Tissue- and tumor-type-specific expression of internal-promoter-driven YEATS-domain-devoid isoforms of MLLT1 and MLLT3

Microvesicles are released by different cell types and shuttle mRNAs and microRNAs which have the possibility to transfer genetic information to a target cell and alter its function. Acute myeloid leukemia is a malignant disorder, and leukemic cells occupy all the bone marrow microenvironment. In this study, we investigate the effect of leukemia microvesicles on healthy umbilical cord blood hematopoietic stem cells to find evidence of cell information transferring. Leukemia microvesicles were isolated from acute myeloid leukemia patients and were co-incubated with healthy hematopoietic stem cells. After 7 days, cell count, hematopoietic stem cell-specific cluster of differentiation (CD) markers, colony-forming unit assay, and some microRNA gene expressions were assessed. Data showed a higher number of hematopoietic stem cells after being treated with leukemia microvesicles compared with control (treated with no microvesicles) and normal (treated with normal microvesicles) groups. Also, increased levels of microRNA-21 and microRNA-29a genes were observed in this group, while colony-forming ability was still maintained and high ranges of CD34+, CD34+CD38-, CD90+, and CD117+ phenotypes were observed as stemness signs. Our results suggest that leukemia microvesicles are able to induce some effects on healthy hematopoietic stem cells such as promoting cell survival and some microRNAs deregulation, while stemness is maintained.

FLT3 OR CD33 NOT EMCN Logic Gated CAR-NK Cell Therapy (SENTI-202) for Precise Targeting of AML

Rabbit Anti-Thymocyte Globulin Kills Leukemic Stem Cells to a Greater Degree Than Healthy Hematopoietic Stem Cells

Anti-Thymocyte Globulin Kills Leukemic Stem Cells in Vitro

For over 40 years the standard treatment for acute myeloid leukemia (AML) patients has been a combination of chemotherapy consisting of cytarabine and an anthracycline such as daunorubicin. This standard treatment results in complete remission (CR) in the majority of AML patients. However, despite these high CR rates, only 30–40% (<60 years) and 10–20% (>60 years) of patients survive five years after diagnosis. The main cause of this treatment failure is insufficient eradication of a subpopulation of chemotherapy resistant leukemic cells with stem cell-like properties, often referred to as “leukemic stem cells” (LSCs). LSCs co-exist in the bone marrow of the AML patient with residual healthy hematopoietic stem cells (HSCs), which are needed to reconstitute the blood after therapy. To prevent relapse, development of additional therapies targeting LSCs, while sparing HSCs, is essential. As LSCs are rare, heterogeneous and dynamic, these cells are extremely difficult to target by single gene therapies. Modulation of miRNAs and consequently the regulation of hundreds of their targets may be the key to successful elimination of resistant LSCs, either by inducing apoptosis or by sensitizing them for chemotherapy. To address the need for specific targeting of LSCs, miRNA expression patterns in highly enriched HSCs, LSCs, and leukemic progenitors, all derived from the same patients’ bone marrow, were determined and differentially expressed miRNAs between LSCs and HSCs and between LSCs and leukemic progenitors were identified. Several of these miRNAs are specifically expressed in LSCs and/or HSCs and associated with AML prognosis and treatment outcome. In this review, we will focus on the expression and function of miRNAs expressed in normal and leukemic stem cells that are residing within the AML bone marrow. Moreover, we will review their possible prospective as specific targets for anti-LSC therapy.

Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell disorder characterized by features of both myelodysplastic syndrome (MDS) and myeloproliferative neoplasms (MPN). Patients with CMML have dismal outcomes partially due to their poor responses to the standard treatment with hypomethylating agents, highlighting a need for new treatments. RAS mutations are detected in roughly 50% of CMML patients and in even higher frequency among patients who progress to acute myeloid leukemia (AML). These mutations contribute to a poor prognosis and are also among the most commonly acquired resistance mutations in AML patients treated with targeted therapies. The current strategy to overcome RAS in myeloid malignancies is to target downstream effectors such as MEK, but these therapies are hindered with short-lived clinical response and resistance emergence. However, recent breakthroughs in the direct inhibition of RAS have renewed enthusiasm for targeting RAS-mutant tumors. Currently, there has been little exploration of the potential efficacy and safety of RAS inhibitors in the context of myeloid malignancies. We hypothesize that the RASmulti(ON) inhibitor RMC-7977, a highly selective inhibitor of the active forms of KRAS, HRAS and NRAS, will be effective against RAS-mutated AML cell lines arising from chronic myeloid neoplasms including CMML. We first characterized the effect of RMC-7977 in various AML cell lines with mutated RAS and wild-type RAS that is activated by alternative mutations. RMC-7977 treatment suppressed proliferation in the low nanomolar range in both RAS-mutated and wild type models. The AML cell lines arising from MDS/CMML were particularly sensitive to the RAS inhibitor, with these cells also displaying increased apoptosis when treated with low nanomolar levels of RMC-7977. Cell signaling experiments revealed that low doses of RMC-7977 significantly decreased phosphorylation of MEK and ERK, as well as ERK transcriptional output. RMC-7977 treatment significantly induced apoptosis and reduced AML blasts from patients with RAS-mutated AML that arose from MDS or MDS/MPN including CMML and prolonged survival as a monotherapy in a RAS mutated secondary AML xenograft model. Additionally, treatment with RMC-7977 showed no effect on the survival and differentiation of healthy hematopoietic stem and progenitor cells taken from human donors, strengthening its potential as an effective therapy in CMML and AML with low toxicity. We have demonstrated that the pan-RAS inhibitor RMC-7977 is highly effective against RAS mutated AML cell lines arising from chronic myeloid neoplasms including CMML while sparing healthy hematopoietic stem and progenitor cells. This work provides the rationale to continue evaluating RAS inhibitors as a targeted therapy in RAS-mutated myeloid malignancies, which is a significant unmet need in the treatment of these conditions. Citation Format: Tessa Seale, Brandy Perkins, Theodora Chatzilygeroudi, Soren Cole, Kareem Battah, Santiago Borrego Garcia, Maximilian Stahl, Mark J. Levis, Alexander Ambinder, Theodoros Karantanos, Sandra Misale. Pan-RAS inhibitor RMC-7977 overcomes oncogenic RAS signaling and exerts antileukemic effects in CMML/AML cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3809.

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are blood cancers with limited treatment options and poor outcomes. The bone marrow (BM) microenvironment significantly influences disease progression; conversely, MDS/AML remodels the niche to its advantage. Indeed, AML cells utilize the Kynurenine-HTR1B-SAA1 axis to reshape the BM niche, creating a pro-inflammatory environment that supports leukemic stem cell (LSC) proliferation. Given these insights, this study aims to investigate the role of SAA1 (Serum amyloid A1) in the progression of MDS and AML and explore potential therapeutic strategies to inhibit its activity. The quantification of bone marrow plasma levels of SAA1 in MDS and AML patients showed a strong correlation with disease severity and progression. Survival analysis revealed that SAA1 levels &amp;gt;4.0 µg/mL were associated with significantly poorer outcomes across all IPSS-R categories, including Very Low/Low-risk groups, highlighting its prognostic value. Functional in vitro and in vivo assays demonstrated that SAA1 selectively stimulates the proliferation of leukemic cells while sparing healthy hematopoietic stem cells (HSCs). Similarly, SAA1 enhanced the clonogenic potential of ASXL1- and TET2-mutant cells, suggesting its role in promoting stem-like properties in genetically altered hematopoietic cells, driving malignancy. Single-cell RNAseq analysis revealed that treatment of primary AML cells with SAA1 induces upregulation of pro-inflammatory cytokines, including IFN-ϒ, IL-6, TNF-α, and IL-1β, chemokines CXCL1 and CXCL8, and activates NFκB signaling, pathways promoting expansion of malignant and pre-malignant clones. In addition, it upregulates the expression of several genes associated with chemoresistance. A mouse monoclonal antibody was developed against SAA1 - 18A3. This antibody significantly inhibited the proliferation of leukemic cells and reduced NFκB activity in vitro. EdU incorporation assays demonstrated that SAA1 selectively promotes the proliferation of malignant cells in both leukemic and solid tumor lines such as T-ALL, B-ALL and pancreatic. The monoclonal antibody suppressed this effect. An in vivo study in an MLL-AF9 AML mouse model validated the therapeutic potential of anti-SAA1 antibody. Mice treated with the antibody showed reduced leukemic burden, as evidenced by bioluminescence imaging, and improved survival compared to untreated controls. Treated mice also maintained body weight and exhibited reduced disease progression, demonstrating the efficacy of the monoclonal antibody in mitigating leukemic proliferation. These findings establish SAA1 as a critical driver of leukemic progression and a valuable prognostic biomarker. The development of SAA1-neutralizing antibodies offers a promising therapeutic strategy for SAA1-targeted therapies to disrupt the inflammatory niche and improve outcomes in MDS and AML. Citation Format: Pallavi Budgude, Brian Chernak, Marta Galan Diez, Abdullah M. Ali, Ziwei Chen, Raul Rabadan, Martin Caroll, Azra Raza, Stavroula Kousteni. Niche driven inflammatory regulation in the pathogenesis and treatment of myeloid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3783.

Pan-RAS Inhibitor RMC-7977 Overcomes Oncogenic RAS Signaling and Exerts Antileukemic Effects in CMML/AML Cells

CAR-based cell therapies have revolutionized cancer treatment, however, applications beyond targeting lineage antigens are challenging due to expression of targeted antigens in healthy cells posing a risk for on-target off-tissue toxicities. This presents an opportunity to leverage synthetic biological logic gated gene circuits, such as NOT gate, to expand cancer targets for CAR-based cell therapies. We have constructed a first-in-class NOT gate in CAR-NK cells to protect healthy cells from CAR-mediated cytotoxicity. An inhibitory CAR (iCAR) recognizes a safety antigen expressed on healthy cells and suppresses activating CAR (aCAR) functions, significantly reducing NK cell activity. Multiple iCARs with intracellular co-inhibitory domains containing immunoreceptor tyrosine-based inhibitory motifs have shown to suppress over 50% of aCAR-mediated killing (p&amp;lt;0.05) and significantly reduce TNFa secretion (p&amp;lt;0.0005) in an antigen-specific manner. Here we describe a robust cancer and safety antigen pairing discovery method for the development of NOT gated CAR-NK therapies. A bioinformatics pipeline uses transcriptomics data to discover and prioritize tumor and healthy tissue antigens. We have identified genes differentially expressed in healthy vs tumor tissue and selected leads based on antigens' co-expression in healthy tissue, subcellular localization, antigen topology (presence of extracellular domain(s)), and antibody availability. Such antigen pairs have been validated in primary tissue samples. In AML, targeting the critical leukemic stem cell (LSC) population via antigens such as FLT3 leads to hematopoietic toxicity due to expression in healthy hematopoietic stem cells (HSCs). Comparative bioinformatic analysis between AML and healthy human bone marrow mononuclear cell (BMMC) samples identified 10 surface antigens differentially expressed between HSCs and AML cells that could be used as NOT gate targets to protect HSCs from CAR-mediated toxicity. We further validated one of the top candidate targets, EMCN, by flow cytometry and confirmed significant differential protein expression between HSCs and LSCs. Similarly, in CEA+ tumors, significant on-target off-tissue toxicities occur in healthy epithelium resulting in colitis and lung damage. We prioritized 3 healthy tissue antigens preferentially expressed in intestinal and lung epithelial cells compared to cancer cells: VSIG2, CPM and SLC26A2. IHC analysis confirmed that these targets are expressed at higher levels in the healthy tissues compared to CEA+ tumor, making them attractive candidates to use in a NOT gate circuit to improve the therapeutic window of CEA CAR-NK cells. Using a bioinformatics discovery and validation pipeline coupled with NOT logic gated CAR-NK cells, we can selectively target tumor antigens, while protecting healthy tissues, to create cell therapies with greater efficacy, precision and control. Citation Format: Alba Gonzalez, Assen Roguev, Nicholas W. Frankel, Brian S. Garrison, Derrick Lee, Marcus Gainer, Alyssa Mullenix, Russell M. Gordley, Kathryn A. Loving, Jenny Chien, Gary Lee. Development of logic-gated CAR-NK cells to reduce target-mediated healthy tissue toxicities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB028.

Introduction: In patients with acute myeloid leukemia (AML), CD123 has been shown to have high expression on blast cells and leukemic stem cells (LSC) compared with normal hematopoietic stem cells (HSCs) and other more mature CD34+ subsets (Zhou J, World J Stem Cells 2014). LSCs are inherently resistant to standard of care chemotherapeutics and LSC persistence after chemotherapy is associated with disease relapse. VIP943 is an ADC consisting of an anti-CD123 antibody, a unique linker cleaved intracellularly by legumain which is required for release, and activation of a novel kinesin spindle protein inhibitor (KSPi) payload that accumulates inside the cell. Herein, we evaluated the potential of VIP943 to target LSC and progenitor cells. Methods: Cytotoxicity assay: Bone marrow aspirates (BMA) and peripheral blood from previously untreated patients with primary or transformed from myelodysplastic syndrome AML were used in this experiment. Cells were treated with VIP943 (66 nM or 330 nM). After the end of the 48 h or 72 h incubation, the CD123+ cells and the CD34+CD38- LSC were detected by flow cytometry. Characterization of progenitor cell population: Fresh BM samples from healthy volunteers (HVBM) were stained with specific monoclonal antibodies to identify progenitor cell populations. Depletion assay: The effect of VIP943 on HVBM was evaluated by measuring the depletion of CD34+ progenitor cell populations in comparison to the anti-CD33-ADC, gemtuzumab ozogamicin (gem-oz). At the end of the incubation time, red blood cells were lysed; the remaining cells were stained with a cocktail of antibodies to discriminate between progenitor cells and analyzed by flow cytometry. Results: Treatment of patient-derived CD123+ leukemic blasts with VIP943 resulted in a 50% reduction after 48 h and up to 80% at 72 h. A reduction of &amp;gt;70% of CD123+CD34+CD38- LSCs derived from BM aspirates of untreated patients with AML was achieved after incubation with VIP943 up to 72h. In addition, HVBM samples derived from five healthy volunteers were treated with VIP943 or gem-oz in a depletion assay, where gem-oz was toxic to CD34+ cells with an EC50 of 0.16 µM in contrast to VIP943 with an EC50 value of 8.83 µM. Conclusions: In this in vitro analysis of primary samples from patients with AML, activity of VIP943 is observed in AML patient-derived CD123+ blasts as well as chemoresistant LSCs. At anticipated pharmacologic levels, VIP943 shows no adverse effects on HSCs unlike gem-oz suggesting an improved therapeutic index. In the ongoing first-in-human dose-escalation study in subjects with advanced CD123+ hematologic malignancies, VIP943 demonstrates a promising safety profile (NCT06034275). Citation Format: Beatrix Stelte-Ludwig, Tibor Schomber, Melanie M. Frigault, Joseph Birkett, Amy J. Johnson, Anne-Sophie Rebstock, Sebastian Ludwig, Raquel Izumi, Ahmed Hamdy. Activity of VIP943 on AML patient-derived leukemic blasts and healthy donor-derived bone marrow hematopoietic stem cells [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 629.

Inhibition of Drp1, a mitochondrial fission GTPase disrupts mitochondrial quality control and impairs stem-like properties in Acute Myeloid Leukemia

Disruption of the Akt-FoxO Axis As a Safeguard Mechanism for the Hematopoietic Stem Cell Compartment during Chronic Metabolic Stress

JUN and ATF3 Regulate the Transcriptional Output of the Unfolded Protein Response to Support Acute Myeloid Leukemia

IGFBP7 Induces Differentiation and Loss of Survival of Human Acute Myeloid Leukemia Stem Cells without Affecting Normal Hematopoiesis

MP0533: A Multispecific Darpin CD3 Engager Targeting CD33, CD123, and CD70 for the Treatment of AML and MDS Designed to Selectively Target Leukemic Stem Cells