopoietic stem and progenitor cell emergence, and cell-autonomous myeloid cell apoptosis. In a sterile acute inflammation model, Peds1-deficient larvae exhibited impaired inflammation resolution and tissue regeneration, increased interleukin-1β and NF-κB expression, and elevated ROS levels at the wound site. Abnormal immune cell recruitment, neutrophil persistence, and fewer but predominantly pro-inflammatory macrophages were observed. Chronic skin inflammation worsened in Peds1-deficient larvae but was mitigated by exogenous plasmalogen, which also alleviated hyper-susceptibility to bacterial infection, as did pharmacological inhibition of caspase-3 and colony-stimulating factor 3-induced myelopoiesis. Overall, our results highlight an important role for plasmalogens in myeloid cell biology and inflammation.

Overlapping Stromal Alterations in Myeloid and Lymphoid Neoplasms.

Myeloid and lymphoid neoplasms share the characteristics of potential bone marrow infiltration as a primary or secondary effect, which readily leads to hematopoietic insufficiency. The mechanisms by which clonal malignant cells inhibit normal hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM) have not been unraveled so far. Given the pivotal role of mesenchymal stromal cells (MSCs) in the regulation of hematopoiesis in the BM niche it is assumed that MSCs also play a relevant role in the pathogenesis of hematological neoplasms. We aimed to identify overlapping mechanisms in MSCs derived from myeloid and lymphoid neoplasms contributing to disease progression and suppression of HSPCs to develop interventions that target these mechanisms. MSCs derived from healthy donors (n = 44) and patients diagnosed with myeloproliferative neoplasia (n = 11), myelodysplastic syndromes (n = 16), or acute myeloid leukemia (n = 25) and B-Non-Hodgkin lymphoma (n = 9) with BM infiltration and acute lymphoblastic leukemia (n = 9) were analyzed for their functionality and by RNA sequencing. A reduced growth and differentiation capacity of MSCs was found in all entities. RNA sequencing distinguished both groups but clearly showed overlapping differentially expressed genes, including major players in the BMP/TGF and WNT-signaling pathway which are crucial for growth, osteogenesis, and hematopoiesis. Functional alterations in healthy MSCs were inducible by exposure to supernatants from malignant cells, implicating the involvement of these factors in disease progression. Overall, we were able to identify overlapping factors that pose potential future therapeutic targets.

CAR-T Cells in Acute Myeloid Leukemia: Where Do We Stand?

Despite recent advances, the prognosis of acute myeloid leukemia (AML) remains unsatisfactory due to disease recurrence and the development of resistance to both conventional and novel therapies. Engineered T cells expressing chimeric antigen receptors (CARs) on their cellular surface represent one of the most promising anticancer agents. CAR-T cells are increasingly used in patients with B cell malignancies, with remarkable clinical results despite some immune-related toxicities. However, at present, the role of CAR-T cells in myeloid neoplasms, including AML, is extremely limited, as specific molecular targets for immune cells are generally lacking on AML blasts. Besides the paucity of dispensable targets, as myeloid antigens are often co-expressed on normal hematopoietic stem and progenitor cells with potentially intolerable myeloablation, the AML microenvironment is hostile to T cell proliferation due to inhibitory soluble factors. In addition, the rapidly progressive nature of the disease further complicates the use of CAR-T in AML. This review discusses the current state of CAR-T cell therapy in AML, including the still scanty clinical evidence and the potential approaches to overcome its limitations, including genetic modifications and combinatorial strategies, to make CAR-T cell therapy an effective option for AML patients.

Single cell and bulk RNA expression analyses identify enhanced hexosamine biosynthetic pathway and O-GlcNAcylation in acute myeloid leukemia blasts and stem cells.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults with an overall poor prognosis and high relapse rate. Multiple factors including genetic abnormalities, differentiation defects and altered cellular metabolism contribute to AML development and progression. Though the roles of oxidative phosphorylation and glycolysis are defined in AML, the role of the hexosamine biosynthetic pathway (HBP), which regulates the O-GlcNAcylation of cytoplasmic and nuclear proteins, remains poorly defined. We studied the expression of the key enzymes involved in the HBP in AML blasts and stem cells by RNA sequencing at the single-cell and bulk level. We performed flow cytometry to study OGT protein expression and global O-GlcNAcylation. We studied the functional effects of inhibiting O-GlcNAcylation on transcriptional activation in AML cells by Western blotting and real time PCR and on cell cycle by flow cytometry. We found higher expression levels of the key enzymes in the HBP in AML as compared to healthy donors in whole blood. We observed elevated O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) expression in AML stem and bulk cells as compared to normal hematopoietic stem and progenitor cells (HSPCs). We also found that both AML bulk cells and stem cells show significantly enhanced OGT protein expression and global O-GlcNAcylation as compared to normal HSPCs, validating our in silico findings. Gene set analysis showed substantial enrichment of the NF-κB pathway in AML cells expressing high OGT levels. Inhibition of O-GlcNAcylation decreased NF-κB nuclear translocation and the expression of selected NF-κB-dependent genes controlling cell cycle. It also blocked cell cycle progression suggesting a link between enhanced O-GlcNAcylation and NF-κB activation in AML cell survival and proliferation. Our study suggests the HBP may prove a potential target, alone or in combination with other therapeutic approaches, to impact both AML blasts and stem cells. Moreover, as insufficient targeting of AML stem cells by traditional chemotherapy is thought to lead to relapse, blocking HBP and O-GlcNAcylation in AML stem cells may represent a novel promising target to control relapse.

Abstract 5228: Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile

Abstract Acute myeloid leukemia (AML) is an aggressive hematological malignancy that, despite advances in treatment, has an overall 5-year survival rate of less than 30%. There is an urgent unmet need for new and effective therapies for AML. Chimeric antigen receptor (CAR)-modified cell therapies are emerging as promising options. However, the selection of suitable antigens is challenging, due to the heterogeneous expression of potential antigen targets in AML. Most antigens, such as CD123, are expressed on both leukemic cells and normal hematopoietic stem and progenitor cells. In addition, conventional autologous or allogeneic T cell therapies present many limitations, such as cytokine release syndrome (CRS) and graft-vs-host disease (GvHD). We therefore evaluated CD123-targeting CAR designs in vδ1-enriched γδT cells, an innate immune cell platform that is inherently non-MHC restricted, has low risks of CRS and GvHD, and is capable of discriminating between malignant and healthy cells in preclinical models. We engineered vδ1-enriched γδT cells from peripheral blood of healthy individuals to express various CD123-targeting CAR constructs. The CD123-targeting CAR-engineered vδ1-enriched γδT cells expressed high levels of natural cytotoxicity receptors, suggesting capability to target innate receptor ligands on AML cell surface. CD123-targeting CAR-expressing vδ1-enriched γδT cells demonstrated enhanced cytotoxicity in vitro against multiple AML cell lines, in both short-term co-culture and repeated antigen stimulation assays. CD123-targeting CAR-expressing vδ1-enriched γδT cells also showed enhanced efficacy and survival in vivo against mouse models of human AML. Interestingly, attenuation of CAR activity via novel designs preserved the inherent selectivity of vδ1-enriched γδT cells against AML cells relative to normal hematopoietic stem or progenitor cells, which also express CD123. Together, our results demonstrated through rational CAR designing and engineering in vδ1-enriched γδT cells, an allogeneic cell therapy targeting CD123 can be generated with selective cytotoxicity against AML cells while sparing normal hematopoietic stem cells. Such product designs have the potential to be a transformative therapeutic option for a broad population of AML patients. Citation Format: Mei Rosa Ng, Sarah L. Hayward, Di Zhang, Gary Gu, Bri Paulhus, Donjeta Gjuka, Collin Crowley, Sophie McLaughlin, Anya Lublinsky, Angel Mathew, Evans Berreondo Giron, Doanh Mai, Madisen Lee, Megha Wal, Rebecca Alade, Andre Simoes, Robin Lytle, James Gavin, Shira Cramer, Zhong-hua Yan, Taylor Hickman, Rashmi Choudhary, Cori Abikoff, Yana Wang, Istvan Kovacs, Nandhu M. Sobhana, Lan Cao. Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile [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 5228.

Increased expression of CD70 in relapsed acute myeloid leukemia after hypomethylating agents.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. While induction chemotherapy leads to remission in most patients, a significant number will experience relapse. Therefore, there is a need for novel therapies that can improve remission rates in patients with relapsed and refractory AML. CD70 is the natural ligand for CD27 (a member of the TNF superfamily) and appears to be a promising therapeutic target. Consequently, there is considerable interest in developing chimeric antigen receptor (CAR) T-cell therapy products that can specifically target CD70 in various neoplasms, including AML. In this study, we employed routine diagnostic techniques, such as immunohistochemistry and flow cytometry, to investigate the expression of CD70 in bone marrow samples from treatment-naïve and relapsed AML patients after hypomethylating agents (HMA). Also, we evaluated the impact of HMA on CD70 expression and examined CD70 expression in various leukemic cell subsets and normal hematopoietic progenitors.

Pyrimidine Starvation Is a Targetable Cancer Vulnerability: Mechanisms of Nucleotide Homeostasis

Introduction: Acute myeloid leukemia (AML) is a heterogeneous disease, though all AML exhibit a characteristic block in differentiation. Hence, much effort has been made to develop small molecules that promote leukemic cell differentiation in the treatment of AML. In 2016 we identified the enzyme dihydroorotate dehydrogenase (DHODH) as a therapeutic target in AML. DHODH is a mitochondrial enzyme that converts dihydroorotate to orotate in the 4th step of de novo pyrimidine synthesis. Without DHODH enzymatic activity, a cell is forced to rely on autophagy or extracellular salvage to maintain its intracellular pyrimidine pool. DHODH inhibitor therapy has shown broad preclinical efficacy across multiple cancer types including, glioma, neuroblastoma, breast, small cell lung and pancreatic cancer suggesting that malignant cells are specifically impaired in their ability to maintain nucleotide homeostasis during periods of limited availability. Despite these very encouraging pre-clinical results, the translation into human clinical trials has been met with limited anti-cancer activity and therapeutic success. This discordance between pre-clinical and clinical benefit likely speaks to our limited understanding of how normal and malignant cells respond to pyrimidine starvation. Methods: Using in vitro and in vivo assays to compare normal hematopoietic progenitor and leukemic cells we interrogated key nodes in pyrimidine synthesis to identify the mechanisms behind this selective, anti-leukemic activity in the context of nucleotide starvation: [1] Nucleotide measurement by HPLC, [2] Flux experiments by isotopic tracing, [3] Polysome profiling to compare the transcriptome and translatome, [4] Quantification of ribosome recycling by examining the lysosomes following ultracentrifugation and [5] Direct visualization of lysosomes using new techniques in cryogenic electron microscopy. Results: Treatment of leukemia cells with brequinar blocked de novo synthesis and led to the rapid depletion of intracellular pyrimidines. In comparison, normal hematopoietic CD34 positive progenitor cells had less depletion, suggesting a selective vulnerability of leukemic cells over normal cells. We used isotopically labeled glutamine (15N) and uridine (13C) to quantify the contribution of de novo synthesis and extracellular salvage under control and starvation conditions. The leukemia cell line THP1 showed a higher reliance on de novo synthesis than normal CD34 positive hematopoietic progenitors ( Figure 1A, left graphs). Furthermore, following DHODH inhibition, THP1 cells were less capable of extracellular salvage compared to normal cells ( Figure 1A, right graphs). Polysome profiling analysis in THP1 cells showed a dramatic decrease in translating ribosomes as well as dramatic decrease in ribosomal RNA. We identified a short-list of 22 genes whose translation is prioritized during nucleotide deprivation. Following DHODH inhibition and nucleotide starvation, cells upregulate their number of lysosomes, as quantified by flow cytometry. In addition, the contents of the lysosome include ribosomal proteins, whose abundance increased during nucleotide starvation, suggesting an ongoing recycling of ribosomes to meet the nucleotide demand of leukemic cells during starvation. Finally, for the first time we have been able to directly visualize (by cryogenic electron microscopy) ribosomes within lysosomes during periods of nucleotide starvation, a finding that was completely absent in leukemia cells cultured under normal conditions ( Figure 1B). Conclusion: We hypothesized that the transformation from normal to malignant cell is accompanied by changes in nucleotide metabolism that render the transformed cells more reliant on de novo synthesis, establishing a metabolic vulnerability (and inherent therapeutic window) in the use of DHODH inhibitors. Our work confirms that leukemic blasts differ in their dependence on de novo nucleotide synthesis and its ability to garner nucleotides via salvage or autophagy pathways. This balance appears to be disturbed in the setting of malignancy such that leukemic cells are empirically more sensitive to treatment targeting pyrimidine synthesis. Understanding the differences in how leukemic and normal cells handle their nucleotide pools at baseline, and under starvation conditions, will guide the clinical utility of this class of inhibitors.

PDZD2 Is Required for Normal Erythropoiesis and Its 37-KDa Secreted Product, sPDZD2, Functions As a Soluble Tumor Suppressor in AML

PDZ domain-containing protein 2 (PDZD2) is expressed in normal hematopoietic stem and progenitor cells (HSPC), specifically HSC, MPP, and erythroid progenitors, however, it is almost universally lost through hypermethylation in acute myeloid leukemia (AML). PDZD2 encodes for a 300-kDa protein containing 6 PDZ domains. The full-length protein is cleaved in the cytoplasm in a caspase 3-dependent manner, and the C-terminal fragment containing PDZ domains 5 and 6 is secreted to the tissue microenvironment as a 37-kDa protein known as secreted PDZD2 (sPDZD2). sPDZD2 has been reported to function as a tumor suppressor in some solid tumors, including breast, prostate, and liver cancer. Thus, we hypothesized that PDZD2 is required for normal hematopoiesis and that sPDZD2 functions as a soluble tumor suppressor in the hematopoietic system. We first sought to determine if sPDZD2 is secreted to the bone marrow (BM) microenvironment. Using an ELISA to quantify sPDZD2 levels in BM plasma isolated from healthy donors (n=6) and primary AMLs (n=20), we confirmed its secretion by normal CD34+ HSPCs (NBM) and downregulation in AMLs (NBM: 169 ± 17.53 vs AML: 46.12 ± 6.70 ng/ml; p<0.0001). In addition, AML cell lines (n=7) secreted significantly lower levels of sPDZD2 into the culture medium than normal HSPCs (n=2) (NBM: 28.71 ± 1.76 vs AML: 4.62 ± 1.19 ng/mL; p<0.0001). Next, to study the role of PDZD2 in normal hematopoiesis we performed in vitro myeloid and erythroid differentiation and colony-forming unit assays in CRISPR-Cas9-edited human HSPCs. CD34+ cells from healthy donors were electroporated with recombinant Cas9-sgRNA ribonucleoprotein complexes targeting PDZD2 and editing confirmed by T7I endonuclease assay and Sanger sequencing. PDZD2-edited cells (PDZD2 KO) were plated either on methylcellulose or liquid differentiation cultures (n=6). PDZD2 KO resulted in decreased colony number of all types (Control: 172.5 ± 34.37 vs PDZD2 KO: 137.0 ± 18.25, p<0.05), indicating reduced colony-forming potential. In addition, loss of PDZD2 resulted in a delay in terminal erythroid differentiation with an accumulation of pro-erythroblasts (CD71+CD235a lowCD105 high, p<0.05) and late basophilic erythroblasts (CD71+CD235a highCD105 int, p<0.01) as well as decreased orthochromatic erythroblasts (CD71+CD235a highCD105 neg, p<0.01) in a tri-phasic liquid culture system. By contrast, PDZD2 KO had no impact on myeloid differentiation. To determine the role of Pdzd2 in vivo, we took advantage of a Pdzd2 Gt/Gt gene-trap (GT) mouse model and performed peripheral blood (PB) and BM analysis compared to wild-type (WT) controls. PB counts at 2 months of age (n=6-9 per genotype) showed anemia, with a significant decrease in both red blood cell counts (WT: 9.04 ± 0.26 vs GT: 7.58 ± 0.47 x10 6/mL; p=0.026) and hemoglobin levels (WT: 14.88 ± 0.46 vs GT: 12.30 ± 0.81 g/dL; p=0.036), an observation compatible with the impaired erythropoiesis seen in vitro after PDZD2 KO in human HSPCs. In addition, we observed a trend to decrease megakaryocyte-erythroid progenitors (MEPs) and short-term HSCs in the BM of 3 months-old mice (n=3), though the small size of the cohort prevented robust statistical analysis. Next, we sought to evaluate the tumor suppressor role of sPDZD2 in the context of AML. We treated a panel of AML cell lines (n=9) and primary human AML specimens (n=4) with recombinant sPDZD2 ( r-sPDZD2; dose: 100nM-300nM/day) for a period of 8 days. AML cells showed dose-dependent growth inhibition (CellTiter-Glo, p<0.05 for all) and increased expression of either CD11b or CD15 differentiation markers. Cell cycle analysis on day 8 revealed that AML cells treated with r-sPDZD2 were arrested at G0/G1 phase compared to control. In summary, our study is the first to shed light on a previously unrecognized role for PDZD2 in hematopoiesis. Loss of PDZD2 in HSPC resulted in impaired erythroid differentiation both in vitro and in vivo. Moreover, our findings validate a soluble tumor suppressor role for sPDZD2 in AML, similar to that seen in solid tumors. Importantly, we further demonstrate a potential role for r-sPDZD2 as a novel therapeutic approach for AML.

Polyamines Are Required for AML LSC Function through Their Role in Regulating eIF5A Dependent Protein Synthesis

Outcomes for patients with acute myeloid leukemia (AML) remain poor due to the inability of current therapies to fully eradicate disease initiating leukemia stem cells (LSCs) (Shlush et al., 2017). Many studies have demonstrated that LSCs have unique metabolic properties compared to AML blasts and normal hematopoietic stem and progenitor cells (HSPCs) (Jones et al., 2021). In this study we sought to quantify metabolite levels in LSCs compared to HSPCs using an unbiased metabolites screen. To examine metabolite levels in enriched LSCs and HSPCs we used mass spectrometry-based metabolomics analysis on enriched LSCs from 18 AML patients and HSPCs from 5 normal bone marrow (BM) specimens. Pathway analysis showed that arginine metabolism and biosynthesis were the most significantly enriched pathways in LSCs compared to HSPCs. Clinical studies have shown limited efficacy of targeting arginine levels using arginine degrading enzymes; therefore, we sought to target pathways downstream of arginine. To identify the pathways that arginine metabolism supports in LSCs, we used stable isotope labeled tracing analysis which showed arginine was metabolized through the urea cycle into polyamines in LSCs. Interestingly, the polyamine spermidine was the most significantly enriched metabolite in LSCs compared to HSPCs in our metabolomics analysis. Moreover, expression of SAT1, the enzyme catalyzing the export of polyamines from the cells, was decreased in AML compared to normal BM. Based on these observations, we sought to determine if the polyamine pathway represents a metabolic vulnerability that can be exploited to target LSCs. Polyamines are cationic compounds which have been previously shown to be important for the growth of various cancers but their role in AML LSCs has yet to be determined. To investigate if polyamines are essential for LSC survival and function, we treated LSCs and HSPCs with N 1, N 11-diethylnorspermine (DENSpm), a polyamine analog causing polyamine depletion through increased SAT1 expression and subsequent cellular export. DENSpm treatment decreased the viability of LSCs in 10 out of 12 of patients tested but had no impact on HSPCs viability. Further, treatment with DENSpm resulted in decreased engraftment potential of 5 AML specimens but did not alter the engraftment of HSPCs from 3 normal BM specimens, suggesting that targeting polyamines represents a promising approach to kill LSCs while sparing HSPCs. We then investigated the mechanisms by which polyamines are essential in LSCs. Using RNA-sequencing analysis on enriched LSCs from 5 AML patients and HSPCs from 3 normal BM specimens we observed an enrichment in myeloid differentiation signatures in DENSpm-treated LSCs but not in DENSpm-treated HSPCs. In line with this, expression of myeloid markers CD11b and CD15 were increased in patient-derived xenograft models engrafted with DENSpm-treated LSCs. As we did not observe such dysregulation in mice engrafted with DENSpm-treated HSPCs, these data suggest polyamine depletion promotes differentiation of LSCs but not HSPCs. Our transcriptomic analysis also unveiled decreased expression of genes involved in protein synthesis in DENSpm-treated LSCs but not in normal HSPCs. Protein synthesis and its regulation are critical for AML survival (Chen et al., 2019; Messling et al., 2022) but the role of polyamines in protein synthesis in LSCs has not been explored. Polyamines stimulate translation notably by serving as precursors for the hypusination of eukaryotic translation initiation factor 5A (eIF5A) (Park et al., 2010). Accordingly, DENSpm led to decreased hypusination of eIF5A in AML cells. This was associated with reduced protein synthesis level in AML cell lines and LSCs but not in normal HSPCs. Strikingly, eIF5A hypusination, protein synthesis and viability were rescued upon co-treatment with spermidine. Moreover, knockdown of eIF5A using siRNA resulted in impaired colony forming potential of AML and altered engraftment in patient-derived xenograft models. Altogether, these results suggest that DENSpm alters AML cell and LSC viability and function at least in part through decreased eIF5A-mediated protein translation. Altogether, our data demonstrates that depletion of polyamines decreased LSC function while sparing normal HSPCs. Mechanistically, our data suggest that LSC targeting is accomplished through decreased eIF5A-mediated synthesis of proteins crucial for LSCs.

Pre-Existing Chromatin States Regulate KLF4 Binding to Eliminate Leukemia Stem Cells

Transcription factors collaborate with chromatin's epigenetic states to regulate cell fate decisions. While recent in vitro work using cryo-EM (Sinha et al., Nature, 2023) suggested that histone modifications could regulate the activity of pioneer transcription factors, it was not clear whether these findings applied in vivo, or if chromatin states could influence transcription factor binding to affect cell fate. Our previous work (Liu et al., Leukemia,2014; Wang et al., Nat Commun,2019) showed that reprogramming factors (Oct4, Sox2, Klf4, and cMyc, collectively known as OSKM) selectively eliminated leukemia cells in vivo, with minimal impact on normal Hematopoietic Stem and Progenitor Cells (HSPCs). This suggested that cell fates induced by OSKM could be influenced by pre-existing chromatin states. Building on this hypothesis, we profiled the pre-existing chromatin states of leukemia cells and HSPCs using MNase-ChIP-seq, ATAC-seq, and WGBS-seq. In our Tet-on-induced models, Klf4 activation resulted in the targeting of distinct gene sets and exhibited similar dynamic changes in chromatin accessibility, despite these regions sharing the same active chromatin state. Using machine learning algorithms, we identified H3K18ac –a feature of the active chromatin states preferred by Klf4– as a dominant factor influencing Klf4's genomic binding. We then altered H3K18ac through an enzymatically deficient SIRT7, which is an H3K18ac deacetylase, and examined where Klf4 binds. We observed an increase in Klf4 binding at sites where H3K18ac levels were elevated. This result suggested that Klf4 binding was influenced by changes in H3K18ac. We exposed human CD34+ cells, several leukemia cell lines, and samples from leukemia patients to ATPO-253. This small molecule, currently in phase 1 clinical trials, has been found to enhance the levels of the KLF4 protein. This treatment led to an increase in KLF4 protein levels and we observed activation of apoptosis genes in the tested leukemia cell lines that possessed high pre-existing H3K18ac levels. In addition, we observed a decline across all tested leukemia samples, while the population of CD34+ cells essentially remained stable. In conclusion, our work demonstrated that pre-existing chromatin states, such as H3K18ac, regulate Klf4's genomic binding, thereby influencing distinct cell fates in leukemia cells and HSPCs. This opens potential avenues for clinical applications in targeted leukemia cell clearance using KLF4 or ATPO-253.

Functional Characterization and Optimization of Switchable Allogeneic Chimeric Antigen Receptor T Cells for Targeting CD19 and CD20 in B Cell Malignancies

Chimeric antigen receptor (CAR) T cell therapy has demonstrated significant efficacy in B cell malignancies. However, despite clinically manageable on-target/off-tumor toxicity when targeting antigens like CD19 and BCMA, severe cytokine release and neurotoxicity remain a clinical challenge. Additionally, patients may require immunoglobulin replacement therapy, and antigen-loss is recognized as a cause of relapse (PMID: 30275569, 31798590, 36263838, 37051246 and 37122700). To overcome these limitations, we developed a novel switchable CAR-T cell therapy that simultaneously targets both CD19 and CD20 in B cell malignancies. This innovative therapy is based on our reverse universal chimeric antigen receptor platform (RevCAR), a 2-component CAR-T technology (Figure 1; PMID: 34638268 and 32923149). The first component is a universal CAR-T cell, incorporating a CAR based on a short, non-immunogenic peptide motif derived from the human nuclear La/SSB autoantigen. The CAR itself does not recognize any human cell surface antigen. However, it is specifically targeted by an antibody-based binder in the second component, a soluble adaptor called targeting module (TM). This TM confers specificity against the desired cancer antigens of choice. Consequently, the availability of the TM effectively controls the activity of RevCAR-T cells. We have recently reported preclinical development of an allogeneic RevCAR-T cell therapy for CD123-positive hematologic malignancies (AVC-201; Ehninger et al. 2023, AACR Abstract 4095; NCT05949125). To target CD123, which is also expressed on normal hematopoietic progenitor cells, a TM with a short half-life was selected (R-TM123), enabling a rapid switch-off of the RevCAR system by TM withdrawal to avoid acute toxicity and continued aplasia. As CD19 and CD20 have a substantially more favorable safety profile and autologous conventional CAR-T cells and bispecific T cell engagers with respective target-specificities are approved in certain B cell malignancies, a half-life extended TM was developed to allow for dosing every 1-2 weeks. After an extensive screen for an optimized TM targeting CD19 and CD20, we screened multiple RevCAR variants differing in hinge, transmembrane and costimulatory domains for optimal anti-tumor potency using both preclinical in vitro and in vivo models. Profound efficacy and complete tumor control could be observed for optimized RevCAR constructs (Figure 2). While short-term in vitro cytotoxicity assays already gave a small hint towards in vivo efficacy, surprisingly, long-term in vitro rechallenge assay results did not correlate with in vivo efficacy. A summary of functional characterization data will be presented at the meeting with a focus on the lack of predictability of certain in vitro models and underlying hypotheses. Insights from these studies are now flowing into preclinical development of a novel switchable allogeneic CAR-T cell product candidate targeting CD19 and CD20 in B cell malignancies engineered to fully overcome graft-versus-host disease as well as graft rejection by host T and NK cells.

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.

Venetoclax Synergizes with IMGN632, a Novel CD123-Targeting Antibody Conjugated to a DNA Alkylating Payload, By Suppressing DNA Damage Response and Potentiating Apoptosis in Acute Myeloid Leukemia in Vitro Models

CD123, a component of interleukin-3 receptor, is overexpressed on the surface of acute myeloid leukemia (AML) blasts and leukemic stem cells compared with normal hematopoietic progenitors, which makes this antigen an attractive therapeutic target. IMGN632 is a novel antibody-drug-conjugate comprising a high affinity CD123-targeting antibody and DNA alkylating cytotoxic payload. In preclinical studies, IMGN632 demonstrated high potency and selective cytotoxicity against AML leukemic cells vs normal myeloid progenitors, suggesting reduced likelihood of myelosuppression (Kovtun, 2018). Importantly, we previously showed high synergy of IMGN632 combination with BCL-2 inhibitor venetoclax (VEN) and DNA hypomethylating azacytidine (AZA) in AML cell lines and xenograft models (ASH 2020, #617). IMGN632 demonstrated encouraging single agent activity and favorable tolerability in relapsed/refractory AML patients (ASH 2019 #734, ASCO 2020 TPS7563, NCT03386513) and is currently tested in combination with VEN and/ or AZA in relapsed and frontline CD123-positive AML patients (NCT04086264). In this study, we provide new insight into molecular mechanisms underlying the synergistic efficacy of these combinations using AML models in vitro. AML cells with FLT3-ITD mutations expressed higher levels of CD123, as previously reported (Han 2013). FLT3-ITD cell lines were particularly sensitive to IMGN632 with IC 50 values at low ng/ml range. Correspondingly, as evidenced by large scale drug combination screen using BLISS independence model, triple IMGN632/VEN/AZA treatment was synergistic at multiple dosage levels and ratios, suggesting a favorable potency of triple combination in FLT3-mutated subtypes of AML associated with poor prognosis. The efficacy of IMGN632/VEN/AZA combination was however decreased in cells lacking functional TP53. shRNA-mediated knockdown of TP53 in FLT3-ITD MOLM13 cells resulted in increased cell viability, reduced apoptosis and downregulation of cleaved caspase-3 and PARP. While both treated wild-type and TP53 KD cells showed robust overexpression of phosphorylated histone H2AX (p-H2AX), TP53 KD cells expressed higher levels of anti-apoptotic MCL-1 and lower levels of pro-apoptotic BAX, indicative of their intrinsic inability to fully execute apoptosis and resulting in suppressed response to triple combination. Importantly, in sensitive AML cells, IMGN632 induced profound DNA damage, G 1 (MOLM13 and MOLM14 cells) or G 2/M (MV4-11 cells) phase cell cycle arrest and decline in S-phase population, all above augmented by VEN/AZA co-treatment. Observed increase in p-H2AX was accompanied by apoptotic caspase-3 and PARP cleavage and was fully prevented by QVD-OPh caspase inhibitor, suggesting that double-stranded DNA damage and p-H2AX triggered by triple combination were secondary events associated with apoptosis but not the initial cause of cell cycle arrest. Thus, given that cytotoxic payload of IMGN632 is a DNA alkylator, we next tested its impact on mediators of the DNA damage response (DDR). IMGN632 alone induced phosphorylation of p53 at Ser15, a step required for p53 transcriptional activity, and triggered activating phosphorylation of checkpoint kinase Chk1 (Ser345), known to facilitate cell cycle arrest and DNA damage repair, thus preventing treated cells from progressing through S-phase but also increasing the potential of repairing IMGN632-induced DNA damage (Fig. 1A). Upon combined IMGN632+VEN treatment, p53 phosphorylation was sustained, but p-ChK1 decreased, suggesting that VEN may impair protective DDR triggered by IMGN632, resulting in greater cell death. To support this hypothesis, we showed that IMGN632 treatment prior to VEN produced significant synergistic activity (Fig. 1B). Together, these results suggest that VEN, apart from its canonical inhibitory effect on anti-apoptotic BCL-2, exerts previously unrecognized ability to suppress DDR program in AML and augments activity of DNA damaging IMGN632. Failure of cells to sustain DDR in the presence of VEN constitutes a key aspect of high efficacy of IMGN/VEN/AZA combination in AML.

Novel CD123xCD3 Bispecific Igm Antibody, Igm-2537, Potently Induces T-Cell Mediated Cytotoxicity of Acute Myeloid Leukemia Cells In Vivo and in Vitro with Minimal Cytokine Release

Introduction: New therapeutics are needed for the effective treatment of acute myeloid leukemia (AML), as standard chemotherapeutics are poorly tolerated and ~50% of patients relapse primarily due to the incomplete elimination of leukemia stem cells (LSCs). CD123, the IL-3Rα chain, is highly expressed on leukemic blasts and LSCs, and is further increased in patients with poor prognostic factors. Bispecific T-cell engager (TCE) antibodies and CAR-T cells targeting CD123 have shown promising clinical efficacy in AML patients, but cytokine release syndrome limits their therapeutic window and remains a major safety concern. IGM-2537 is a novel pentameric IgM bispecific TCE antibody engineered with ten anti-CD123 binding sites, and an anti-CD3ε single chain Fv domain fused to the joining chain to engage T-cells. Here, we report that IGM-2537 not only potently induces T-cell mediated cytolytic killing of AML cells in vitro and in vivo but also demonstrates minimal cytokine release, and little to no effect on normal hematopoietic progenitor cells and human vascular endothelial cells (HUVECs) with in vitro and ex vivo studies. Additionally, an improved tolerability, excellent pharmacodynamic (PD) response and lower cytokine release was observed with a cynomolgus cross-reactive IgM TCE in monkeys. Materials and Methods:Surface plasma resonance (SPR), cell membrane protein array, and alanine scanning were utilized to characterize human CD123 binding affinity, specificity and epitope mapping, respectively. T-cell dependent cellular cytotoxicity (TDCC) assays were employed to evaluate the potency of IGM-2537 target cytotoxicity, T cell activation and cytokine release. Ex vivo AML colony formation assays were used to assess the activity on AML blasts. Human MV-4-11 and MOLM-13 xenograft tumor models in humanized NSG dKO mice were utilized to determine in vivo anti-tumor activity of IGM-2537. Healthy human PBMCs and bone marrow (BM) samples as well as HUVECs were used in ex vivo assays to address potential safety concerns of IGM-2537 mediated effects on hematopoietic progenitor cells and HUVECs, respectively. Lastly, to confirm that a CD123 directed IgM-based TCE molecule can potentially provide better safety profile in vivo, a cynomolgus-cross reactive IgM TCE comparator was assessed in monkeys for tolerability and PK/PD responses. Results and Conclusions: IGM-2537 bound with high affinity and avidity to CD123. In vitro, IGM-2537 engaged both CD123 and CD3 to induce potent T-cell activation and T-cell mediated cytotoxicity of CD123 + AML cells, and autologous CD123 + basophils and plasmacytoid dendritic cells (pDCs). Though IGM-2537 demonstrated comparable maximal killing activity to a comparator IgG TCE, IGM-2537 demonstrated minimal cytokine release. In ex vivo patient-derived AML or normal BM colony formation assays, IGM-2537 eliminated AML colony forming cells at physiologically relevant effector/target (E/T) ratios but had little or no effect on normal hematopoietic progenitors. In addition, IGM-2537 demonstrated no cytotoxicity against HUVECs regardless of their CD123 expression at basal levels or at maximal levels upregulated by TNF-a and IFN-g, in contrast to a comparator IgG TCE. In vivo, IGM-2537 completely inhibited tumor growth in two AML xenograft tumor models, a subcutaneous MV-4-11 model, and a systemic MOLM-13 model in humanized NSG dKO mice. To further evaluate the potency and safety of the CD123xCD3 IgM bispecific TCE format in vivo, a cynomolgus cross-reactive CD123xCD3 IgM bispecific TCE was evaluated for tolerability and PD responses in cynomolgus monkeys using single doses. All animals tolerated the cross-reactive bispecific IgM TCE well at doses up to 10 mg/kg (the maximal dose level evaluated), a dose 100-fold greater than published doses of a comparator IgG TCE. Complete depletion of CD123 + basophils and substantial reductions of pDCs were seen in blood and BM with minimal to no cytokine induction. In summary, IGM-2537 demonstrated potent in vitro and in vivo T-cell mediated cytotoxicity of AML cell lines with minimal cytokine induction. The encouraging preclinical safety profiles were further supported by little/no effect on normal hematopoietic progenitor cells and HUVECs. These preclinical data support the clinical development of IGM-2537 for the treatment of AML and other hematological malignancies.

BGS-2456 Is a Novel Potent Covalent Inhibitor of FLT3 That Highly Discriminates Against KIT and Is Not Toxic Toward Normal Hematopoiesis in Vitro

Introduction: Class III receptor tyrosine kinases include FLT3, KIT, PDGFRA/B and CSF1R. Activating mutations in FLT3 represent the most common genetic anomalies in AML (30%). While there are numerous examples of KIT TKIs that do not inhibit FLT3 (imatinib, avapritinib, dasatinib), to date, all clinically active FLT3 TKIs (quizartinib, gilteritinib, midostaurin, sorafenib) fail to spare KIT inhibition. Preclinical studies suggest that coinhibition of FLT3 and KIT can result in non-specific myelosuppression, which can limit dose intensification and the ability to safely achieve maintenance dosing. A potent FLT3 TKI that is non-myelosuppressive may enable prolonged treatment in conjunction with chemotherapy and thereby substantially improve clinical outcomes, similar to BCR::ABL1 TKIs in Ph+ ALL (Slayton et al, JCO 2018). FF-10101 is a recently described covalent inhibitor to irreversibly bind FLT3. In vitro and in vivo studies have revealed that FF-10101 exhibits substantial activity against FLT3 and resistant mutants F691L and D835V/Y. However, it does not effectively discriminate against KIT inhibition (Yamara et al, Blood 2018). In clinical experience, a majority of responses were associated with persistent myelosuppression (Levis et al, JCO 2021 39:15_suppl, 7008). Further clinical development of FF-10101 has been abandoned. Here we describe a novel covalent inhibitor (BGS-2456) that potently targets FLT3 with outstanding selectivity against KIT. Results: To confirm that BGS-2456 is a covalent FLT3 inhibitor, we tested various Cys→Ser substitutions in FLT3-ITD. Of these, only substitution of C695 resulted in loss of activity, suggesting that this residue is critical for covalent bond formation with BGS-2456, as has also been demonstrated for FF-10101 (Yamara et al, Blood 2018). Additionally, the compound without the covalent warhead (BGS-2457) was found to be unimpacted by the C695S substitution and substantially less potent than BGS-2456 (Figure). To characterize in vitro properties of BGS-2456 and whether it achieves selectively against FLT3 relative to KIT, we performed cell viability assays of the patient-derived FLT-ITD-positive AML Molm14 cell line as well as the gastrointestinal stromal tumor cell line GIST-T1, which contains an activating juxtamembrane mutation in KIT. We compared EC50 ratios with other FLT3 TKIs and found that BGS-2456 is highly potent, with an EC50 value of ~0.2nM. Moreover, BGS-2456 displayed remarkable selectivity toward FLT3, >10-fold greater than all other TKIs tested. Kinome profiling confirmed FLT3 as the kinase that was most highly inhibited by BGS-2456 among the 300 kinases assessed, with BMX being a distant second (Figure). To assess whether the selectivity of BGS-2456 results in less myelosuppression in vitro, we performed CFU assays of healthy bone marrow. Compared to FF-10101 and gilteritinib, BGS-2456 exhibited the least amount of hematologic toxicity, facilitating in vitro proliferation and differentiation of normal hematopoietic progenitor cells even at 100x EC50 concentration against Molm14 cells. Finally, we assessed BGS-2456's ability to retain activity against the known clinically problematic drug-resistant FLT3-ITD mutants D835Y and F691L. Encouragingly, BGS-2456 retained activity against both D835Y and F691L mutants in the low nanomolar range. Conclusion: This is the first description of a potent and exquisitely specific FLT3 inhibitor that spares KIT inhibition and displays no myelosuppression in vitro at >100x EC50 concentration. The potent inhibitory effects of BGS-2456 on both D835Y and F691L mutants support its promise as a best-in-class TKI for the treatment of FLT3-mutant AML. Efforts to molecularly dissect the basis of the high degree of selectivity of BGS-2456 are ongoing.

CD72 is a pan-tumor antigen associated to pediatric acute leukemia.

In the development of novel immunotherapeutic approaches, the step of target identification is a challenging process, because it aims at identifying robust tumor-associated antigens (TAAs) specific for the pathological population and causing no off-target effects. Here we propose CD72 as a novel and robust TAA for pediatric acute leukemias. We provided an outline of CD72 expression assessed by flow cytometry on a variety of cancer cell lines and primary samples, including normal bone marrow (BM) samples and hematopoietic stem and progenitor cells. We analyzed CD 72 expression on a cohort of 495 pathological pediatric BM aspirates, including: 215 B-cell precursor acute lymphoblastic leukemias (BCP-ALL), 156 acute myeloid leukemias (AMLs), 88 T-lineage ALLs or lymphoblastic lymphomas with BM infiltration, 13 B-lineage lymphoblastic lymphomas with BM infiltration, 9 myelodysplastic syndromes with increased blasts (5%-9% blasts on BM: MDS-IB1) and 14 non-hematopoietic solid tumors infiltrating BM. Results showed that CD72 is highly expressed in almost all BCP-ALL and the majority of AML at diagnosis, including BCP-ALL cases characterized by CD19 loss. These findings support a potential role for advanced diagnostics and novel immunotherapy approaches, providing a pan-ALL and AML target.

Making normal hematopoiesis invisible to CAR T cells

Two recent studies, by Casirati et al. and Wellhausen et al., report genetically engineering normal hematopoietic stem and progenitor cells (HSPCs) to be resistant to chimeric antigen receptor (CAR)-T cells, by changing a single amino acid in the target protein that abrogates CAR binding, without compromising protein function. This allows for selective targeting of cancer cells without harming normal hematopoietic cells.

Abnormal adipogenic signaling in the bone marrow mesenchymal stem cells contributes to supportive microenvironment for leukemia development

BackgroundAcute myeloid leukemia (AML) is an aggressive hematological malignancy, associated with unfavorable patient outcome, primarily due to disease relapse. Mesenchymal stem cells (MSCs) residing in the bone marrow (BM) niche are the source of mesenchyma-derived subpopulations, including adipocytes, and osteocytes, that are critical for normal hematopoiesis. This study aimed to characterize BM-derived adipocyte/osteocyte fractions and their crosstalk with AML cells as a potential mechanism underlying leukemogenesis.MethodsBM cell subpopulations derived from primary AML patients were evaluated using humanized ex-vivo and in-vivo models, established for this study. The models comprised AML blasts, normal hematopoietic stem and progenitor cells and mesenchymal stromal subpopulations. ELISA, FACS analysis, colony forming unit assay, whole exome sequencing and real-time qPCR were employed to assess the differentiation capacity, genetic status, gene expression and function of these cell fractions. To explore communication pathways between AML cells and BM subpopulations, levels of signaling mediators, including cytokines and chemokines, were measured using the ProcartaPlex multiplex immunoassay.ResultsThe study revealed deficiencies in adipogenic/osteogenic differentiation of BM-MSCs derived from AML patients, with adipocytes directly promoting survival and clonogenicity of AML cells in-vitro. In whole exome sequencing of BM-MSC/stromal cells, the AHNAK2 gene, associated with the stimulation of adipocyte differentiation, was found to be mutated and significantly under-expressed, implying its abnormal function in AML. The evaluation of communication pathways between AML cells and BM subpopulations demonstrated pronounced alterations in the crosstalk between these cell fractions. This was reflected by significantly elevated levels of signaling mediators cytokines/chemokines, in AML-induced adipocytes/osteocytes compared to non-induced MSCs, indicating abnormal hematopoiesis. Furthermore, in-vivo experiments using a fully humanized 3D scaffold model, showed that AML-induced adipocytes were the dominant component of the tumor microenvironment, providing preferential support to leukemia cell survival and proliferation.ConclusionsThis study has disclosed direct contribution of impaired functional, genetic and molecular properties of AML patient-derived adipocytes to effective protection of AML blasts from apoptosis and to stimulation of their growth in vitro and in vivo, which overall leads to disease propagation and relapse. The detected AHNAK2 gene mutations in AML-MSCs point to their involvement in the mechanism underlying abnormal adipogenesis.AaFEVfq6u-1YqH7bnQ85uaVideo