Therapeutic Target In Acute Myeloid Leukemia Research Articles

PRDM16 Induces Methylation of FLT3 to Promote FLT3-ITD Signaling and Leukemia Progression.

Internal tandem duplication (ITD) in the FMS-like receptor tyrosine kinase-3 (FLT3) is one of the most frequent mutations in acute myeloid leukemia (AML) and is associated with poor prognosis. FLT3-ITD mutations result in endoplasmic reticulum (ER) retention and constitutive autophosphorylation of FLT3. The PR/SET domain 16 (PRDM16) is highly expressed in FLT3-ITD+ AML patients, suggesting it might play a role in leukemogenesis. Here, we revealed that genetic and pharmacological suppression of PRDM16 greatly slowed the progression of FLT3-ITD-driven leukemia, sensitized leukemic cells to tyrosine kinase inhibitors (TKIs), and extended the survival of leukemic mice. PRDM16 enhanced activation of oncogenic FLT3-ITD and ligand-dependent activation of wild-type FLT3 in leukemic cells. Mechanistically, PRDM16 mediated monomethylation of FLT3-ITD at lysine 614 and promoted its ER localization, resulting in enhanced FLT3 signaling in leukemia cells. Moreover, pharmacological suppression of FLT3-ITD methylation in combination with TKIs increased the elimination of FLT3-ITD+ AML cells. Altogether, these results suggest that PRDM16 boosts oncogenic FLT3 signaling in leukemic cells by prompting FLT3-ITD methylation. Therefore, PRDM16 may serve as a therapeutic target for AML.

Expanding the therapeutic horizon in acute myeloid leukemia: The role of phosphoinositide-3-kinase-γ inhibition

Abstract Acute myeloid leukemia (AML) remains a formidable challenge due to its high relapse rate and limited therapeutic options. This study investigates the role of phosphoinositide-3-kinase-γ (PI3Kγ) in AML and explores the therapeutic potential of the PI3Kγ inhibitor, eganelisib, in preclinical models. Using genome-wide CRISPR interference screening, we identified PI3Kγ as a critical survival pathway in certain AML subsets. We demonstrated that inhibition of PI3Kγ with eganelisib disrupts oxidative phosphorylation, thereby impeding AML cell survival and proliferation. Notably, the combination of eganelisib with cytarabine showed enhanced efficacy, suggesting a synergistic interaction that improves treatment outcomes. These findings indicate that PI3Kγ is a viable therapeutic target in AML and that PI3Kγ inhibitors could be pivotal in overcoming treatment resistance and reducing relapse.

Mitochondrial dynamics as a potential therapeutic target in acute myeloid leukemia.

Acute myeloid leukemia (AML) cells are highly dependent on oxidative phosphorylation and the mitochondrial dynamics regulated by fusion-related genes MFN1, MFN2, and OPA1 and fission-related genes DNM1L and MFF. An analysis of previously published gene expression datasets showed that high expression of MFF was significantly associated with poor prognosis in patients with AML. Based on this finding, we investigated the impact of mitochondrial dynamics in AML. Transduction of shRNA against fission-related genes, DNM1L and MFF, inhibited growth and increased the mitochondrial area in AML cell lines. Extracellular flux analysis showed that deletion of mitochondrial dynamic regulators reduced mitochondrial respiration without significantly affecting glycolysis, except in shDNM1L-transfected cells. Immunodeficient NOG mice transplanted with DNM1L- or MFF-knockdown AML cells survived significantly longer than controls. Treatment of AML cell lines with Mdivi-1, which inhibits the DRP1 encoded by DNM1L, inhibited cell proliferation and oxidative phosphorylation. Our results show that mitochondrial dynamics play an important role in AML, and provide novel biological insights. The inhibition of mitochondrial dynamics induces unique mitochondrial alterations, which may be explored as a potential therapeutic target in AML.

The Role of Epithelial-to-Mesenchymal Transition Transcription Factors (EMT-TFs) in Acute Myeloid Leukemia Progression.

Acute myeloid leukemia (AML) is a diverse malignancy originating from myeloid progenitor cells, with significant genetic and clinical variability. Modern classification systems like those from the World Health Organization (WHO) and European LeukemiaNet use immunophenotyping, molecular genetics, and clinical features to categorize AML subtypes. This classification highlights crucial genetic markers such as FLT3, NPM1 mutations, and MLL-AF9 fusion, which are essential for prognosis and directing targeted therapies. The MLL-AF9 fusion protein is often linked with therapy-resistant AML, highlighting the risk of relapse due to standard chemotherapeutic regimes. In this sense, factors like the ZEB, SNAI, and TWIST gene families, known for their roles in epithelial-mesenchymal transition (EMT) and cancer metastasis, also regulate hematopoiesis and may serve as effective therapeutic targets in AML. These genes contribute to cell proliferation, differentiation, and extramedullary hematopoiesis, suggesting new possibilities for treatment. Advancing our understanding of the molecular mechanisms that promote AML, especially how the bone marrow microenvironment affects invasion and drug resistance, is crucial. This comprehensive insight into the molecular and environmental interactions in AML emphasizes the need for ongoing research and more effective treatments.

PIWI pathway: bridging acute myeloid leukemia stemness and cellular differentiation.

PIWI proteins are stem cell-associated RNA-binding proteins crucial for survival of germ stem cells. In cancer, PIWI proteins are overexpressed. Specifically, PIWIL4 is highly expressed in multiple cancers with the highest levels found in acute myeloid leukemia (AML), an aggressive malignancy propagated by a population of leukemia stem cells (LSCs). Bamezai et al. (Blood Journal, blood, 2023, 142, 90-105) demonstrated that PIWIL4 supports AML blasts and LSCs but is not necessary for healthy human hematopoietic progenitor stem cells (HSPCs) function in vivo. PIWIL4 in AML acts by preventing the accumulation of R-loops in key genes for LSCs persistence implicated in: DNA damage, replicative stress, and transcription arrest. We report that PIWIL4 expression significantly decreases in THP-1 monocytes exposed to a differentiating agent, suggesting a potential role for PIWIL4 in maintaining the undifferentiated state of myeloid cells. PIWIL4 overexpression could lead to the emergence of LSCs, driving leukemia propagation and maintenance. Our findings correlate with the persistent overexpression of PIWIL4 in myeloid cancers as reported by Bamezai et al., and suggest that PIWIL4 may be involved in myeloid cell differentiation. In this perspective, we highlight recent findings on the implication of PIWI pathway in maintaining AML stemness. Additionally, we propose further investigation on the role of PIWI pathway in oncogenesis and cellular differentiation as a strategy to identify biomarkers and therapeutic targets for AML.

Integrin-α9 overexpression underlies the niche-independent maintenance of leukemia stem cells in acute myeloid leukemia

Leukemia stem cells (LSCs) are widely believed to reside in well-characterized bone marrow (BM) niches; however, the capacity of the BM niches to accommodate LSCs is insufficient, and a significant proportion of LSCs are instead maintained in regions outside the BM. The molecular basis for this niche-independent behavior of LSCs remains elusive. Here, we show that integrin-α9 overexpression (ITGA9 OE) plays a pivotal role in the extramedullary maintenance of LSCs by molecularly mimicking the niche-interacting status, through the binding with its soluble ligand, osteopontin (OPN). Retroviral insertional mutagenesis conducted on leukemia-prone Runx-deficient mice identified Itga9 OE as a novel leukemogenic event. Itga9 OE activates Akt and p38MAPK signaling pathways. The elevated Myc expression subsequently enhances ribosomal biogenesis to overcome the cell integrity defect caused by the preexisting Runx alteration. The Itga9-Myc axis, originally discovered in mice, was further confirmed in multiple human acute myeloid leukemia (AML) subtypes, other than RUNX leukemias. In addition, ITGA9 was shown to be a functional LSC marker of the best prognostic value among 14 known LSC markers tested. Notably, the binding of ITGA9 with soluble OPN, a known negative regulator against HSC activation, induced LSC dormancy, while the disruption of ITGA9-soluble OPN interaction caused rapid cell propagation. These findings suggest that the ITGA9 OE increases both actively proliferating leukemia cells and dormant LSCs in a well-balanced manner, thereby maintaining LSCs. The ITGA9 OE would serve as a novel therapeutic target in AML.

LILRB4 in acute myeloid leukemia: From prognostic biomarker to immunotherapeutic target.

Leukocyte immunoglobulin-like receptor (LILR) B4 (also known as ILT3/CD85k) is an immune checkpoint protein that is highly expressed in solid tumors and hematological malignancies and plays a significant role in the pathophysiology of cancer. LILRB4 is highly expressed in acute myeloid leukemia (AML), and this phenotype is associated with adverse patient outcomes. Its differential expression in tumors compared to normal tissues, its presence in tumor stem cells, and its multifaceted roles in tumorigenesis position it as a promising therapeutic target in AML. Currently, several immunotherapies targeting LILRB4 are undergoing clinical trials. This review summarizes advancements made in the study of LILRB4 in AML, focusing on its structure, ligands, expression, and significance in normal tissues and AML; its protumorigenic effects and mechanisms in AML; and the application of LILRB4-targeted therapies in AML. These insights highlight the potential advantages of LILRB4 as an immunotherapeutic target in the context of AML.

ITGAM sustains MAPK signaling and serves as an adverse prognostic factor and therapeutic target in acute myeloid leukemia.

Acute myeloid leukemia (AML) is the second most frequently occurring type of leukemia in adults. Despite breakthroughs in genetics, the prognosis of AML patients remains dismal. The aim of this study is to find new therapeutic targets and diagnostic markers for AML and to explore their mechanisms of action. The expression patterns of integrin subunit alpha M (ITGAM) were investigated across different cell types using the Human Protein Atlas (HPA) database. The ITGAM levels across cancer types were analyzed using the Gene Expression Profiling Interactive Analysis (GEPIA) database. Prognostic correlations in AML individuals were evaluated using The Cancer Genome Atlas (TGCA) database. ITGAM-associated functions were evaluated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The AML cells were transfected with short-hairpin RNA targeting ITGAM or a control, and subsequently subjected to analysis in order to ascertain the impact of ITGAM on proliferation and apoptosis. The expression of ITGAM was significantly higher in the AML patient samples compared to the control samples. High ITGAM expression was significantly associated with poor overall survival (OS). The knockdown of ITGAM in the AML cells resulted in a decrease in proliferation and an increase in apoptosis. This was accompanied by cell cycle arrest at the G1 phase and a downregulation of protein production for cyclin D1, cyclin E1, cyclin-dependent kinase 2 (CDK2), and cyclin-dependent kinase 4 (CDK4). A pathway analysis and a western blot analysis revealed that ITGAM positively regulated mitogen-activated protein kinase (MAPK) signaling by silencing attenuated p38 MAPK (P38), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) phosphorylation, while the total protein levels remained unchanged. ITGAM can serve as a potential prognostic biomarker and therapeutic target for AML. ITGAM production was elevated in AML and indicated poor survival. Silencing ITGAM suppressed AML cell viability and induced apoptosis by blocking cell cycle progression, likely by impeding the activation of the MAPK pathway. Further investigations that directly target the ITGAM-MAPK axis may offer novel strategies for mitigating AML pathogenesis and overcoming chemotherapy resistance.

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.

Recent advances in CAR-T therapy for the treatment of acute myeloid leukemia.

Chimeric antigen receptor T-cell (CAR-T) therapy, which has demonstrated notable efficacy against B-cell malignancies and is approved by the US Food and Drug Administration for clinical use in this context, represents a significant milestone in cancer immunotherapy. However, the efficacy of CAR-T therapy for the treatment of acute myeloid leukemia (AML) is poor. The challenges associated with the application of CAR-T therapy for the clinical treatment of AML include, but are not limited to, nonspecific distribution of AML therapeutic targets, difficulties in the production of CAR-T cells, AML blast cell heterogeneity, the immunosuppressive microenvironment in AML, and treatment-related adverse events. In this review, we summarize the recent findings regarding various therapeutic targets for AML (CD33, CD123, CLL1, CD7, etc.) and the results of the latest clinical studies on these targets. Thereafter, we also discuss the challenges related to CAR-T therapy for AML and some promising strategies for overcoming these challenges, including novel approaches such as gene editing and advances in CAR design.

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.

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.

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.