Mouse Model Of Acute Myeloid Leukemia Research Articles

Effects of Hypomethylating Agents on Gene Modulation in the Leukemic Microenvironment and Disease Trajectory in a Mouse Model of AML.

Hypomethylating agents (HMAs), such as decitabine and 5-azacytidine (AZA), are valuable treatment options for patients with acute myeloid leukemia that are ineligible for intensive chemotherapy. Despite providing significant extensions in survival when used alone or in combination, eventual relapse and resistance to HMAs are observed. The mechanisms leading to these outcomes are still not well defined and may, in part, be due to a focus on leukemic populations with limited information on the effects of HMAs on non-leukemic cells in the blood and other tissue compartments. In this study, we elucidated effects on immune-related gene expression in non-leukemic blood cells and the spleen during AZA treatment in leukemia-challenged mice. We observed significant changes in pathways regulating adhesion, thrombosis, and angiogenesis as well as a dichotomy in extramedullary disease sites that manifests during relapse. We also identify several genes that may contribute to the anti-leukemic activity of AZA in blood and spleen. Overall, this work has identified novel gene targets and pathways that could be further modulated to augment efficacy of HMA treatment.

Dual specific STAT3/5 degraders effectively block acute myeloid leukemia and natural killer/T cell lymphoma.

The transcription factors STAT3, STAT5A, and STAT5B steer hematopoiesis and immunity, but their enhanced expression and activation promote acute myeloid leukemia (AML) or natural killer/T cell lymphoma (NKCL). Current therapeutic strategies focus on blocking upstream tyrosine kinases to inhibit STAT3/5, but these kinase blockers are not selective against STAT3/5 activation and frequent resistance causes relapse, emphasizing the need for targeted drugs. We evaluated the efficacy of JPX-0700 and JPX-0750 as dual STAT3/5 binding inhibitors promoting protein degradation. JPX-0700/-0750 decreased the mRNA and protein levels of STAT3/5 targets involved in cancer survival, metabolism, and cell cycle progression, exhibiting nanomolar to low micromolar efficacy. They induced cell death and growth arrest in both AML/NKCL cell lines and primary AML patient blasts. We found that both AML/NKCL cells hijack STAT3/5 signaling through either upstream activating mutations in kinases, activating mutations in STAT3, mutational loss of negative STAT regulators, or genetic gains in anti-apoptotic, pro-proliferative, or epigenetic-modifying STAT3/5 targets. This emphasizes a vicious cycle for proliferation and survival through STAT3/5. Both JPX-0700/-0750 treatment reduced leukemic cell growth in human AML or NKCL xenograft mouse models significantly, being well tolerated by mice. Synergistic cell death was induced upon combinatorial use with approved chemotherapeutics in AML/NKCL cells.

Systemic Multifunctional Nanovaccines for Potent Personalized Immunotherapy of Acute Myeloid Leukemia.

Hematological malignancies (HM) like acute myeloid leukemia (AML) are often intractable. Cancer vaccines possibly inducing robust and broad anti-tumor immune responses may be a promising treatment option for HM. Few effective vaccines against blood cancers are, however, developed to date partly owing to insufficient stimulation of dendritic cells (DCs) in the body and lacking appropriate tumor antigens (Ags). Here it is found that systemic multifunctional nanovaccines consisting of nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and Toll-like receptor 9 (TLR9) agonists - muramyl dipeptide (MDP) and CpG, and tumor cell lysate (TCL) as Ags (MCA-NV) induce potent and broad immunity against AML. MCA-NV show complementary stimulation of DCs and prime homing to lymphoid organs following systemic administration. Of note, in orthotopic AML mouse models, intravenous infusion of different vaccine formulations elicits substantially higher anti-AML efficacies than subcutaneous administration. Systemic MCA-NV cure 78% of AML mice and elicit long-term immune memory with 100% protection from rechallenging AML cells. Systemic MCA-NV can also serve as prophylactic vaccines against the same AML. These systemic nanovaccines utilizing patient TCL as Ags and dual adjuvants to elicit strong, durable, and broad immune responses can provide a personalized immunotherapeutic strategy against AML and other HM.

A novel bispecific T-cell engager using the ligand-target csGRP78 against acute myeloid leukemia

Current medical therapies for treating acute myeloid leukemia (AML) remain unmet, and AML patients may benefit from targeted immunotherapy approaches that focus on specific tumor antigens. GRP78, which is upregulated in various malignant tumors such as AML, is partially expressed as cell surface GRP78 (csGRP78) on the cell membrane, making it an ideal target for redirecting T cells, including T-cell engagers. However, considering the conventional approach of using two scFv segments to construct a bispecific T-cell engager (BiTE), we have undertaken the development of a novel BiTE that utilizes a cyclic peptide ligand to specifically target csGRP78, which we refer to as GRP78-CD3/BiTE. We studied the effects of GRP78-CD3/BiTE on treatments for AML in vitro and in vivo and assessed the pharmacokinetics of this engager. Our findings demonstrated that GRP78-CD3/BiTE could not only effectively mediate the cytotoxicity of T cells against csGRP78-expressing AML cells but also specifically eliminate primary AML tumor cells in vitro. Furthermore, GRP78-CD3/BiTE exhibited a longer half-life despite having a lower molecular weight than CD19-CD3/BiTE. In a xenograft mouse model of AML, treatment with GRP78-CD3/BiTE prolonged the survival time of the mice. Our findings demonstrate that GRP78-CD3/BiTE is effective and selective for eliminating csGRP78-expressing AML cells and suggest that this approach to targeted immunotherapy could lead to effective new treatments for AML.

Genetic deletion of JAM-C in preleukemic cells rewires leukemic stem cell gene expression program in AML

AbstractThe leukemic stem cell (LSC) score LSC-17 based on a stemness–related gene expression signature is an indicator of poor disease outcome in acute myeloid leukemia (AML). However, it is not known whether “niche anchoring” of LSC affects disease evolution. To address this issue, we conditionally inactivated the adhesion molecule JAM-C (Junctional Adhesion Molecule-C) expressed by hematopoietic stem cells (HSCs) and LSCs in an inducible mixed-lineage leukemia (iMLL)-AF9–driven AML mouse model. Deletion of Jam3 (encoding JAM-C) before induction of the leukemia–initiating iMLL-AF9 fusion resulted in a shift from long-term to short-term HSC expansion, without affecting disease initiation and progression. In vitro experiments showed that JAM-C controlled leukemic cell nesting irrespective of the bone marrow stromal cells used. RNA sequencing performed on leukemic HSCs isolated from diseased mice revealed that genes upregulated in Jam3-deficient animals belonged to activation protein-1 (AP-1) and tumor necrosis factor α (TNF-α)/NF-κB pathways. Human orthologs of dysregulated genes allowed to identify a score that was distinct from, and complementary to, the LSC-17 score. Substratification of patients with AML using LSC-17 and AP-1/TNF-α genes signature defined 4 groups with median survival ranging from <1 year to a median of “not reached” after 8 years. Finally, coculture experiments showed that AP-1 activation in leukemic cells was dependent on the nature of stromal cells. Altogether, our results identify the AP-1/TNF-α gene signature as a proxy of LSC anchoring in bone marrow niches, which improves the prognostic value of the LSC-17 score. This trial was registered at www.ClinicalTrials.gov as #NCT02320656.

Inhibiting AGTR1 reduces AML burden and protects the heart from cardiotoxicity in mouse models.

Clinical treatment of acute myeloid leukemia (AML) largely relies on intensive chemotherapy. However, the application of chemotherapy is often hindered by cardiotoxicity. Patient sequence data revealed that angiotensin II receptor type 1 (AGTR1) is a shared target between AML and cardiovascular disease (CVD). We found that inhibiting AGTR1 sensitized AML to chemotherapy and protected the heart against chemotherapy-induced cardiotoxicity in a human AML cell-transplanted mouse model. These effects were regulated by the AGTR1-Notch1 axis in AML cells and cardiomyocytes from mice. In mouse cardiomyocytes, AGTR1 was hyperactivated by AML and chemotherapy. AML leukemogenesis increased the expression of the angiotensin-converting enzyme and led to increased production of angiotensin II, the ligand of AGTR1, in an MLL-AF9-driven AML mouse model. In this model, the AGTR1-Notch1 axis regulated a variety of genes involved with cell stemness and chemotherapy resistance. AML cell stemness was reduced after Agtr1a deletion in the mouse AML cell transplant model. Mechanistically, Agtr1a deletion decreased γ-secretase formation, which is required for transmembrane Notch1 cleavage and release of the Notch1 intracellular domain into the nucleus. Using multiomics, we identified AGTR1-Notch1 signaling downstream genes and found decreased binding between these gene sequences with Notch1 and chromatin enhancers, as well as increased binding with silencers. These findings describe an AML/CVD association that may be used to improve AML treatment.

Targeting a lineage-specific PI3Kɣ-Akt signaling module in acute myeloid leukemia using a heterobifunctional degrader molecule.

Dose-limiting toxicity poses a major limitation to the clinical utility of targeted cancer therapies, often arising from target engagement in nonmalignant tissues. This obstacle can be minimized by targeting cancer dependencies driven by proteins with tissue-restricted and/or tumor-restricted expression. In line with another recent report, we show here that, in acute myeloid leukemia (AML), suppression of the myeloid-restricted PIK3CG/p110γ-PIK3R5/p101 axis inhibits protein kinase B/Akt signaling and compromises AML cell fitness. Furthermore, silencing the genes encoding PIK3CG/p110γ or PIK3R5/p101 sensitizes AML cells to established AML therapies. Importantly, we find that existing small-molecule inhibitors against PIK3CG are insufficient to achieve a sustained long-term antileukemic effect. To address this concern, we developed a proteolysis-targeting chimera (PROTAC) heterobifunctional molecule that specifically degrades PIK3CG and potently suppresses AML progression alone and in combination with venetoclax in human AML cell lines, primary samples from patients with AML and syngeneic mouse models.

Anlotinib synergizes with venetoclax to induce mitotic catastrophe in acute myeloid leukemia

Venetoclax is a BCL2-targeted drug employed in treating various cancers, particularly hematologic malignancies. Venetoclax combination therapies are increasingly recognized as promising treatment strategies for acute myeloid leukemia (AML). In this study, we conducted an unbiased drug screen and identified anlotinib, a promising multi-targeted receptor tyrosine kinase inhibitor with oral activity currently utilized in the treatment of solid tumor, as a potent enhancer of venetoclax's anticancer activity in AML. Our investigation encompassed AML cell lines, primary cells, and mouse models, demonstrating effective low-dose combination therapy of anlotinib and venetoclax with minimal cytopenia or organ damage. Proteomic analysis revealed abnormal mitotic signals induced by this combination in AML cells. Mechanistically, anlotinib synergized with venetoclax by suppressing ARPP19 protein, leading to sustained activation of PP2A-B55δ. This inhibited AML cells from entering the mitotic phase, culminating in mitotic catastrophe and apoptosis. Additionally, we identified a specific synthetic lethal vulnerability in AML involving an ARPP19 mutation at S62 phosphorylation. These findings underscore the therapeutic potential of anlotinib and venetoclax combination therapy in AML, warranting further clinical investigation.

A chemically adjustable BMP6-IL6 axis in mesenchymal stem cells drives acute myeloid leukemia cell differentiation

Chemotherapy alone or in combination with allogeneic stem cell transplantation has been the standard of care for acute myeloid leukemia (AML) for decades. Leukemia relapse with limited treatment options remains the main cause of treatment failure. Therefore, an effective and safe approach to improve treatment outcomes is urgently needed for most AML patients. Mesenchymal stem cells (MSCs) have been reported to efficiently induce apoptosis and shape the fate of acute myeloid leukemia cells. Here, we identified LG190155 as a potent compound that enhances the antileukemia efficiency of MSCs. Pretreatment of MSCs with LG190155 significantly provoked differentiation in both AML patient-derived primary leukemia cells and AML cell lines and reduced the tumor burden in the AML mouse model. Using the quantitative proteomic technique, we discovered a pivotal mechanism that mediates AML cell differentiation, in which autocrine bone morphogenetic protein 6 (BMP6) in MSCs boosted IL-6 secretion and further acted on leukemic cells to trigger differentiation. Furthermore, the activity of the BMP6-IL6 axis was dramatically enhanced by activating vitamin D receptor (VDR) in MSCs. Our data illustrated an effective preactivated approach to reinforcing the antileukemia effect of MSCs, which could serve as an effective therapeutic strategy for AML.

Age-specific induction of mutant p53 drives clonal hematopoiesis and acute myeloid leukemia in adult mice

The investigation of the mechanisms behind p53 mutations in acute myeloid leukemia (AML) has been limited by the lack of suitable mouse models, which historically have resulted in lymphoma rather than leukemia. This study introduces two new AML mouse models. One model induces mutant p53 and Mdm2 haploinsufficiency in early development, showing the role of Mdm2 in myeloid-biased hematopoiesis and AML predisposition, independent of p53. The second model mimics clonal hematopoiesis by inducing mutant p53 in adult hematopoietic stem cells, demonstrating that the timing of p53 mutation determines AML vs. lymphoma development. In this context, age-related changes in hematopoietic stem cells (HSCs) collaborate with mutant p53 to predispose toward myeloid transformation rather than lymphoma development. Our study unveils new insights into the cooperative impact of HSC age, Trp53 mutations, and Mdm2 haploinsufficiency on clonal hematopoiesis and the development of myeloid malignancies.

The CD33xCD123xCD70 Multispecific CD3-Engaging DARPin MP0533 Induces Selective T Cell-Mediated Killing of AML Leukemic Stem Cells.

The prognosis of patients with acute myeloid leukemia (AML) is limited, especially for elderly or unfit patients not eligible for hematopoietic stem cell (HSC) transplantation. The disease is driven by leukemic stem cells (LSCs), which are characterized by clonal heterogeneity and resistance to conventional therapy. These cells are therefore believed to be a major cause of progression and relapse. We designed MP0533, a multispecific CD3-engaging designed ankyrin repeat protein (DARPin) that can simultaneously bind to three antigens on AML cells (CD33, CD123, and CD70), aiming to enable avidity-driven T cell-mediated killing of AML cells coexpressing at least two of the antigens. In vitro, MP0533 induced selective T cell-mediated killing of AML cell lines, as well as patient-derived AML blasts and LSCs, expressing two or more target antigens, while sparing healthy HSCs, blood, and endothelial cells. The higher selectivity also resulted in markedly lower levels of cytokine release in normal human blood compared to single antigen-targeting T-cell engagers. In xenograft AML mice models, MP0533 induced tumor-localized T-cell activation and cytokine release, leading to complete eradication of the tumors while having no systemic adverse effects. These studies show that the multispecific-targeting strategy used with MP0533 holds promise for improved selectivity toward LSCs and efficacy against clonal heterogeneity, potentially bringing a new therapeutic option to this group of patients with a high unmet need. MP0533 is currently being evaluated in a dose-escalation phase 1 study in patients with relapsed or refractory AML (NCT05673057).

Self-Stimulated Photodynamic Nanoreactor in Combination with CXCR4 Antagonists for Antileukemia Therapy.

The treatment of acute myeloid leukemia (AML) remains unsatisfactory, owing to the absence of efficacious therapy regimens over decades. However, advances in molecular biology, including inhibiting the CXCR4/CXCL12 biological axis, have introduced novel therapeutic options for AML. Additionally, self-stimulated phototherapy can solve the poor light penetration from external sources, and it will overcome the limitation that traditional phototherapy cannot be applied to the treatment of AML. Herein, we designed and manufactured a self-stimulated photodynamic nanoreactor to enhance antileukemia efficacy and suppress leukemia recurrence and metastasis in AML mouse models. To fulfill our design, we utilized the CXCR4/CXCL12 biological axis and biomimetic cell membranes in conjunction with self-stimulated phototherapy. This nanoreactor possesses the capability to migrate into the bone marrow cavity, inhibit AML cells from infiltrating into the visceral organ, significantly enhance the antileukemia effect, and prolong the survival time of leukemic mice. Therefore, this nanoreactor has significant potential for achieving high success rates and low recurrence rates in leukemia treatment.

Abstract 5092: Development of a priming approach to enhance antigen-directed therapies in hematological malignancies and solid tumors

Abstract Clinical success of antigen-directed therapies in cancer has been limited by heterogeneous antigen expression and a narrow therapeutic window. In many patients, failure of antigen-directed therapies such as CAR T-cell therapies, antibody-drug conjugates (ADC) or radioligand therapies (RLT) has less to do with a lack of potency or specificity of the agent and more to do with variable and suboptimal antigen expression in the patient. We are developing combination therapies that introduce a priming agent designed to optimize antigen expression in the patient and improve the therapeutic window of existing antigen-directed therapies. Our high-throughput platform utilizes machine learning and automation to scale functional combinatorial screening for small molecule priming agents using cell lines and patient samples. Using this platform, we are identifying priming therapies for CD123, CD33, CD22, and CD19 in hematological malignancies including acute myeloid leukemia (AML), and HER2, HER3, and TROP2 in solid tumors including breast cancer.For our CD123 program, we have identified priming agents that selectively upregulate CD123 expression ≥2-fold on AML cells without increasing expression on normal myeloid progenitor cells, which also express CD123. AML cells treated with a CD123 priming agent in combination with a CD123 ADC showed greater tumor-cell killing compared to the ADC alone. In comparison, normal myeloid progenitor cells from healthy bone marrow treated with the same CD123 priming-ADC combination treatment did not show an increase in ADC-mediated cell killing. From our mechanistic studies, we have identified a somatic mutation associated with CD123 upregulation that may lead to selective upregulation on cancer cells and not on normal cells. Additionally, this marker can be used for patient selection. We have also observed CD123 upregulation in vivo using mouse models of AML and are currently conducting additional in vivo proof of concept studies to demonstrate that CD123 priming therapy enhances efficacy.There are two distinct advantages of a priming approach paired with an antigen-directed therapy, which include: (1) improving the durability of patient responses and (2) expanding the patient population. Our priming combination approach has the potential to overcome antigen heterogeneity and thereby improve the clinical success of antigen-directed therapies. Citation Format: Kathryn E. Vanderlaag, Anukriti Dhar, Molly Volkman, Erik Werner, Transon Nguyen, Kamran Ali. Development of a priming approach to enhance antigen-directed therapies in hematological malignancies and solid tumors [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 5092.

Disruption of polyunsaturated fatty acid biosynthesis drives STING-dependent acute myeloid leukemia cell maturation and death

The role of polyunsaturated fatty acid (PUFA) biosynthesis in acute myeloid leukemia (AML) remains largely undefined. A comparative expression analysis of 35 genes encoding fatty acid biosynthesis enzymes showed that fatty acid desaturase 1 (FADS1) was highly expressed across multiple AML subtypes relative to healthy controls and that elevated FADS1 expression correlates with worse overall AML patient survival. Functionally, shRNA-mediated inhibition of FADS1 reduced AML cell growth invitro and significantly delayed leukemia onset in an AML mouse model. AML cell lines depleted of FADS1 arrested in the G1/S-phase of the cell cycle, acquired characteristics of myeloid maturation and subsequently died. To understand the molecular consequences of FADS1 inhibition, a combination of mass spectrometry-based analysis of complex lipids and gene expression analysis (RNA-seq) was performed. FADS1 inhibition caused AML cells to exhibit significant lipidomic remodeling, including depletion of PUFAs from the phospholipids, phosphatidylserine, and phosphatidylethanolamine. These lipidomic alterations were accompanied by an increase induction of inflammatory and stimulator of interferon genes (STING)-mediated type-1 interferon signaling. Remarkably, genetic deletion of STING largely prevented the AML cell maturation and death phenotypes mediated by FADS1 inhibition. Highlighting the therapeutic implications of these findings, pharmacological blockade of PUFA biosynthesis reduced patient-derived AML cell numbers exvivo but not that of healthy donor cells. Similarly, STING agonism attenuated patient-derived-AML survival; however, STING activation also reduced healthy granulocyte numbers. Collectively, these data unveil a previously unrecognized importance of PUFA biosynthesis in leukemogenesis and that imbalances in PUFA metabolism can drive STING-mediated AML maturation and death.

Abstract 3883: Cryo-EM-guided enhancement of target selectivity of a novel p97 inhibitor for treating multiple myeloma and acute myeloid leukemia

Abstract The AAA ATPase p97, also known as valosin-containing protein (VCP), serves to regulate protein homeostasis by facilitating the translocation of ubiquitinated proteins from membranes or chromatin to the proteasome for degradation. Certain cancers, including multiple myeloma (MM) and acute myeloid leukemia (AML) are known for the intracellular overexpression of proteins, and are thus susceptible to the inhibition of p97 and resulting proteotoxic stress. A known p97 inhibitor, CB-5083 entered human phase 1 clinical trials in 2015, but trials were terminated due to side effects that were later traced to strong interaction of the compound with the phosphodiesterase PDE6. To address this problem, we devised a subtractive approach that leveraged high resolution structures of CB-5083 bound to PDE6 and p97. Using this information, we designed a p97 inhibitor that no longer displayed significant off-target binding but retained strong on-target binding. The optimization process started when we identified the 7-azaindole core from an in silico screen as a suitable hit pharmacophore and confirmed the binding mode of several variants of this scaffold in p97 by cryo-EM. Through a structure-guided medicinal chemistry program, we identified GND-135, a potent, on-target binder through the synthesis of less than 100 compounds. The design was supported with about 50 cryo-EM structures throughout the optimization process. Compared to CB-5083, our compound GND-135 displays greatly reduced PDE6 activity (&amp;lt; 1% inhibition at 10 μM versus 89% inhibition for CB-5083) and more potent p97 ATPase inhibition (3 nM IC50 versus 12 nM for CB-5083). We also evaluated the pharmacokinetic profile of GND-135 in mice; the half-life and clearance of 6.6 h and 15.9 mL/min/kg respectively were deemed suitable for daily administration. Based on the initial profiling, we carried out an efficacy study in a cell derived xenograft mouse model of AML using a U937 cell line with subcutaneous implantation. In this model, where GND-135 was administered IP (40 mg/kg QD) and CB-5083 was administered orally (40 mg/kg QD) the compounds showed statistically comparable efficacy, which was differentiated from untreated control. Thus, GND-135 is a novel p97 inhibitor lead compound with on-target selectivity powered by cryo-EM driven rational drug design. Citation Format: Jason Crawford, Ravi Munuganti, Charles Leung, Kriti Singh, Ellen Gates, Xing Zhu, Marcel Bally, Nancy Dos Santos, Maryam Sharifiaghdam, Zeynab Nosrati, Peter Axerio-Cilies, Alison Berezuk, Spencer Cholak, Sriram Subramaniam. Cryo-EM-guided enhancement of target selectivity of a novel p97 inhibitor for treating multiple myeloma and acute myeloid leukemia [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 3883.

Abstract 263: RNA Editing ADAR1 Deaminase Activation Responsive (READAR) platforms for dynamic detection of clonal myeloproliferative neoplasm stem cell fitness

Abstract Introduction Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell (HSC) derived disorders that can progress to acute myeloid leukemia (AML) at variable rates depending on macroenvironmental stressors and microenvironmental inflammatory cytokines. The total symptom score (TSS) and variant allele frequency (VAF) of mutations have proven valuable predictors of therapeutic response. Recently, we discovered inflammatory-cytokine-responsive ADAR1 RNA editing enzyme overexpression during MPN progression. However, dynamic prediction of clonal MPN stem cell fitness and progression to AML remains challenging. Methods We performed longitudinal 150-gene next-generation sequencing (NGS) analyses for 129 MPN patients. We developed single-cell RNA Editing ADAR1 Deaminase Activation Responsive (READAR) platforms to quantify ADAR1 activity with a novel ADAR1 nanoluc-GFP reporter assay, whole transcriptome RNA editome analyses, and MPN stem cell replating assays. To identify novel RNA editing sites, we performed whole genome sequencing on CD34+ cells from 43 MPN patients and whole transcriptome sequencing on FACS-purified HSC and progenitor cells from 32 MPN patients and 24 non-MPN controls. Results Sequential NGS analysis was performed with a median follow-up of 958 days (range 0-4214; interquartile range (IQR) 314-1350). On average, 6 NGS analyses (range 1-19; IQR 3-9) were performed for each patient. In our cohort, 58.9% of patients were JAK2 V617F+, while mutations in CALR and ASXL1 were observed in 11.6% and 14.0% of cases, respectively. In total, 44.2% (N=57) of patients in our cohort received Inrebic, with 56% reaching the maximum dose of 400mg. Notably, most patients on Inrebic were JAK2 V617F+ (N=38, 67.7%). In JAK2 V617F+ patients on Inrebic, 50% (n=19) experienced a reduction in the JAK2 V617F VAF from their peak value with a median drop of 16% (range 4-57). Our analyses using READAR platforms revealed increased RNA editing levels in JAK2 V617F+ patients from whole transcriptome sequencing analyses compared with JAK2 wild-type patients. Transduction of our ADAR1 nanoluc-GFP reporter into CD34+ cells from MPN patients (N=7), enabled us to quantify ADAR1 activity in an ex vivo setting. Moreover, CD34+ cells from MPN patient samples (n=7) treated in stromal co-cultures with ADAR1 inhibitor, Rebecsinib, demonstrated inhibition of self-renewal in replating assays. A significant reduction in ADAR1-GFP reporter activity was observed in MPN CD34+ cells following Rebecsinib treatment of stromal co-cultures. Also, humanized ADAR1-GFP-luciferase reporter AML mouse models showed a significant reduction in ADAR1 activity and leukemia stem cell self-renewal following Rebecsinib treatment. Conclusion These READAR platform results suggest that ADAR1 activity can be utilized to predict clonal MPN stem cell fitness and progression. Citation Format: Inge van der Werf, Larisa Balaian, Jessica Pham, Wenxue Ma, Athena Mohebbi, Emma Klacking, Antonio Ruiz, Karla Mack, John Mascarenhas, Thomas Whisenant, Ludmil Alexandrov, Sheldon Morris, Catriona Jamieson. RNA Editing ADAR1 Deaminase Activation Responsive (READAR) platforms for dynamic detection of clonal myeloproliferative neoplasm stem cell fitness [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 263.

CLPs-miR-103a-2-5p inhibits proliferation and promotes cell apoptosis in AML cells by targeting LILRB3 and Nrf2/HO-1 axis, regulating CD8 + T cell response

BackgroundLILRB3, a member of the leukocyte immunoglobulin-like receptor B (LILRB) family, has immunosuppressive functions and directly regulates cancer development, which indicates that LILRB3 is an attractive target for cancer diagnosis and therapy. Novel therapeutic treatments for acute myeloid leukemia (AML) are urgent and important, and RNA therapeutics including microRNAs (miRNAs) could be an effective option. Here, we investigate the role of dysregulated miRNA targeting LILRB3 in the AML microenvironment.MethodsPotential miRNAs binding to the 3ʹ-untranslated region (3ʹ-UTR) of the LILRB3 mRNA were predicted by bioinformatics websites. Then, we screened miRNAs targeting LILRB3 by quantitative real-time PCR, and the dual luciferase reporter assay. The expression of LILRB3 and microRNA (miR)-103a-2-5p in AML were determined and then their interactions were also analyzed. In vitro, the effects of miR-103a-2-5p were determined by CCK8, colony formation assay, and transwell assay, while cell apoptosis and cell cycle were analyzed by flow cytometry. Cationic liposomes (CLPs) were used for the delivery of miR-103a-2-5p in the AML mouse model, which was to validate the potential roles of miR-103a-2-5p in vivo.ResultsLILRB3 was upregulated in AML cells while miR-103a-2-5p was dramatically downregulated. Thus, a negative correlation was found between them. MiR-103a-2-5p directly targeted LILRB3 in AML cells. Overexpressed miR-103a-2-5p significantly suppressed the mRNA and protein levels of LILRB3, thereby inhibiting AML cell growth and reducing CD8 + T cell apoptosis. In addition, overexpressed miR-103a-2-5p reduced both the relative expression of Nrf2/HO-1 pathway-related proteins and the ratio of GSH/ROS, leading to the excessive intracellular ROS that may promote AML cell apoptosis. In the mouse model, the delivery of miR-103a-2-5p through CLPs could inhibit tumor growth.ConclusionsMiR-103a-2-5p serves as a tumor suppressor that could inhibit AML cell proliferation and promote their apoptosis by downregulating LILRB3 expression, suppressing the Nrf2/HO-1 axis, and reducing the ratio of GSH/ROS. Besides, our findings indicate that miR-103a-2-5p may enhance the CD8 + T cell response by inhibiting LILRB3 expression. Therefore, the delivery of miR-103a-2-5p through CLPs could be useful for the treatment of AML.

Abstract P07: Identification of the unique preleukemic bone marrow niche in acute myeloid leukemia

Abstract P07: Identification of the unique preleukemic bone marrow niche in acute myeloid leukemia

PHF6 loss reduces leukemia stem cell activity in an acute myeloid leukemia mouse model

Acute myeloid leukemia (AML) is a malignant hematologic disease caused by gene mutations and genomic rearrangements in hematologic progenitors. The PHF6 (PHD finger protein 6) gene is highly conserved and located on the X chromosome in humans and mice. We found that PHF6 was highly expressed in AML cells with MLL rearrangement and was related to the shortened survival time of AML patients. In our study, we knocked out the Phf6 gene at different disease stages in the AML mice model. Moreover, we knocked down PHF6 by shRNA in two AML cell lines and examined the cell growth, apoptosis, and cell cycle. We found that PHF6 deletion significantly inhibited the proliferation of leukemic cells and prolonged the survival time of AML mice. Interestingly, the deletion of PHF6 at a later stage of the disease displayed a better anti-leukemia effect. The expressions of genes related to cell differentiation were increased, while genes that inhibit cell differentiation were decreased with PHF6 knockout. It is very important to analyze the maintenance role of PHF6 in AML, which is different from its tumor-suppressing function in T-cell acute lymphoblastic leukemia (T-ALL). Our study showed that inhibiting PHF6 expression may be a potential therapeutic strategy targeting AML patients.

Cancer-Stem-Cell Phenotype-Guided Discovery of a Microbiota-Inspired Synthetic Compound Targeting NPM1 for Leukemia.

The human microbiota plays an important role in human health and disease, through the secretion of metabolites that regulate key biological functions. We propose that microbiota metabolites represent an unexplored chemical space of small drug-like molecules in the search of new hits for drug discovery. Here, we describe the generation of a set of complex chemotypes inspired on selected microbiota metabolites, which have been synthesized using asymmetric organocatalytic reactions. Following a primary screening in CSC models, we identified the novel compound UCM-13369 (4b) whose cytotoxicity was mediated by NPM1. This protein is one of the most frequent mutations of AML, and NPM1-mutated AML is recognized by the WHO as a distinct hematopoietic malignancy. UCM-13369 inhibits NPM1 expression, downregulates the pathway associated with mutant NPM1 C+, and specifically recognizes the C-end DNA-binding domain of NPM1 C+, avoiding the nucleus-cytoplasm translocation involved in the AML tumorological process. The new NPM1 inhibitor triggers apoptosis in AML cell lines and primary cells from AML patients and reduces tumor infiltration in a mouse model of AML with NPM1 C+ mutation. The disclosed phenotype-guided discovery of UCM-13369, a novel small molecule inspired on microbiota metabolites, confirms that CSC death induced by NPM1 inhibition represents a promising therapeutic opportunity for NPM1-mutated AML, a high-mortality disease.