Growth Of Acute Myeloid Leukemia Cells Research Articles

Abstract A013: Prioritization of Eleven-Nineteen-Leukemia (ENL) inhibitors as orally available drug candidates for acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is the second most prevalent and fatal form of leukemia. The growth of AML cells harboring oncogenic MLL rearrangements relies on The YEATS domain-containing protein ENL. Many small molecule inhibitors targeting ENL have been developed. To prioritize these inhibitors for in vivo studies, a NanoBRET system was introduced to evaluate their cellular permeability and potency. This screening identified Inhibitor 13 as a promising candidate. This inhibitor has remarkable metabolic stability and potent anti-proliferative effects on MLL-fusion leukemia cell lines. In AML-xenografted mice, Inhibitor 13 significantly improved survival. Subsequent optimization efforts led to the development of SR-C-107 (R), which exhibited strong activity against AML both at the cellular level (CC50 (MOLM-13): 1.25 ± 0.18 μM; CC50 (MV4-11): 0.81 ± 0.15 μM) and in vivo. These findings establish SR-C-107 (R) as a compelling candidate for AML treatment and lay the groundwork for the development of next-generation AML inhibitors. Citation Format: Wenshe R. Liu, Xuejiao Guo, Sandeep Alta, Shiqing Xu. Prioritization of Eleven-Nineteen-Leukemia (ENL) inhibitors as orally available drug candidates for acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr A013

The co-receptor Neuropilin-1 enhances proliferation in inv(16) acute myeloid leukemia via VEGF signaling.

Oncogenic programs regulate the proliferation and maintenance of cancer stem cells, and can define pharmacologic dependencies. In acute myeloid leukemia (AML) with the chromosome inversion 16 (inv(16)), the fusion oncoproteinCBFβ::MYH11 regulates pathways associated with leukemia stem cell activity. Here we demonstrate that expression of Neuropilin-1 (NRP1) is regulated by the fusion oncoprotein, and promotes AML expansion. Mechanistically, we show that the NRP1 locus has open chromatin in inv(16) AML, and that CBFβ::MYH11 modulates the local function of the transcription factors ERG, GATA2 and RUNX1 to sustain NRP1 levels. We found that ERG activates NRP1 expression, and that CBFβ::MYH11 knockdown represses ERG expression, thereby allowing the repressive activity of GATA2/RUNX1 at three NRP1 enhancers. Functionally, we demonstrate that NRP1 enhances the expansion of leukemic cells in vitro and in mice, and that this activity is dependent on its VEGFR-associated FV/FVIII domain. Finally, we show that treatment with VEGF inhibitor axitinib reduces AML cell growth and delays median leukemia latency in vivo. Our findings reveal that the NRP1/VEGF axis mediates proliferation in inv(16) AML blasts, and suggest that targeting NRP1 function could be promising in combination AML therapy.

Prioritization of Eleven-Nineteen-Leukemia Inhibitors as Orally Available Drug Candidates for Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the second most prevalent and fatal form of leukemia. The growth of AML cells harboring oncogenic MLL rearrangements relies on the YEATS domain-containing protein ENL. Many small molecule inhibitors targeting ENL have been developed. To prioritize these inhibitors for in vivo studies, a NanoBRET system was introduced to evaluate their cellular permeability and potency. This screening identified inhibitor 13 as a promising candidate. This inhibitor has remarkable metabolic stability and potent antiproliferative effects on MLL-fusion leukemia cell lines. In AML-xenografted mice, inhibitor 13 significantly improved survival. Subsequent optimization efforts led to the development of SR-C-107 (R), which exhibited strong activity against AML both at the cellular level (CC50(MOLM-13): 1.25 ± 0.18 μM; CC50(MV4-11): 0.81 ± 0.15 μM) and in vivo. These findings establish SR-C-107 (R) as a compelling candidate for AML treatment and lay the groundwork for the development of next-generation AML inhibitors.

Screening potential antileukemia agents from duckweed: Integration of chemical profiling, network pharmacology, and experimental validation.

The identification of active dietary flavonoids in food is promising for novel drug discovery. The active ingredients of duckweed (a widely recognized food and herb with abundant flavonoids) that are associated with acute myeloid leukemia (AML) have yet to be identified, and their underlying mechanisms have not been elucidated. The objective of this study was to identify novel constituents exhibiting antileukemia activity in duckweed through the integration of chemical profiling, network pharmacology, and experimental validation. First, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used to characterize the primary constituents of duckweed. Subsequently, AML cell-xenograft tumor models were used to validate the anticancer effect of duckweed extract. Furthermore, network pharmacology analysis was conducted to predict the potential active compounds and drug targets against AML. Lastly, based on these findings, two monomers (apiin and luteoloside) were selected for experimental validation. A total of 17 compounds, all of which are apigenin and luteolin derivatives, were identified in duckweed. The duckweed extract significantly inhibited AML cell growth in vivo. Furthermore, a total of 88 targets for duckweed against AML were predicted, with key targets including PTGS2, MYC, MDM2, VEGFA, CTNNB1, CASP3, EGFR, TP53, HSP90AA1, CCND1, MMP9, TNF, and MAPK1. GO and KEGG pathway enrichment analyses indicated that these targets were primarily involved in the apoptotic signaling pathway. Lastly, both apiin and luteoloside effectively induced apoptosis through CASP3 activation, and this effect could be partially reversed by a caspase inhibitor (Z-VAD). Duckweed extract has an antileukemic effect, and apiin derived from duckweed shows potential as a treatment for AML.

Pseudolaric Acid B Targets CD147 to Selectively Kill Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is an aggressive blood cancer. With low survival rates, new drug targets are needed to improve treatment regimens and patient outcomes. Pseudolaric acid B (PAB) is a plant-derived bioactive compound predicted to interact with cluster of differentiation 147 (CD147/BSG). CD147 is a transmembrane glycoprotein overexpressed in various malignancies with suggested roles in regulating cancer cell survival, proliferation, invasion, and apoptosis. However, the detailed function of PAB in AML remains unknown. In this study, AML cell lines and patient-derived cells were used to show that PAB selectively targeted AML (IC50: 1.59 ± 0.47 µM). Moreover, proliferation assays, flow cytometry, and immunoblotting confirmed that PAB targeting of CD147 resulted in AML cell apoptosis. Indeed, the genetic silencing of CD147 significantly suppressed AML cell growth and attenuated PAB activity. Overall, PAB imparts anti-AML activity through transmembrane glycoprotein CD147.

Clinical Significance of USP5 Expression Level in Acute Myeloid Leukemia and Its Regulatory Effects on AKT/mTOR/4EBP1 Signaling Pathway

To investigate the clinical significance, functional role and potential downstream mechanism of USP5 in acute myeloid leukemia (AML). The expression of USP5 in AML and normal tissues and its correlation with patients' survival were analyzed based on TCGA database. USP5 was knocked down and overexpressed in Jurkat and HL-60 cells using lentivirus. USP5 mRNA and protein expression were detected by RT-qPCR and Western blot, respectively. Cell proliferation and growth were measured by CCK-8 and methylcellulose colony-forming assay. Flow cytometry was used to analyze cell cycle and apoptosis. USP5 was highly expression in AML compared with normal tissues. Up-regulation of USP5 was negatively correlated with the survival of AML patients. USP5 knockdown and overexpression inhibited and promoted the proliferation and colony growth of AML cells, respectively. Cell cycle arrest and apoptosis were induced in USP5 knockdown Jurkat and HL-60 cells. Furthermore, USP5 knockdown inhibited the phosphrylation of AKT, mTOR and 4EBP1. Overexpression of USP5 predicts poor survival of AML patients. Targeting USP5 suppresses AKT/mTOR/4EBP1 signaling and reduces the proliferation and growth of AML cells.

Preclinical efficacy of BRG1/BRM ATPase inhibitor in B lymphoblastic leukemia.

10030 Background: Acute lymphoblastic leukemia (ALL) is the most common blood cancer in children and young adults. While ALL has a high cure rate, approximately 20% of patients who initially respond to treatment relapse and prognosis post relapse remains poor. New therapeutic treatments are needed to reduce relapse rates in B-ALL pediatric patients and to minimize the toxic side effects seen with current therapies. We and others have previously shown that BRG1/BRM ATPase inhibitors have efficacy in targeting acute myeloid leukemia (AML) and one, FHD-286, is currently being evaluated for its use in AML in an ongoing clinical trial (NCT04891757). BRG1 and BRM are the ATP-dependent catalytic subunits of the SWI/SNF chromatin remodeling complex which has a critical role in controlling gene expression, cell growth and survival of AML cells. Our prior work revealed that BRG1/BRM ATPase inhibition leads to selective transient reduction of normal B cells. Further, data from Depmap also nominates SMARCA4, the gene encoding BRG1, as a dependency in B-ALL cell lines, leading us to hypothesize that BRG1/BRM inhibition may be an effective therapy for B-ALL. Methods: We treated B-ALL cell lines with varying genetic lesions ( KMT2A-rearranged: RS4;11, KOPN-8, SEM; ETV6::RUNX1 fusion: REH and DUX4-rearranged: Nalm6) with dose titrations of FHD-286 and measured cell growth over the course of six days. In addition, at 48- and 72-hours post-treatment, we assayed cells for viability, cell cycle kinetics, and apoptosis induction via flow cytometry to provide insight into FHD-286’s mechanism of action in B-ALL. Results: We found that FHD-286 treatment resulted in decreased cell growth in a dose-dependent manner across all B-ALL cell lines. While all cell lines were displayed reduced proliferation at nanomolar doses, sensitivity varied. Nalm6, REH and SEM cells were the most sensitive with low nanomolar IC50s (2-7nM). In contrast, two of the KMT2Ar B-ALL lines, RS4;11 and KOPN-8, required higher doses to achieve growth inhibition (40-70nM). To better understand the mechanism by which FHD-286 is affecting cell growth, we evaluate whether FHD-286 is targeting cell growth through the apoptotic pathway or through cell cycle arrest. Our preliminary data indicates cell growth inhibition is only slightly attributable to cell cycle arrest and apoptosis induction, leading us to explore terminal differentiation as a mechanism of slowed proliferation. Conclusions: Our results show FHD-286 can strongly inhibit B-ALL cell growth in vitro, indicating FHD-286 as a potential therapeutic agent to treat B-ALL. In vivo validation is ongoing. Further directions include defining the effect of FHD-286 on chromatin remodeling, gene expression, and exploring efficacy in combination therapy, which has been shown to increase FHD-286 efficacy against AML. Our data may ultimately justify inclusion of B-ALL patients in clinical studies of BRG1/BRM inhibitors.

Circ_0012152 Accelerates Acute Myeloid Leukemia Progression through the miR-652-3p/SOX4 Axis.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by abnormal myeloid blast expansion. Recent studies have demonstrated that circular RNAs play a role in AML pathogenesis. In this study, we aimed to investigate the clinical significance of circ_0012152 in AML and elucidate its underlying molecular mechanism in the pathogenesis of this condition. Circ_0012152 expression was detected by quantitative real-time polymerase chain reaction in samples obtained from 247 patients with AML and 40 healthy controls. A systematic analysis of clinical characteristics and prognostic factors was also conducted. Cell growth was assessed using the Cell Counting Kit-8 (CCK-8) assay, and apoptosis and cell cycle progression were evaluated by flow cytometry. Moreover, RNA pull-down was performed to identify target microRNAs, and transcriptome RNA sequencing and bioinformatics analyses were utilized to identify downstream mRNA targets. Circ_0012152 was significantly upregulated in samples from patients with AML and served as an independent adverse prognostic factor for overall survival (OS) (hazard ratio: 2.357; 95% confidence interval 1.258-4.415). The circ_0012152 knockdown reduced cell growth, increased apoptosis, and inhibited cell cycle progression in AML cell lines. RNA pull-down and sequencing identified miR-652-3p as a target microRNA of circ_0012152. Cell growth inhibition by circ_0012152 knockdown was significantly relieved by miR-652-3p inhibitors. We suggested that miR-652-3p targeted SOX4, as the decrease in SOX4 expression resulting from circ_0012152 knockdown was upregulated by miR-652-3p inhibitors in AML cells. Circ_0012152 is an independent poor prognostic factor for OS in AML, and it promotes AML cell growth by upregulating SOX4 through miR-652-3p.

Discovery of Highly Potent and Efficient CBP/p300 Degraders with Strong In Vivo Antitumor Activity.

The transcriptional coactivator cAMP response element binding protein (CREB)-binding protein (CBP) and its homologue p300 have emerged as attractive therapeutic targets for human cancers such as acute myeloid leukemia (AML). Herein, we report the design, synthesis, and biological evaluation of a series of cereblon (CRBN)-recruiting CBP/p300 proteolysis targeting chimeras (PROTACs) based on the inhibitor CCS1477. The representative compounds 14g (XYD190) and 14h (XYD198) potently inhibited the growth of AML cells with low nanomolar IC50 values and effectively degraded CBP and p300 proteins in a concentration- and time-dependent manner. Mechanistic studies confirmed that 14g and 14h can selectively bind to CBP/p300 bromodomains and induce CBP and p300 degradation in bromodomain family proteins in a CRBN- and proteasome-dependent manner. 14g and 14h displayed remarkable antitumor efficacy in the MV4;11 xenograft model (TGI = 88% and 93%, respectively). Our findings demonstrated that 14g and 14h are useful lead compounds and deserve further optimization and activity evaluation for the treatment of human cancers.

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.

Discovery of Selective and Potent ATR Degrader for Exploration its Kinase‐Independent Functions in Acute Myeloid Leukemia Cells

AbstractATR has emerged as a promising target for anti‐cancer drug development. Several potent ATR inhibitors are currently undergoing various stages of clinical trials, but none have yet received FDA approval due to unclear regulatory mechanisms. In this study, we discovered a potent and selective ATR degrader. Its kinase‐independent regulatory functions in acute myeloid leukemia (AML) cells were elucidated using this proteolysis‐targeting chimera (PROTAC) molecule as a probe. The ATR degrader, 8 i, exhibited significantly different cellular phenotypes compared to the ATR kinase inhibitor 1. Mechanistic studies revealed that ATR deletion led to breakdown in the nuclear envelope, causing genome instability and extensive DNA damage. This would increase the expression of p53 and triggered immediately p53‐mediated apoptosis signaling pathway, which was earlier and more effective than ATR kinase inhibition. Based on these findings, the in vivo anti‐proliferative effects of ATR degrader 8 i were assessed using xenograft models. The degrader significantly inhibited the growth of AML cells in vivo, unlike the ATR inhibitor. These results suggest that the marked anti‐AML activity is regulated by the kinase‐independent functions of the ATR protein. Consequently, developing potent and selective ATR degraders could be a promising strategy for treating AML.

Discovery of Selective and Potent ATR Degrader for Exploration its Kinase-Independent Functions in Acute Myeloid Leukemia Cells.

ATR has emerged as a promising target for anti-cancer drug development. Several potent ATR inhibitors are currently undergoing various stages of clinical trials, but none have yet received FDA approval due to unclear regulatory mechanisms. In this study, we discovered a potent and selective ATR degrader. Its kinase-independent regulatory functions in acute myeloid leukemia (AML) cells were elucidated using this proteolysis-targeting chimera (PROTAC) molecule as a probe. The ATR degrader, 8 i, exhibited significantly different cellular phenotypes compared to the ATR kinase inhibitor 1. Mechanistic studies revealed that ATR deletion led to breakdown in the nuclear envelope, causing genome instability and extensive DNA damage. This would increase the expression of p53 and triggered immediately p53-mediated apoptosis signaling pathway, which was earlier and more effective than ATR kinase inhibition. Based on these findings, the in vivo anti-proliferative effects of ATR degrader 8 i were assessed using xenograft models. The degrader significantly inhibited the growth of AML cells in vivo, unlike the ATR inhibitor. These results suggest that the marked anti-AML activity is regulated by the kinase-independent functions of the ATR protein. Consequently, developing potent and selective ATR degraders could be a promising strategy for treating AML.

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.

Epigenetic control over the cell-intrinsic immune response antagonizes self-renewal in acute myeloid leukemia

AbstractEpigenetic modulation of the cell-intrinsic immune response holds promise as a therapeutic approach for leukemia. However, current strategies designed for transcriptional activation of endogenous transposons and subsequent interferon type-I (IFN-I) response, show limited clinical efficacy. Histone lysine methylation is an epigenetic signature in IFN-I response associated with suppression of IFN-I and IFN-stimulated genes, suggesting histone demethylation as key mechanism of reactivation. In this study, we unveil the histone demethylase PHF8 as a direct initiator and regulator of cell-intrinsic immune response in acute myeloid leukemia (AML). Site-specific phosphorylation of PHF8 orchestrates epigenetic changes that upregulate cytosolic RNA sensors, particularly the TRIM25-RIG-I-IFIT5 axis, thereby triggering the cellular IFN-I response-differentiation-apoptosis network. This signaling cascade largely counteracts differentiation block and growth of human AML cells across various disease subtypes in vitro and in vivo. Through proteome analysis of over 200 primary AML bone marrow samples, we identify a distinct PHF8/IFN-I signature in half of the patient population, without significant associations with known clinically or genetically defined AML subgroups. This profile was absent in healthy CD34+ hematopoietic progenitor cells, suggesting therapeutic applicability in a large fraction of patients with AML. Pharmacological support of PHF8 phosphorylation significantly impairs the growth in samples from patients with primary AML. These findings provide novel opportunities for harnessing the cell-intrinsic immune response in the development of immunotherapeutic strategies against AML.

Abstract P29: Zotatifin, the eukaryotic translation initiation factor 4A (eIF4A) inhibitor, synergizes with Venetoclax to inhibit acute myeloid leukemia cell growth

Abstract New targeted therapies such as the selective and potent BCL-2 inhibitor Venetoclax have revolutionized the treatment of acute myeloid leukemia (AML). However, due to the cellular heterogeneity of the disease, drug-resistant cell types often emerge and drive relapse. We found that the eukaryotic translation initiation factor 4A (EIF4A) is highly expressed in multiple primitive AML cell types compared to their healthy counterparts, suggesting its potential as a therapeutic target. By unwinding complex structures in the 5’ UTR of transcripts, eIF4A promotes the translation of oncogene transcripts. A first-in-class drug, Zotatifin, inhibits the helicase activity of eIF4A and curtails tumor growth in receptor tyrosine kinase-driven tumors. Zotatifin is currently being evaluated in phase 1/2 trials across multiple solid tumors (NCT04092673). However, its effectiveness in blood cancers such as AML remains unexplored, and it is unknown whether Zotatifin may work synergistically with Venetoclax. Here, we treated AML cell lines in vitro and primary healthy or AML bone marrow ex vivo with Zotatifin or Venetoclax alone and in combination, and demonstrate synergistic killing of AML cells by the combination therapy (ZIP synergy score = 57.0 in MOLM-13, 19.3 in MV4-11, P < 0.0001). Mechanistically, we show that Zotatifin decreases eIF4A and AKT expression, and its combination with Venetoclax drives increased BAK and cleaved BAK expression, as well as increased cleavage of MCL-1 and BCL-2, consistent with drug synergy by convergence on the apoptotic pathway. In pilot colony-forming unit (CFU) assays, we found the combination to be tolerable in healthy CD34+ human hematopoietic stem and progenitor cells (HSPCs). To test drug efficacy in vivo, we generated a patient-derived xenograft (PDX) AML model from a patient who experienced relapse with complex cytogenetics, FLT3 and NPM1 mutations. Only the combination of Zotatifin and Venetoclax but not monotherapies effectively suppressed tumor burden as assessed by human CD45 engraftment in the peripheral blood. Further, the combination of Zotatifin and Venetoclax significantly prolonged survival of xenografted mice (median survival after treatment: vehicle 11 days, Venetoclax 13 days, Zotatifin 15 days, combination 42 days, P = 0.0002). Together, our data indicates that Zotatifin is synergistic with Venetoclax and may represent an effective treatment option for patients who are either primary refractory or have developed resistance to existing Venetoclax-containing regimens. Citation Format: Yoke Seng Lee, Jonathan Good, Noha Awais, Benjamin Eschle, Jenny Noel, Jean Acosta, Erica DePasquale, David Cucchi, Prafulla Gokhale, Fadi Najm, Peter van Galen. Zotatifin, the eukaryotic translation initiation factor 4A (eIF4A) inhibitor, synergizes with Venetoclax to inhibit acute myeloid leukemia cell growth [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P29.

Deciphering Acute Myeloid Leukemia Associated Transcription Factors in Human Primary CD34+ Hematopoietic Stem/Progenitor Cells.

Hemato-oncological diseases account for nearly 10% of all malignancies and can be classified into leukemia, lymphoma, myeloproliferative diseases, and myelodysplastic syndromes. The causes and prognosis of these disease entities are highly variable. Most entities are not permanently controllable and ultimately lead to the patient's death. At the molecular level, recurrent mutations including chromosomal translocations initiate the transformation from normal stem-/progenitor cells into malignant blasts finally floating the patient's bone marrow and blood system. In acute myeloid leukemia (AML), the so-called master transcription factors such as RUNX1, KMT2A, and HOX are frequently disrupted by chromosomal translocations, resulting in neomorphic oncogenic fusion genes. Triggering ex vivo expansion of primary human CD34+ stem/progenitor cells represents a distinct characteristic of such chimeric AML transcription factors. Regarding oncogenic mechanisms of AML, most studies focus on murine models. However, due to biological differences between mice and humans, findings are only partly transferable. This review focuses on the genetic manipulation of human CD34+ primary hematopoietic stem/progenitor cells derived from healthy donors to model acute myeloid leukemia cell growth. Analysis of defined single- or multi-hit human cellular AML models will elucidate molecular mechanisms of the development, maintenance, and potential molecular intervention strategies to counteract malignant human AML blast cell growth.

Hyperactive Natural Killer cells in Rag2 knockout mice inhibit the development of acute myeloid leukemia

Immunotherapy has attracted considerable attention as a therapeutic strategy for cancers including acute myeloid leukemia (AML). In this study, we found that the development of several aggressive subtypes of AML is slower in Rag2−/− mice despite the lack of B and T lymphocytes, even compared to the immunologically normal C57BL/6 mice. Furthermore, an orally active p53-activating drug shows stronger antileukemia effect on AML in Rag2−/− mice than C57BL/6 mice. Intriguingly, Natural Killer (NK) cells in Rag2−/− mice are increased in number, highly express activation markers, and show increased cytotoxicity to leukemia cells in a coculture assay. B2m depletion that triggers missing-self recognition of NK cells impairs the growth of AML cells in vivo. In contrast, NK cell depletion accelerates AML progression in Rag2−/− mice. Interestingly, immunogenicity of AML keeps changing during tumor evolution, showing a trend that the aggressive AMLs generate through serial transplantations are susceptible to NK cell-mediated tumor suppression in Rag2−/− mice. Thus, we show the critical role of NK cells in suppressing the development of certain subtypes of AML using Rag2−/− mice, which lack functional lymphocytes but have hyperactive NK cells.

Dinaciclib inhibits the growth of acute myeloid leukemia cells through either cell cycle-related or ERK1/STAT3/MYC pathways

Although immature differentiation and uncontrolled proliferation of hematopoietic stem cells are thought to be the primary mechanisms of acute myeloid leukemia (AML), the pathophysiology in most cases remains unclear. Dinaciclib, a selective small molecule targeting multiple cyclin-dependent kinases (CDKs), is currently being evaluated in oncological clinical trials. Despite the proven anticancer potential of dinaciclib, the differential molecular mechanisms by which it inhibits the growth of different AML cell lines remain unclear. In the current study, we treated HL-60 and KG-1 AML cell lines with dinaciclib and investigated the potential mechanisms of dinaciclib-induced AML cell growth inhibition using flow cytometry and western blotting assays. Data from HL-60 and KG-1 AML cells were validated using human primary AML cells. The results showed that the growth inhibitory effect of dinaciclib was more sensitive in HL-60 cells (IC50: 8.46 nM) than in KG-1 cells (IC50: 14.37 nM). The protein decline in Cyclin A/B and CDK1 and cell cycle arrest in the G2/M phase were more profound in HL-60 cells, corresponding to its growth inhibition. Although the growth inhibition of KG-1 cells by dinaciclib was still pronounced, the cell cycle-associated proteins were relatively insensitive. In addition to cell cycle regulation, the activation/expression of ERK1/STAT3/MYC signaling was significantly reduced by dinaciclib in KG-1 cells compared with that in HL-60 cells. Regarding the results of primary AML cells, we observed ERK1/STAT3/MYC inhibition and cell cycle regulation in different patients. These findings suggest that the cell cycle-associated and ERK1/STAT3/MYC signaling pathways might be two distinct mechanisms by which dinaciclib inhibits AML cells, which could facilitate the development of combination therapy for AML in the future.

AML alters bone marrow stromal cell osteogenic commitment via Notch signaling.

Acute myeloid leukemia (AML) is a highly heterogeneous malignancy caused by various genetic alterations and characterized by the accumulation of immature myeloid blasts in the bone marrow (BM). This abnormal growth of AML cells disrupts normal hematopoiesis and alters the BM microenvironment components, establishing a niche supportive of leukemogenesis. Bone marrow stromal cells (BMSCs) play a pivotal role in giving rise to essential elements of the BM niche, including adipocytes and osteogenic cells. Animal models have shown that the BM microenvironment is significantly remodeled by AML cells, which skew BMSCs toward an ineffective osteogenic differentiation with an accumulation of osteoprogenitors. However, little is known about the mechanisms by which AML cells affect osteogenesis. We studied the effect of AML cells on the osteogenic commitment of normal BMSCs, using a 2D co-culture system. We found that AML cell lines and primary blasts, but not normal hematopoietic CD34+ cells, induced in BMSCs an ineffective osteogenic commitment, with an increase of the early-osteogenic marker tissue non-specific alkaline phosphatase (TNAP) in the absence of the late-osteogenic gene up-regulation. Moreover, the direct interaction of AML cells and BMSCs was indispensable in influencing osteogenic differentiation. Mechanistic studies identified a role for AML-mediated Notch activation in BMSCs contributing to their ineffective osteogenic commitment. Inhibition of Notch using a γ-secretase inhibitor strongly influenced Notch signaling in BMSCs and abrogated the AML-induced TNAP up-regulation. Together, our data support the hypothesis that AML infiltration produces a leukemia-supportive pre-osteoblast-rich niche in the BM, which can be partially ascribed to AML-induced activation of Notch signaling in BMSCs.

Abstract A053: The dual BRG1/BRM (SMARCA4/2) inhibitor FHD-286 induces functional differentiation and splicing defects in preclinical models of acute myeloid leukemia (AML)

Abstract The BRG/BRM associated factor (BAF) complex is a chromatin remodeler critical for maintenance of cell viability in many cancers. This includes acute myeloid leukemia (AML), where BAF maintains the stem-like transcriptional state of blast cells. FHD-286 is a dual inhibitor of the BAF ATPase subunits BRG1 and BRM and is currently being investigated in relapsed or refractory AML and myelodysplastic syndrome. We have shown previously that FHD-286 induces the expression of the myeloid differentiation marker CD11b in preclinical models after treatment with sub-cytoreductive doses at long timepoints. Here we further characterized the differentiation phenotype induced by low-dose FHD-286 in AML cell lines through RNA-seq and a genome-wide CRISPR-Cas9 knockout screen. These datasets and mechanistic follow-up studies suggested AML cell lines treated with FHD-286 can functionally differentiate to gain the ability to produce superoxide anion and perform phagocytosis. We also found that FHD-286 treatment disrupts mRNA splicing and that this is a likely contributor to AML cell growth defects induced by BRG1/BRM inhibition. This study suggests multiple mechanisms by which FHD-286 is able to disrupt the stem-like transcriptional state of AML blasts to cause differentiation and ultimately cell death. Citation Format: Ashley K. Gartin, David N. Mayhew, David L. Lahr, GiNell Elliott, Heena Gandevia, Molly M. Wilson, Richard Centore, Mike Collins, Astrid Thomsen, Jessica Piel, Gabriel J. Sandoval, Martin Hentemann. The dual BRG1/BRM (SMARCA4/2) inhibitor FHD-286 induces functional differentiation and splicing defects in preclinical models of acute myeloid leukemia (AML) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A053.