Therapeutic Target In Acute Myeloid Leukemia Research Articles

Abstract 7089: Proteomic evaluation of combination data in acute myeloid leukemia to inform mechanism of action and new targets

Abstract Background: Acute myeloid leukemia (AML) is a prevalent adult leukemia. In the US, around 20,380 individuals were diagnosed with AML in 2023. Despite standard therapies, relapse and resistance persist. Cyclin-dependent kinase 9 (CDK9) has been recognized an actionable therapeutic target in AML. The potent inhibitor, AZD4573, induces durable apoptosis and suppresses tumor growth. Our study reveals that combining AZD4573 with standard-of-care (SoC) treatments deepens cell death responses in multiple AML cell models. We also used proteomics tools to understand the mechanisms underlying the vulnerability and resistance in SoC and CDK9 combinations. Methods: In the drug combination study, three SoC agents (cytarabine, decitabine, and venetoclax) were evaluated in combination using a 6 × 6 dose-response matrix in four AML cell models (MOLM13, MOLM16, MV411, and OCI-AML2). The HSA synergy score and Emax drug activity were used to assess the benefits of the drug combinations. In the proteomics evaluation study, MOLM13 cells treated for 7 days under different conditions - (1) vehicle control; (2) cytarabine alone; (3) decitabine alone; (4) venetoclax alone; (5) AZD4573 alone; (6) AZD4573+cytarabine; (7) AZD4573+decitabine; (8) AZD4573+venetoclax - were collected for proteomics analysis using LC-MS/MS. Differential expression and missing pattern analyses were used to identify dysregulated proteins. Functional enrichment analysis was employed to identify dysregulated biological processes and pathways in response to different treatments. Results: Our drug combination screens demonstrated the efficacy of AZD4573 in combination with cytarabine, decitabine, and venetoclax in 3 of 4 AML cell models. In our proteomic analysis, we took two approaches. First, we compared the proteomes of AML cells treated with SoC agents, identifying shared downregulated pathways such as serine biosynthesis, notably pointing to the rate-limiting enzyme PHGDH as a potential therapeutic target in AML. Cytarabine and decitabine induced an inflammatory response, while venetoclax increased the expression of cell division regulators like Aurora kinase B (AURKB). Interestingly, targeting AURKB with AZD2811 has been shown to overcome venetoclax resistance in AML. Second, we compared proteome changes induced by various AZD4573 combinations. We identified IRF2BP2 as a shared vulnerable protein across the three AZD4573 combinations. POUF2F2 expression was significantly induced by cytarabine but suppressed when combined with AZD4573. Given that POUF2F2 activates the PDK1-Akt pathway, our findings suggest that AZD4573-induced POUF2F2 downregulation suppresses the PDK1-AKT pathway, inhibiting tumor progression. Conclusions: Our proteomics analysis identifies vulnerable and resistant pathways and proteins, shedding light on the mechanism of action and revealing potential new targets for AML. Citation Format: Lori Chan, Zuleyha Ozen, Anthony Iannetta, Eric Miele, Funda Kar, Andrew Jarnuczak, Geoff Nelson, Aleksandra Markovets, Justine Roderick-Richardson, Jelena Urosevic, Lisa Drew, Jerome Mettetal. Proteomic evaluation of combination data in acute myeloid leukemia to inform mechanism of action and new targets [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 7089.

The MYC-NFATC2 axis maintains the cell cycle and mitochondrial function in acute myeloid leukaemia cells.

Acute myeloid leukaemia (AML) is a clonal haematological malignancy affecting the myeloid lineage, with generally poor patient outcomes owing to the lack of targeted therapies. The histone lysine demethylase 4A (KDM4A) has been established as a novel therapeutic target in AML, due to its selective oncogenic role within leukaemic cells. We identify that the transcription factor nuclear factor of activated T cells 2 (NFATC2) is a novel binding and transcriptional target of KDM4A in the human AML THP-1 cell line. Furthermore, cytogenetically diverse AML cell lines, including THP-1, were dependent on NFATC2 for colony formation in vitro, highlighting a putative novel mechanism of AML oncogenesis. Our study demonstrates that NFATC2 maintenance of cell cycle progression in human AML cells was driven primarily by CCND1. Through RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq), NFATc2 was shown to bind to the promoter region of genes involved in oxidative phosphorylation and subsequently regulate their gene expression in THP-1 cells. Furthermore, our data show that NFATC2 shares transcriptional targets with the transcription factor c-MYC, with MYC knockdown phenocopying NFATC2 knockdown. These data suggest a newly identified co-ordinated role for NFATC2 and MYC in the maintenance of THP-1 cell function, indicative of a potential means of therapeutic targeting in human AML.

The multi-CDK inhibitor dinaciclib reverses bromo- and extra-terminal domain (BET) inhibitor resistance in acute myeloid leukemia via inhibition of Wnt/β-catenin signaling

Acute myeloid leukemia (AML) is a highly aggressive hematologic cancer with poor survival across a broad range of molecular subtypes. Development of efficacious and well-tolerable therapies encompassing the range of mutations that can arise in AML remains an unmet need. The bromo- and extra-terminal domain (BET) family of proteins represents an attractive therapeutic target in AML due to their crucial roles in many cellular functions, regardless of any specific mutation. Many BET inhibitors (BETi) are currently in pre-clinical and early clinical development, but acquisition of resistance continues to remain an obstacle for the drug class. Novel methods to circumvent this development of resistance could be instrumental for the future use of BET inhibitors in AML, both as monotherapy and in combination. To date, many investigations into possible drug combinations of BETi with CDK inhibitors have focused on CDK9, which has a known physical and functional interaction with the BET protein BRD4. Therefore, we wished to investigate possible synergy and additive effects between inhibitors of these targets in AML. Here, we describe combination therapy with the multi-CDK inhibitor dinaciclib and the BETi PLX51107 in pre-clinical models of AML. Dinaciclib and PLX51107 demonstrate additive effects in AML cell lines, primary AML samples, and in vivo. Further, we demonstrate novel activity of dinaciclib through inhibition of the canonical/β-catenin dependent Wnt signaling pathway, a known resistance mechanism to BETi in AML. We show dinaciclib inhibits Wnt signaling at multiple levels, including downregulation of β-catenin, the Wnt co-receptor LRP6, as well as many Wnt pathway components and targets. Moreover, dinaciclib sensitivity remains unaffected in a setting of BET resistance, demonstrating similar inhibitory effects on Wnt signaling when compared to BET-sensitive cells. Ultimately, our results demonstrate rationale for combination CDKi and BETi in AML. In addition, our novel finding of Wnt signaling inhibition could have potential implications in other cancers where Wnt signaling is dysregulated and demonstrates one possible approach to circumvent development of BET resistance in AML.

Regulatory function and mechanism research for m6A modification WTAP via SUCLG2-AS1- miR-17-5p-JAK1 axis in AML

Acute myeloid leukemia (AML), characterized by the abnormal accumulation of immature marrow cells in the bone marrow, is a malignant tumor of the blood system. Currently, the pathogenesis of AML is not yet clear. Therefore, this study aims to explore the mechanisms underlying the development of AML. Firstly, we identified a competing endogenous RNA (ceRNA) SUCLG2-AS1-miR-17-5p-JAK1 axis through bioinformatics analysis. Overexpression of SUCLG2-AS1 inhibits proliferation, migration and invasion and promotes apoptosis of AML cells. Secondly, luciferase reporter assay and RIP assay validated that SUCLG2-AS1 functioned as ceRNA for sponging miR-17-5p, further leading to JAK1 underexpression. Additionally, the results of MeRIP-qPCR and m6A RNA methylation quantification indicted that SUCLG2-AS1(lncRNA) had higher levels of m6A RNA methylation compared with controls, and SUCLG2-AS1 is regulated by m6A modification of WTAP in AML cells. WTAP, one of the main regulatory components of m6A methyltransferase complexes, proved to be highly expressed in AML and elevated WTAP is associated with poor prognosis of AML patients. Taken together, the WTAP-SUCLG2-AS1-miR-17-5p-JAK1 axis played essential roles in the process of AML development, which provided a novel therapeutic target for AML.

Oncogenic lncRNA FAM215A promotes the malignant cell phenotypes of acute myeloid leukemia (AML) cell lines.

Acute myeloid leukemia (AML) is a form of blood cancer that arise as a result of clonal proliferation of malignant myeloid precursors acquiring genetic abnormalities. Primary resistance to initial treatment and disease recurrence continues to be huge challenge in treating AML. Herein, GSE114868 was analyzed for differentially-expressed lncRNAs between AML patients' mononucleated cells and healthy normal control mononucleated cells and 191 lncRNAs were significantly deregulated in AML patients' mononucleated cells. The correlation between candidate lncRNAs and AML patients' overall survival was analyzed and 6 lncRNAs, including MIR181A1HG, TRAF3IP2-AS1, STARD4-AS1, E2F3-IT1, FAM215A, and HHIP-AS1 were dramatically linked to AML patients' OS. Using a Cox proportional-hazards model, we identified risk factors and found FAM215A as a risk factor for AML patients' prognosis. The expression level of FAM215A showed to be upregulated within blood samples and cells. Genes correlated with FAM215A were correlated to cell division, modulation of cell apoptosis, and modulation of programmed cell death. FAM215A knockdown inhibited AML cell viability, elicited G0/G1-phase arrest of cell cycle, enhanced cell apoptosis, increased proapoptotic Bax and cleaved-caspase3 levels, and decreased antiapoptotic Bcl2. FAM215A overexpression exerted opposite effects on AML cells. Conclusively, FAM215A serves as an oncogenic lncRNA in AML, promoting cell viability, relieving cell cycle arrest, and suppressing cell apoptosis. FAM215A might be un underlying biological prognostic marker and therapeutic target for AML.

Suppression of DNMT1 combined with ATM or ATR inhibitor as a therapeutic combination of acute myeloid leukemia.

The potential treatment option of targeting DNA methyltransferase 1 (DNMT1) has been explored, but further investigation is required to assess the efficacy of combination therapy in acute myeloid leukemia (AML). In this study, bioinformatics and online databases were utilized to select the combined therapeutic targets. The potential kinases associated with DNMT1-related genes in AML were analyzed using the Cancer Genome Atlas (TCGA) database and X2K Appyter (Expression2Kinases) database. In-vitro evaluations were conducted to assess the synergistic effects between DNMT1 and ATR/ATM in five AML cell lines (MOLM-16, NB-4, HEL 92.1.7, HEL, EOL-1). In our study, ATR and ATM are primarily the kinases associated with DNMT1-related genes in AML. We observed a significant upregulation of DNMT1, ATR, and ATM expression in AML tissues and cell lines. The five AML cell lines demonstrated sensitivity to monotherapy with GSK-368, AZD-1390, or AZD-6738 (EC50 value ranges from 5.461 to 7.349 nM, 5.821 to 10.120 nM, and 7.618 to 10.100 nM, respectively). A considerable synergistic effect was observed in AML cell lines when combining GSK-368 and AZD-1390, GSK-368 and AZD-6738, or AZD-1390 and AZD-6738, resulting in induced cell apoptosis and inhibited cell growth. DNMT1, ATM, and ATR possess potential as therapeutic targets for AML. Both individual targeting and combination targeting of these molecules have been confirmed as promising therapeutic approaches for AML.

Reduced Proteolipid Protein 2 promotes endoplasmic reticulum stress-related apoptosis and increases drug sensitivity in acute myeloid leukemia.

The Proteolipid Protein 2 (PLP2), a protein in the Endoplasmic Reticulum (ER) membrane, has been reported to be highly expressed in various tumors. Previous studies have demonstrated that the reduced PLP2 can induce apoptosis and autophagy through ER stress-related pathways, leading to a decreased proliferation and aggressiveness. However, there is no research literature on the role of PLP2 in Acute Myeloid Leukemia (AML). PLP2 expression, clinical data, genetic mutations, and karyotype changes from GEO, TCGA, and timer2.0 databases were analyzed through the R packages. The possible functions and pathways of cells were explored through GO, KEGG, and GSEA enrichment analysis using the clusterProfiler R package. Immuno-infiltration analysis was conducted using the Cibersort algorithm and the Xcell R package. RT-PCR and western blot techniques were employed to identify the PLP2 expression, examine the knockdown effects in THP-1 cells, and assess the expression of genes associated with endoplasmic reticulum stress and apoptosis. Flow cytometry was utilized to determine the apoptosis and survival rates of different groups. PLP2 expression was observed in different subsets of AML and other cancers. Enrichment analyses revealed that PLP2 was involved in various tumor-related biological processes, primarily apoptosis and lysosomal functions. Additionally, PLP2 expression showed a strong association with immune cell infiltration, particularly monocytes. In vitro, the knockdown of PLP2 enhanced endoplasmic reticulum stress-related apoptosis and increased drug sensitivity in THP-1 cells. PLP2 could be a novel therapeutic target in AML, in addition, PLP2 is a potential endoplasmic reticulum stress regulatory gene in AML.

Immunoproteasome function maintains oncogenic gene expression in KMT2A-complex driven leukemia

Pharmacologic targeting of chromatin-associated protein complexes has shown significant responses in KMT2A-rearranged (KMT2A-r) acute myeloid leukemia (AML) but resistance frequently develops to single agents. This points to a need for therapeutic combinations that target multiple mechanisms. To enhance our understanding of functional dependencies in KMT2A-r AML, we have used a proteomic approach to identify the catalytic immunoproteasome subunit PSMB8 as a specific vulnerability. Genetic and pharmacologic inactivation of PSMB8 results in impaired proliferation of murine and human leukemic cells while normal hematopoietic cells remain unaffected. Disruption of immunoproteasome function drives an increase in transcription factor BASP1 which in turn represses KMT2A-fusion protein target genes. Pharmacologic targeting of PSMB8 improves efficacy of Menin-inhibitors, synergistically reduces leukemia in human xenografts and shows preserved activity against Menin-inhibitor resistance mutations. This identifies and validates a cell-intrinsic mechanism whereby selective disruption of proteostasis results in altered transcription factor abundance and repression of oncogene-specific transcriptional networks. These data demonstrate that the immunoproteasome is a relevant therapeutic target in AML and that targeting the immunoproteasome in combination with Menin-inhibition could be a novel approach for treatment of KMT2A-r AML.

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.

Synthetic Lethal Interactions with IRAK4 Inhibition in Myeloid Malignancies

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

A CD38/CD28xCD3 Trispecific T-Cell Engager (TCE) As a Potentially Active Agent for the Treatment of Older Patients with Acute Myeloid Leukemia (AML)

Background: Refractory disease is common and the outcome of elderly AML patients unable to receive intensive chemotherapy with acceptable side effects remains dismal. Thus, the identification of effective and safer drugs in AML is warranted. Immunotherapeutic strategies have shown extraordinary efficacy for the treatment of hematological malignancies, such as anti-CD38 monoclonal antibodies (mAbs) in multiple myeloma. Indeed, CD38 could represent a potential therapeutic target for AML too, not only because it is expressed in AML blasts, but also because AML patients with heterogeneous or low CD38 expression could benefit from T-cell based immunotherapies that are less dependent of the density of the targeted antigen. Aims: To evaluate CD38 as a potential therapeutic target in AML, and to determine the mode of action and preclinical efficacy of isatuximab (an IgG1 anti-CD38 mAb) and SAR442257, which is a new CD38/CD28xCD3 trispecific TCE. Results: We evaluated CD38 expression in bone marrow blasts from 241 newly diagnosed elderly AML patients by flow cytometry. Overall, 99.2% of AML patients expressed CD38, 55.6% with homogeneous pattern and 44.6% with heterogeneous pattern. CD38 expression had no impact in the overall survival of AML patients. Interestingly, CD38 expression increased 2-fold in MRD positive leukemic blasts compared with patient-matched blasts at diagnosis (n=10). At 24h and up to 96h, isatuximab (70 nM) decreased CD38 surface level in 3 AML cell lines expressing low (KG-1), intermediate (MOLM-13) and high or similar (OCI-AML3) basal CD38 density as compared to AML patients. Isatuximab (70 nM) did not induce complement-dependent cytotoxicity; by contrast, we observed a trend to an increased antibody-dependent cellular phagocytosis and significant antibody-dependent cellular cytotoxicity (ADCC) only in OCI-AML3 cells in the presence of purified NK cells. In 17 primary samples from AML patients with homogeneous vs heterogeneous CD38 expression, isatuximab (70 nM) showed a median of 19% and 4.5% tumor-cell death, respectively. Altogether, these findings indicated an association between CD38 density and the mode of action triggered by isatuximab and its efficacy. As such, AML patients with heterogeneous CD38 expression are less likely to respond to isatuximab, but might benefit from TCE that are less dependent of the density of the targeted antigen. Contrary to isatuximab, the CD38/CD28xCD3 TCE did not induce a dose- and time-dependent decrease in CD38 surface levels in any AML cell line measured with a non-competing anti-CD38 antibody, regardless of the CD38 antigen density. When we cultured PBMCs from healthy adults (n=6) with MOLM-13 cells (E:T ratio of 10:1) in the presence of isatuximab or the CD38/CD28xCD3 TCE (700 pM), we observed a significantly higher tumor cell lysis at 48h induced by the latter. The same effect of the CD38/CD28xCD3 TCE was observed with purified T-, but not NK-cells from healthy adults (n=3). To identify the immune cells involved in tumor cell lysis, we confirmed that isatuximab only induced NK-cell activation, whereas the CD38/CD28xCD3 TCE induced both T- and NK-cell activation, measured as percentage of CD69+ cells. Co-culture of purified NK- or T-cells with MOLM-13 cells in the presence of the CD38/CD28xCD3 TCE demonstrated a T-cell dependent bystander activation of NK cells. Furthermore, isatuximab downregulated CD38 in a dose-dependent manner, especially in NK cells, while the CD38/CD28xCD3 TCE did not. Surprisingly, the CD38/CD28xCD3 TCE induced CD38 upregulation on T cells, indicating again T-cell activation. The efficacy of isatuximab and the CD38/CD28xCD3 TCE was analyzed in primary AML samples (n=8) and blast lysis was observed in two and three samples, respectively. Notably, two of the three CD38/CD28xCD3 TCE-responders were resistant to isatuximab. CD38/CD28xCD3 TCE-mediated lysis was independent of CD38 expression and induced no significant off-target toxicity. Conclusions: CD38 is widely present in blasts from older AML patients but nearly half show heterogeneous expression. While isatuximab-driven ADCC in AML cell lines and primary samples is dependent on CD38 density in tumor cells, the CD38/CD28xCD3 TCE exerted its anti-tumor efficacy regardless of CD38 density. Thus, AML patients expressing both high and low/heterogenous levels of CD38 could benefit from T cell based immunotherapeutic strategies targeting CD38.

LSD1 Inhibition Enhances Antigen Presentation and Co-Stimulation in AML to Promote T Cell-Mediated Anti-Leukemia Immune Responses

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can provide a curative treatment option for acute myeloid leukemia (AML). Donor T cells play an important role in the curative potential of allo-HSCT by recognizing and eliminating residual leukemic cells, inducing the desirable graft-versus-leukemia (GVL) effect. Evading from this effect of donor T cell immune surveillance through the downregulation of human leukocyte antigen class II (HLA-II) molecules has been shown to contribute to AML relapse after allo-HSCT. Lysine-specific demethylase 1 (LSD1) is an emerging epigenetic therapeutic target in AML due to its role in regulating myeloid differentiation pathways. We hypothesized that LSD1 inhibition has the potential to enhance anti-leukemic immune responses by differentiating AML cells and inducing the acquisition of markers typical for antigen-presenting cells. We have studied these effects by treating human AML cell lines with the LSD1 inhibitor bomedemstat and investigated the expression of antigen-presenting molecules by flow cytometry and quantitative PCR (qPCR). At a concentration of 100 nM, bomedemstat upregulated the expression of CIITA by a log2 fold change of 2.23 ± 0.68 (p < 0.01) in OCI-AML3, 2.17 ± 0.28 (p < 0.01) in HL60 and 1.80 ± 0.67 (p < 0.05) in MOLM-13. To assess if the observed upregulation of CIITA can lead to a re-expression of HLA-DR in these HLA-DR negative cell lines, we then treated these cell lines with 100 nM of bomedemstat with additional IFN-γ stimulation. Expression of HLA-DR and the co-stimulatory molecule CD86 was determined by flow cytometry. Bomedemstat increased the expression of HLA-DR and CD86 across all three cell lines leading to the generation of 26.63 ± 3.07% (p < 0.0001) HLA-DR +/CD86 + cells in OCI-AML3, 4.19 ± 1.68% (p < 0.001) HLA-DR +/CD86 + cells in HL-60 and 14.67 ± 2.03% (p < 0.001) HLA-DR +/CD86 + cells in MOLM-13. We further assessed the effects of bomedemstat treatment on cytokine production by qPCR. Notably, bomedemstat treatment increased the expression of CXCL-10 by a log2 fold change of 2.59 ± 0.66 (p < 0.001) in MOLM-13 and 4.12 ± 0.88 (p < 0.001) in HL-60 at 100 nM concentration. The upregulation of CXCL-10 was also validated using bead-based ELISA assays. To investigate the molecular mechanism of the observed concomitant upregulation of MHC-II molecules and CD86, we then used murine myeloid progenitor cells transformed by overexpression of Hoxa9 and Meis1 (H9M) or MN1. In H9M-transformed cells, bomedemstat concurrently upregulated MHC-II (I-A/I-E) and CD86 even in the absence of IFN-γ stimulation, leading to an increase in MHC-II +/CD86 + cells from 2.52 ± 1.22% at baseline to 29.95 ± 14.11 (p < 0.05). This effect was not observed in MN1-transformed cells. H9M-transformed progenitor cells from Irf8 knockout mice (H9M- Irf8-KO) did not show any upregulation of MHC-II, pointing towards a role for Irf8 in this process. To interrogate whether the concurrent upregulation of MHC-II and CD86 can enhance CD4 + T cell activation, we used ovalbumin-expressing H9M-transformed cells that were co-cultured with OT-II T cells after bomedemstat treatment. We could show that bomedemstat-treatment enhanced the ability of H9M-OVA cells to activate T cells as demonstrated by an enhanced expression of the T cell activation markers CD25 and CD69. Furthermore, bomedemstat also increased the immune killing of OVA-peptide pulsed H9M cells at an effector-to-target (E:T) ratio of 1:1 from 1.12 ± 4.01 to 19.70 ± 6.20% (p < 0.01). It also enhanced the killing of OVA-expressing H9M cells from 5.80 ± 2.95% to 17.70 ± 3.16% (p < 0.05). No significant killing effect was observed for MN1 cells. In conclusion, we demonstrate both phenotypically and functionally that LSD1 inhibition by bomedemstat can enhance the immunogenicity of human and murine AML models. These findings point towards a potential role of LSD1 inhibition as a maintenance therapy after allo-HSCT.

Prognostic and therapeutic implications of iron-related cell death pathways in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a blood cancer that is diverse in terms of its molecular abnormalities and clinical outcomes. Iron homeostasis and cell death pathways play crucial roles in cancer pathogenesis, including AML. The objective of this study was to examine the clinical significance of genes involved in iron-related cell death and apoptotic pathways in AML, with the intention of providing insights that could have prognostic implications and facilitate the development of targeted therapeutic interventions. Gene expression profiles, clinical information, and molecular alterations were integrated from multiple datasets, including TCGA-LAML and GSE71014. Our analysis identified specific molecular subtypes of acute myeloid leukemia (AML) displaying varying outcomes, patterns of immune cell infiltration, and profiles of drug sensitivity for targeted therapies based on the expression of genes involved in iron-related apoptotic and cell death pathways. We further developed a risk model based on four genes, which demonstrated promising prognostic value in both the training and validation cohorts, indicating the potential of this model for clinical decision-making and risk stratification in AML. Subsequently, Western blot analysis showed that the expression levels of C-Myc and CyclinD1 were significantly reduced after CD4 expression levels were knocked down. The findings underscore the potential of iron-related cell death pathways as prognostic biomarkers and therapeutic targets in AML, paving the way for further research aimed at understanding the molecular mechanisms underlying the correlation between iron balance, apoptosis regulation, and immune modulation in the bone marrow microenvironment.

The prognostic value of 19S ATPase proteasome subunits in acute myeloid leukemia and other forms of cancer.

The ubiquitin-proteasome system (UPS) is an intracellular organelle responsible for targeted protein degradation, which represents a standard therapeutic target for many different human malignancies. Bortezomib, a reversible inhibitor of chymotrypsin-like proteasome activity, was first approved by the FDA in 2003 to treat multiple myeloma and is now used to treat a number of different cancers, including relapsed mantle cell lymphoma, diffuse large B-cell lymphoma, colorectal cancer, and thyroid carcinoma. Despite the success, bortezomib and other proteasome inhibitors are subject to severe side effects, and ultimately, drug resistance. We recently reported an oncogenic role for non-ATPase members of the 19S proteasome in chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and several different solid tumors. In the present study, we hypothesized that ATPase members of the 19S proteasome would also serve as biomarkers and putative therapeutic targets in AML and multiple other cancers. We used data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) available at UALCAN and/or GEPIA2 to assess the expression and prognostic value of proteasome 26S subunit, ATPases 1-6 (PSMC1-6) of the 19S proteasome in cancer. UALCAN was also used to associate PSMC1-6 mRNA expression with distinct clinicopathological features. Finally, cBioPortal was employed to assess genomic alterations of PSMC genes across different cancer types. The mRNA and protein expression of PSMC1-6 of the 19S proteasome were elevated in several cancers compared with normal controls, which often correlated with worse overall survival. In contrast, AML patients demonstrated reduced expression of these proteasome subunits compared with normal mononuclear cells. However, AML patients with high expression of PSMC2-5 had worse outcomes. Altogether, our data suggest that components of the 19S proteasome could serve as prognostic biomarkers and novel therapeutic targets in AML and several other human malignancies.

Apicidin confers promising therapeutic effect on acute myeloid leukemia cells via increasing QPCT expression

ABSTRACT Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by abnormal cell proliferation, apoptosis repression and myeloid differentiation blockade of hematopoietic stem/progenitor cells. Developing and identifying novel therapeutic agents to reverse the pathological processes of AML are of great significance. Here in this study, we found that a fungus-derived histone deacetylase inhibitor, Apicidin, presents promising therapeutic effect on AML by inhibiting cell proliferation, facilitating apoptosis and inducing myeloid differentiation of AML cells. Mechanistic investigation revealed that QPCT is identified as a potential downstream target of Apicidin, which exhibits significantly decreased expression in AML samples compared with the normal controls and is remarkably up-regulated in AML cells upon Apicidin management. Functional study and rescue assay demonstrated that QPCT depletion further promotes cell proliferation, inhibits apoptosis and impairs myeloid differentiation of AML cells, alleviating the anti-leukemic effect of Apicidin on AML. Our findings not only provide novel therapeutic target for AML, but also lay theoretical and experimental foundation for the clinical application of Apicidin in AML patients.

Classification of Acute Myeloid Leukemia by Cell-Free DNA 5-Hydroxymethylcytosine.

Epigenetic abnormality is a hallmark of acute myeloid leukemia (AML), and aberrant 5-hydroxymethylcytosine (5hmC) levels are commonly observed in AML patients. As epigenetic subgroups of AML correlate with different clinical outcomes, we investigated whether plasma cell-free DNA (cfDNA) 5hmC could categorize AML patients into subtypes. We profiled the genome-wide landscape of 5hmC in plasma cfDNA from 54 AML patients. Using an unbiased clustering approach, we found that 5hmC levels in genomic regions with a histone mark H3K4me3 classified AML samples into three distinct clusters that were significantly associated with leukemia burden and survival. Cluster 3 showed the highest leukemia burden, the shortest overall survival of patients, and the lowest 5hmC levels in the TET2 promoter. 5hmC levels in the TET2 promoter could represent TET2 activity resulting from mutations in DNA demethylation genes and other factors. The novel genes and key signaling pathways associated with aberrant 5hmC patterns could add to our understanding of DNA hydroxymethylation and highlight the potential therapeutic targets in AML. Our results identify a novel 5hmC-based AML classification system and further underscore cfDNA 5hmC as a highly sensitive marker for AML.

Prolactin receptor signaling induces acquisition of chemoresistance and reduces clonogenicity in acute myeloid leukemia

BackgroundDevelopment of precision medicine requires the identification of easily detectable and druggable biomarkers. Despite recent targeted drug approvals, prognosis of acute myeloid leukemia (AML) patients needs to be greatly improved, as relapse and refractory disease are still difficult to manage. Thus, new therapeutic approaches are needed. Based on in silico-generated preliminary data and the literature, the role of the prolactin (PRL)-mediated signaling was interrogated in AML.MethodsProtein expression and cell viability were determined by flow cytometry. Repopulation capacity was studied in murine xenotransplantation assays. Gene expression was measured by qPCR and luciferase-reporters. SA-β-Gal staining was used as a senescence marker.ResultsThe prolactin receptor (PRLR) was upregulated in AML cells, as compared to their healthy counterpart. The genetic and molecular inhibition of this receptor reduced the colony-forming potential. Disruption of the PRLR signaling, either using a mutant PRL or a dominant-negative isoform of PRLR, reduced the leukemia burden in vivo, in xenotransplantation assays. The expression levels of PRLR directly correlated with resistance to cytarabine. Indeed, acquired cytarabine resistance was accompanied with the induction of PRLR surface expression. The signaling associated to PRLR in AML was mainly mediated by Stat5, in contrast to the residual function of Stat3. In concordance, Stat5 mRNA was significantly overexpressed at mRNA levels in relapse AML samples. A senescence-like phenotype, measured by SA-β-gal staining, was induced upon enforced expression of PRLR in AML cells, partially dependent on ATR. Similar to the previously described chemoresistance-induced senescence in AML, no cell cycle arrest was observed. Additionally, the therapeutic potential of PRLR in AML was genetically validated.ConclusionsThese results support the role of PRLR as a therapeutic target for AML and the further development of drug discovery programs searching for specific PRLR inhibitors.

Circular RNAs: pivotal role in the leukemogenesis and novel indicators for the diagnosis and prognosis of acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy and affected patients have poor overall survival (OS) rates. Circular RNAs (circRNAs) are a novel class of non-coding RNAs (ncRNAs) with a unique loop structure. In recent years, with the development of high-throughput RNA sequencing, many circRNAs have been identified exhibiting either up-regulation or down-regulation in AML patients compared with healthy controls. Recent studies have reported that circRNAs regulate leukemia cell proliferation, stemness, and apoptosis, both positively and negatively. Additionally, circRNAs could be promising biomarkers and therapeutic targets in AML. In this study, we present a comprehensive review of the regulatory roles and potentials of a number of dysregulated circRNAs in AML.

Abstract 1732: Functional characterization of the impact of CD36 deletion on normal hematopoiesis and the leukemia microenvironment

Abstract CD36 has been identified as a potential therapeutic target both in the leukemic cells and in the tumor immune microenvironment. CD36 also plays a role in lipid metabolism of cancer associated T-cells leading to impaired cytotoxic CD8+ T-cell and enhanced Treg cell function. In acute myeloid leukemia (AML), we found that APOC2 acts with CD36 to promote leukemia growth by activating the LYN-ERK signaling. To establish CD36 as a viable therapeutic target in AML, we recently investigated whether targeting CD36 has any detrimental impact on normal hematopoietic cells. We compared B6.129S1-Cd36tm1Mfe/J Cd36 knockout (Cd36-KO) with C57BL/6J control (WT) mice (N=6 per group). Cd36-KO mice have normal blood counts except for lower red blood cells count (5.85 vs 6.93 (x 106/μL), P = 0.0332), hemoglobin (10.03 vs 11.35 (gr/dL), P = 0.0109), and hematocrit (31.67 vs 35.48 (%), P = 0.02) than WT mice, yet these values remained within the normal range. Cd36-KO mice exhibited similar spleen T cell phenotypes compared with the WT mice and expanded similarly in vitro in the presence IL2 and PHA. The in vitro expansion of hematopoietic stem and progenitor cells (HSPC) in HSPC expansion media containing SCF and TPO cytokines was also similar between Cd36-KO mice and WT mice. However, mouse colony forming cell assay using methylcellulose-based media showed that the HSPC of Cd36-KO mice resulted in 40% fewer colonies than WT mouse HSPC cells (P = 0.0003). However, when HSPC obtained from Cd36-KO and WT mice were transplanted in CD45.1 mice, their percentage of engraftment were not significantly different in the bone marrow (BM: KO vs WT: 64.92% vs 63.45%, P > 0.9999), spleen (40.88% vs 40.05%, P = 0.8413), and blood (83.47% vs 82.88%, P > 0.9999). To assess whether CD36 in the leukemia microenvironment has an impact on leukemia growth and progression, we injected 5 x 106 FLT3-ITD/MLL-PTD mouse leukemic cells via tail vein in either Cd36-KO mice (N = 5) or WT mice (N = 6) and compared the percentage of engraftment in their tissues. Cd36-KO mice exhibited similar leukemia engraftment in the BM (88.92% vs 86.38%, P = 0.0497), spleen (76.48% vs 68.10%, P = 0.1627), liver (16.12% vs 6.70%, P = 0.2521), and blood (16.33% vs 0.96%, P = 0.2504) compared with the WT group. Cd36-KO and WT mice developed similar leukemia burden when less aggressive models are used with the engraftment of 1 x 106 FLT3-ITD/MLL-PTD mouse leukemic cells. Altogether, our preliminary data suggest that loss of Cd36 may influence the hematopoietic stem cell and erythropoiesis yet have limited detrimental overall impact on normal hematopoiesis and leukemia microenvironment. Therefore, targeting CD36 in leukemic cells presents limited toxicity on normal hematopoiesis and therapeutic approaches to target this receptor present safe strategies to treat AML. Citation Format: Yiting Meng, Mateusz Pospiech, Atham Ali, Ritu Chandwani, Sandra Onyemaechi, George Yaghmour, Houda Alachkar. Functional characterization of the impact of CD36 deletion on normal hematopoiesis and the leukemia microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1732.

Loss of IRF8 inhibits the growth of acute myeloid leukemia cells.

The transcription factor interferon regulatory factor 8 (IRF8), as a member of the IRF family, is essential for myeloid cell differentiation. However, the precise role of IRF8 in the pathogenesis of acute myeloid leukemia (AML) remains unknown. By using multivariate analysis, we discovered that high IRF8 expression was an independent poor predictor of overall survival (OS) in AML patients from our clinical follow-up study. The proliferation of three AML cell lines was significantly inhibited by shRNA-mediated knockdown of IRF8, owing to cell cycle S-phase arrest. Furthermore, we demonstrated that knocking down IRF8 could suppress the expression of CyclinA and CyclinB1, resulting in a shift in cell cycle distribution. Loss of IRF8 in AML cells decreased the expression of STAT3 and phosphor-STAT3 (pSTAT3), which are key factors in JAK/STAT signal pathway and are important for AML progression. Using a xenograft mouse model, we discovered the antiproliferative effect of losing IRF8 in vivo. In conclusion, this study found that IRF8 may play a prognostic factor and therapeutic target in AML.