Primary Acute Myeloid Leukemia Cells Research Articles

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.

Induction of NK cell reactivity against acute myeloid leukemia by Fc-optimized CD276 (B7-H3) antibody.

Acute myeloid leukemia (AML) remains a therapeutic challenge despite recent therapeutic advances. Although monoclonal antibodies (mAbs) engaging natural killer (NK) cells via antibody-dependent cellular cytotoxicity (ADCC) hold promise in cancer therapy, almost none have received clinical approval for AML, so far. Recently, CD276 (B7-H3) has emerged as a promising target for AML immunotherapy, due to its high expression on leukemic blasts of AML patients. Here, we present the preclinical development of the Fc-optimized CD276 mAb 8H8_SDIE with enhanced CD16 affinity. We demonstrate that 8H8_SDIE specifically binds to CD276 on AML cell lines and primary AML cells and induces pronounced NK cell activation and degranulation as measured by CD69, CD25, and CD107a. Secretion of IFNγ, TNF, granzyme B, granulysin, and perforin, which mediate NK cell effector functions, was induced by 8H8_SDIE. A pronounced target cell-restricted lysis of AML cell lines and primary AML cells was observed in cytotoxicity assays using 8H8_SDIE. Finally, xenograft models with 8H8_SDIE did not cause off-target immune activation and effectively inhibited leukemia growth in vivo. We here present a novel attractive immunotherapeutic compound that potently induces anti-leukemic NK cell reactivity in vitro and in vivo as treatment option for AML.

FLT3 and IRAK4 Inhibitor Emavusertib in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia.

Targeting the FLT3 receptor and the IL-1R associated kinase 4 as well as the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The FLT3 and IRAK4 inhibitor emavusertib (CA4948), the MCL1 inhibitor S63845, the BCL2 inhibitor venetoclax, and the HSP90 inhibitor PU-H71 were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells in vitro. AML cells represented all major morphologic and molecular subtypes, including FLT3-ITD and NPM1 mutant AML cell lines and a variety of patient-derived AML cells. Emavusertib in combination with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in MOLM-13 cells. In primary AML cells, the response to emavusertib was associated with the presence of the FLT3 gene mutation with an allelic ratio >0.5 and the presence of NPM1 gene mutations. S63845 was effective in all tested AML cell lines and primary AML samples. Blast cell percentage was positively associated with the response to CA4948, S63845, and venetoclax, with elevated susceptibility of primary AML with blast cell fraction >80%. Biomarkers of the response to venetoclax included the blast cell percentage and bone marrow infiltration rate, as well as the expression levels of CD11b, CD64, and CD117. Elevated susceptibility to CA4948 combination treatments with S63845 or PU-H71 was associated with FLT3-mutated AML and CD34 < 30%. The combination of CA4948 and BH3-mimetics may be effective in the treatment in FLT3-mutated AML with differential target specificity for MCL1 and BCL2 inhibitors. Moreover, the combination of CA4948 and PU-H71 may be a candidate combination treatment in FLT3-mutated AML.

Abstract 6341: Targeting myeloid epithelial tyrosine kinase (MERTK) receptor in acute myeloid leukemia using a novel antibody-drug conjugate, RGX-019-MMAE

Abstract Background: Myeloid epithelial reproductive tyrosine kinase (MERTK), a transmembrane protein receptor from the TAM (Tyro3, Axl, Mertk) family, is overexpressed in cancer and associated with reduced apoptosis and chemoresistance, making it an attractive therapeutic target. RGX-019-MMAE, a novel humanized IgG1-MMAE antibody-drug conjugate (ADC) (Inspirna, Inc), selectively binds to MERTK with high affinity on tumor cells, resulting in internalization and degradation of the receptor. It then induces cytotoxicity through the release of the payload, MMAE (monomethyl auristatin E), which disrupts mitosis. We hypothesize that targeting MERTK via RGX-019-MMAE exerts an antileukemic effect. Methods: First, to determine the clinical relevance of MERTK, using the Oregon Health and Science University (OHSU) acute myeloid leukemia (AML) dataset, we divided patients into low and high MERTK mRNA expression based on mean expression and compared their overall survival. To identify the AML subtypes with the highest MERTK expression, we analyzed samples from 810 AML patients at MD Anderson using reverse phase protein arrays (RPPA), as well as 8 AML cell lines and the peripheral blood of 9 AML patients using flow cytometry. Then, the cell lines with the highest MERTK expression were treated with varying doses of RGX-019-MMAE or isotype control for 120 hours and assessed for viability with CellTiter-Glo 2.0. Likewise, we administered RGX-019-MMAE to primary cells from AML patients and determined the anti-leukemic effect. Finally, we investigated the synergistic effect of RGX-019-MMAE with venetoclax, a BCL2 inhibitor, or cytarabine, a chemotherapy agent, in vitro. Results: In the AML OHSU dataset, patients with high MERTK mRNA expression had significantly worse overall survival (p=0.02). RPPA revealed that patients with PTPN11 mutation, t (9;11), and monocytic AML subtypes had significantly higher MERTK protein expression. MERTK protein expression varied in AML cell lines and was highest in Kasumi-1 and OCI-AML3. Treatment of these two cell lines with RGX-019-MMAE resulted in significantly more killing of leukemic cells than the isotype control ADC in a dose-dependent manner (p&amp;lt;0.01). Similarly, in primary AML cells with high MERTK expression, RGX-019-MMAE treatment induced significant cell death compared to the control ADC. In addition, Kasumi-1 and OCl-AML3 cells treated with RGX-019-MMAE combined with venetoclax or cytarabine showed a synergistic cytotoxic effect in a dose-dependent manner. Conclusion: High MERTK expression in monocytic AML suggested that MERTK is a promising therapeutic target in this subtype. RGX-019-MMAE significantly induced AML cell death and produced a synergistic anti-leukemic effect in combination with chemotherapeutic agents and targeted therapy in vitro, suggesting its potential for targeting patients with high-risk AML. Citation Format: Anudishi Tyagi, Maryam Siddiqui, Isabel Kurth, Shugaku Takeda, Amanda Eckstrom, Jenny Borgman, Abhishek Maiti, Venkata Lokesh Battula. Targeting myeloid epithelial tyrosine kinase (MERTK) receptor in acute myeloid leukemia using a novel antibody-drug conjugate, RGX-019-MMAE [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 6341.

Abstract 618: Preclinical assessment of APL-030, a selective and orally bioavailable inhibitor of the integrated stress response regulator GCN2 with activity against acute myeloid leukemia

Abstract GCN2 (EIF2AK4) is an evolutionarily conserved kinase and a pivotal regulator of the Integrated Stress Response (ISR), an adaptive cellular program triggered primarily by amino acid scarcity. Active GCN2 phosphorylates the translation initiation factor eIF2α, resulting in the attenuation of global protein synthesis and a largely ATF4-driven program of resource enhancement. GCN2 promotes cancer cell survival under conditions of microenvironmental or drug-induced intracellular nutrient scarcity. Here, we describe the preclinical development of APL-030, a novel and selective ATP-competitive inhibitor of GCN2. APL-030 was confirmed as a potent inhibitor of GCN2 in an on-target biochemical kinase assay with a Ki of 4.4nM. In a cell-based assay APL-030 decreased eIF2α phosphorylation in a dose-dependent manner via direct GCN2 inhibition with an IC50 of 50.8nM. In acute myeloid leukemia (AML) cell lines GCN2 inhibition resulted in a decrease in gene expression of effector genes CHAC1 and DDIT3. Moreover, treatment with APL-030 dose-dependently decreased cell viability in multiple AML cell lines, which was accompanied by an increase in caspase 3/7 activity. Bulk mRNA sequencing of MOLM-13 AML cells showed that APL-030 inhibited the extensive transcriptome changes triggered by glutamine depletion, in line with a high degree of selectivity of APL-030 for GCN2-mediated ISR signaling. In cells grown in rich medium, APL-030 triggered a largely ISR-independent transcriptome response that was dominated by the enrichment of pathways related to cell cycle as well as RNA and protein metabolism. In vivo, APL-030 treatment of an AML xenograft animal model completely inhibited tumor growth and prolonged survival time. Inhibition of AML tumor growth was shown to be a consequence of decreased cell viability and induction of caspase-dependent apoptosis. APL-030 was also tested on primary diagnostic AML cells, which were assessed for cell death by flow cytometry following staining for Annexin-V, 7-AAD, CD45, CD34, and CD38. Treatment with APL-030 caused a statistically significant increase in AML cell death in all six samples studied. Three samples exhibited a measurable CD34+/CD38- compartment, enabling evaluation of this leukemic stem cell-enriched population. APL-030 caused a statistically significant increase in cell death in the leukemic stem cell-enriched population in all three samples studied. APL-030 is a novel GCN2 inhibitor that that has shown encouraging efficacy in preclinical studies using both AML cell lines and patient-derived AML samples. Based on these preclinical results, a phase 1/2 study is planned in hematological tumors. Citation Format: Monica Roman-Trufero, Gavin Whitlock, Matthew Fuchter, Maxmila Jeyakumar, Panagiota Chaida, Sereina Annik Herzog, Armin Zebisch, Richard Butt, Holger W. Auner, Nadine Clemo. Preclinical assessment of APL-030, a selective and orally bioavailable inhibitor of the integrated stress response regulator GCN2 with activity against 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 618.

Abstract 6322: CCTx-001 CAR T-cells targeting the AML specific antigen IL-1RAP show robust pre-clinical anti-tumor efficacy

Abstract Despite significant advancements in acute myeloid leukemia (AML) treatment, patients with relapsed/refractory (r/r) AML have limited therapeutic options. Novel approaches are urgently needed. CAR T-cell therapy has shown remarkable success in hematological malignancies. However, its application in AML has been hampered by the absence of highly AML-specific antigens, making current CAR T-cell AML therapies under development poorly tolerated and effective. This pre-clinical study introduces a novel CAR T-cell therapy, CCTx-001, specifically designed to target IL-1RAP, a 78-kDa transmembrane glycoprotein. IL-1RAP, is highly expressed on AML cells but not on healthy hematopoietic stem and progenitor cells (CD34+ HSPC). Additionally, transcriptomic analyses reveal a correlation between high IL-1RAP expression and poor prognosis in AML patients. IL-1RAP's predominant presence on myeloid leukemia cells, coupled with their dependence to IL-1RAP-related pathways for their survival, presents a promising avenue for CAR T-cell therapy without the risk of treatment-induced antigen loss. CCTx-001, an autologous CAR T-cell therapy, was developed to target IL-1RAP on AML cells. It comprises CD8+ and CD4+ T-cells transduced with a highly specific anti-IL-1RAP Single-Chain Variable Fragment (scFv), an IgG1 hinge region, CD28, 4-1BB co-stimulatory domains, and a CD3ζ activation domain. In vitro experiments confirmed CCTx-001's efficacy in killing AML cell lines and primary AML cells of varying IL-1RAP expression levels, also demonstrating cytotoxicity and cytokine release that was dependent on the (E:T) ratio and target expression. Furthermore, in vivo studies using immunocompromised NCG mice confirmed CCTx-001's ability to target IL-1RAP and inhibit the growth of IL-1RAP-positive human AML tumor xenografts, leading to increased survival time. Biodistribution analyses confirmed the successful adoptive transfer, expansion and persistence of CCTx-001 CAR T-cells in vivo, only when injected into IL-1RAP-expressing tumor-bearing mice, which correlated with reduced levels of circulating AML cells. The promising preclinical data presented herein warrant the evaluation of CCTx-001 CAR T-cell therapy in clinical trials for r/r AML patients. CCTx-001 CAR T-cell therapy offers a potential breakthrough in addressing the unmet medical needs of AML patients with resistant or refractory disease. Citation Format: Edouard De Dreuzy, Soha Reda El Sayed, Delphine Genin, Clementine Primus, Lucie Bouquet, Alexis Collette, Olivier Favre-Bulle, Erik Rutjens, Jens Hasskarl, Ksenija Pavletic, Marina Deschamps, Christophe Ferrand, Richard C. A. Sainson. CCTx-001 CAR T-cells targeting the AML specific antigen IL-1RAP show robust pre-clinical anti-tumor efficacy [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 6322.

Abstract 2081: Biomarkers for CLK inhibitor CTX-712 treatment response in myeloid neoplasms: Paving the way toward clinical trials

Abstract Dysregulated RNA splicing is a molecular feature that characterizes almost all tumor types. Splicing is altered in cancer owing to both recurrent mutations and deregulated expression of trans-acting factors that catalyze and regulate splicing. Therefore, splicing can be targeted for the development of new drugs to treat cancers, particularly those with mutations in splicing factors. In myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), genes that encode splicing factors are among those with the highest mutation rates. These mutations are heterozygous and largely mutually exclusive, suggesting the possibility of synthetic lethality due to multiple splicing factor mutations. Such synthetic lethality can be applied as an innovative strategy to develop drugs for the treatment of MDS and AML. We have reported the development of a CLK kinase inhibitor, CTX-712, and evaluated its anti-leukemic activities both in vitro and in vivo. The mechanism of action of CTX-712 involves the suppression of the phosphorylation of multiple SR proteins, including SRSF2/3/4/6, alteration of nuclear speckle morphology, and switching of binding partners of SRSF2. RNA-seq analysis revealed that CTX-712 induced global alterations in splicing, typically resulting in exon exclusion (exon skipping) of cassette exons. Notably, the anti-leukemic effects of CTX-712 positively correlated with the degree of altered splicing, suggesting that CTX-712 inhibits RNA splicing. [Cancer Res (2022) 82 (12_Supplement): 5494., Blood (2022) 140 (Supplement 1): 489-490]. However, patient stratification markers for CTX-712 based on the above mechanism are still undefined. To identify biomarkers for CTX-712, we performed a comprehensive analysis by integrating both wet and dry DNA/RNA sequencing on more than 60 ex vivo primary samples from MDS/AML patients and 23 MDS/AML-derived xenografts (PDX). Unexpectedly, regardless of their splicing factor-mutation status, various types of primary AML cells and PDX models were highly sensitive to CTX-712. Both the primary cells and PDX models showed significant responses to CTX-712 and greater changes in splicing. Additionally, we performed biomarker analysis by comparing gene mutations and RNA splicing patterns between the sensitive and insensitive models and associating them with the anti-tumor effect. We identified several potential biomarkers that can indicate sensitivity to CTX-712. Mechanistic studies that aim to determine why these biomarkers could make cancer more sensitive to splicing perturbation by CTX-712 are ongoing. In conclusion, our results demonstrated a significant effect of CTX-712 on MDS/AML-derived models regardless of their splicing factor-mutation status. Additionally, we present novel biomarkers that could predict sensitivity to CTX-712. Citation Format: Akinori Yoda, Daisuke Morishita, Yotaro Ochi, Akio Mizutani, Hirokazu Tozaki, Yoshihiko Satoh, Takuto Mori, June Takeda, Hideki Makishima, Masahiro Nakagawa, Yasuhito Nannya, Seishi Ogawa. Biomarkers for CLK inhibitor CTX-712 treatment response in myeloid neoplasms: Paving the way toward clinical trials [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 2081.

Abstract 3904: Multiplexed flow cytometry based immunophenotyping paired with functional ex vivo (MFLEX) drug profiling informs potential efficacy of therapeutic agents in acute myeloid leukemia

Abstract Introduction: Acute Myeloid Leukemia (AML) is a heterogenous hematological malignancy with a complex clonal architecture that requires in-depth immunophenotyping for disease characterization. Conventional AML ex vivo models for preclinical drug screening lack functional readouts that provide single cell level data to identify drug activity in defined AML subsets with unique immune signatures. Here, we present a novel platform that links Multiparameter Flow cytometry with high-throughput EX vivo drug screening (MFLEX) in AML patient-derived cell models to simultaneously detect phenotypic changes and predict cell-subset specific drug responses in AML. Method: We developed a 21-color high-dimensional immune profiling panel that captures AML heterogeneity by delineating primitive and mature AML blasts along with normal hematopoietic lineages. The panel also includes markers for functional measurement of apoptosis, proliferation, differentiation, and expression of BCL2 family proteins in lymphoid and myeloid cell subsets. Ex vivo culture conditions were optimized using physiologically relevant stroma-free cytokine-rich media that supports primary AML cells with minimal phenotypic shift, allowing for high throughput functional drug sensitivity testing. Timepoints and dosing conditions for AML standard of care (SoC) agents were optimized to support combination screens. Multi-parameter datasets were analysed for functional differences and visualized using dimension reduction methods. Results: We tested MFLEX on clinically annotated AML samples to understand sensitivity profiles to venetoclax and combination partners: 5-azacytidine, cytarabine, fludarabine and gilteritinib. We identified distinct drug sensitivity profiles for venetoclax in AML patients with varying leukemic differentiation states where M0/M1 subtypes were more sensitive to therapy than M4/M5. We also observed cell lineage specific sensitivities, with myeloid blast being more sensitive to venetoclax compared to autologous CD4+ and CD8+ T-cells and this observed difference was correlated with higher expression of BCL2. Our platform demonstrated that patients who previously failed venetoclax therapy could benefit from a combination with 5-azacytidine or fludarabine and this was patient-specific. We further evaluated MFLEX’s predictive value for patient clinical response by systematically comparing venetoclax sensitivity in patient-derived xenograft models in vivo vs ex vivo and found a high correlation between drug responses. Conclusion: We have established a unique, immune drug screening platform, MFLEX, that has high translational value and can be implemented in clinical trials to identify drug response patterns, novel combinations, and potential biomarkers of response for patient stratification in AML. Citation Format: Reecha Shah, Heba Wander, Michelle Hsueh, Muskan Floren, Wei Liu, Patricia Cheung, Courtney Andersen, Michael White, Nathan Kingston, Pablo Morentin Gutierrez, Lisa Drew, Giulia Fabbri, Elena Bibikova, Daniel Auclair. Multiplexed flow cytometry based immunophenotyping paired with functional ex vivo (MFLEX) drug profiling informs potential efficacy of therapeutic agents in 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 3904.

Histone lysine demethylase KDM5B facilitates proliferation and suppresses apoptosis in human acute myeloid leukemia cells through the miR-140-3p/BCL2 axis.

The histone lysine demethylase KDM5B is frequently up-regulated in various human cancer cells. However, its expression and functional role in human acute myeloid leukemia (AML) cells remain unclear. Here, we found that the expression level of KDM5B is high in primary human AML cells. We have demonstrated that knocking down KDM5B leads to apoptosis and impairs proliferation in primary human AML and some human AML cell lines. We further identified miR-140-3p as a downstream target gene of KDM5B. KDM5B expression was inversely correlated with the miR-140-3p level in primary human AML cells. Molecular studies showed that silencing KDM5B enhanced H3K4 trimethylation (H3K4me3) at the promoter of miR-140-3p, leading to high expression of miR-140-3p, which in turn inhibited B-cell CLL/lymphoma 2 (BCL2) expression. Finally, we demonstrate that the defective proliferation induced by KDM5B knockdown (KD) can be rescued with the miR-140-3p inhibitor or enhanced by combining KDM5B KD with a BCL2 inhibitor. Altogether, our data support the conclusion that KDM5B promotes tumorigenesis in human AML cells through the miR-140-3p/BCL2 axis. Targeting the KDM5B/miR-140-3p/BCL2 pathway may hold therapeutic promise for treating human AML.

Inhibition of Enhancer of Zeste Homolog 2 Induces Blast Differentiation, Impairs Engraftment and Prolongs Survival in Murine Models of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the malignant proliferation of immature myeloid cells characterized by a block in differentiation. As such, novel therapeutic strategies to promote the differentiation of immature myeloid cells have been successful in AML, although these agents are targeted to a specific mutation that is only present in a subset of AML patients. In the current study, we show that targeting the epigenetic modifier enhancer of zeste homolog 2 (EZH2) can induce the differentiation of immature blast cells into a more mature myeloid phenotype and promote survival in AML murine models. The EZH2 inhibitor EPZ011989 (EPZ) was studied in AML cell lines, primary in AML cells and normal CD34+ stem cells. A pharmacodynamic assessment of H3K27me3; studies of differentiation, cell growth, and colony formation; and in vivo therapeutic studies including the influence on primary AML cell engraftment were also conducted. EPZ inhibited H3K27me3 in AML cell lines and primary AML samples in vitro. EZH2 inhibition reduced colony formation in multiple AML cell lines and primary AML samples, while exhibiting no effect on colony formation in normal CD34+ stem cells. In AML cells, EPZ promoted phenotypic evidence of differentiation. Finally, the pretreatment of primary AML cells with EPZ significantly delayed engraftment and prolonged the overall survival when engrafted into immunodeficient mice. Despite evidence that EZH2 silencing in MDS/MPN can promote AML pathogenesis, our data demonstrate that the therapeutic inhibition of EZH2 in established AML has the potential to improve survival.

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.

Proteogenomic analysis reveals cytoplasmic sequestration of RUNX1 by the acute myeloid leukemia-initiating CBFB::MYH11 oncofusion protein.

Several canonical translocations produce oncofusion genes that can initiate acute myeloid leukemia (AML). Although each translocation is associated with unique features, the mechanisms responsible remain unclear. While proteins interacting with each oncofusion are known to be relevant for how they act, these interactions have not yet been systematically defined. To address this issue in an unbiased fashion, we fused a promiscuous biotin ligase (TurboID) in-frame with 3 favorable-risk AML oncofusion cDNAs (PML::RARA, RUNX1::RUNX1T1, and CBFB::MYH11) and identified their interacting proteins in primary murine hematopoietic cells. The PML::RARA- and RUNX1::RUNX1T1-TurboID fusion proteins labeled common and unique nuclear repressor complexes, implying their nuclear localization. However, CBFB::MYH11-TurboID-interacting proteins were largely cytoplasmic, probably because of an interaction of the MYH11 domain with several cytoplasmic myosin-related proteins. Using a variety of methods, we showed that the CBFB domain of CBFB::MYH11 sequesters RUNX1 in cytoplasmic aggregates; these findings were confirmed in primary human AML cells. Paradoxically, CBFB::MYH11 expression was associated with increased RUNX1/2 expression, suggesting the presence of a sensor for reduced functional RUNX1 protein, and a feedback loop that may attempt to compensate by increasing RUNX1/2 transcription. These findings may have broad implications for AML pathogenesis.

Mitochondrial STAT3 Plays a Critical Role in Survival of AML Cells

Introduction: Signal transducer and activator of transcription 3 (STAT3) has been shown to be critical to the survival of acute myeloid leukemia (AML) cells. It has been previously shown to mediate oxidative phosphorylation (OXPHOS) in acute myeloid leukemia bulk and stem cells via its transcriptional regulation of the amino acid transporter SLC1A5 and thereby glutamine uptake to AML cells (Amaya et al. Blood 2022). Further, STAT3 has been shown to regulate the transcription of important mitochondrial genes such as MCL-1 (Shastri et al. JCI 2018). Although STAT3 has been previously shown to also translocate to the mitochondria of myeloid malignant cells, its role in the mitochondria is less well defined in this cell population. In this study, we sought to understand the role of mitochondrial STAT3 in AML bulk leukemia and leukemia stem cell population. Methods: Human primary AML samples and MOLM-13 cells were utilized for experiments. To assess STAT3 localization to the mitochondria, mitochondrial fractions were isolated followed by Western Blot analysis. Flow cytometry-based imaging (ImageStream) was also utilized to quantify localization of STAT3 to the mitochondria. Mitochondrial mass was assessed using flow cytometry based MitoTracker assays. Metabolomic assays were performed by mass spectrometry at the University of Colorado metabolomics core. Viability assays were performed with flow cytometry using Annexin V and Dapi stains. Patient derived xenograft models were performed as previously described (Amaya et al. Blood 2022) in NSG-S mouse with Busulfan conditioning. Results: In this study, we show that primary human AML cells have constitutive STAT3 activation via phosphorylation of S727, which has been shown to signal mitochondrial localization. Further, we show that STAT3 is localized in the mitochondria of leukemia cells in primary patient samples and the cell line MOLM-13. Using genetic (siRNA) and pharmacologic (Stattic) inhibition of STAT3, we show a decrease in pSTAT3 at S727, with subsequent decreased translocation of STAT3 to the mitochondria. This leads to mitochondrial dysfunction including decreased TCA cycle metabolites, as well as decreased intracellular ATP upon STAT3 inhibition. STAT3 inhibition also leads to decreased mitochondrial mass suggesting autophagy upon mitochondrial STAT3 (mitoSTAT3) inhibition. To assess whether mitoSTAT3 is functionally important, we performed viability assays in MOLM-13, as well as leukemia stem cells isolated from primary AML cells. Inhibition of mitochondrial STAT3 leads to decreased viability in MOLM-13 and primary AML cells, and reduced engraftment in PDX mouse models using primary AML samples (Figure 1A), suggesting a decrease in leukemia stem cell potential. Interestingly, STAT3 inhibition with Stattic is able to overcome venetoclax resistance for primary AML patient samples in vitro (Figure 1B). Conclusions: In this study, we show mitoSTAT3 plays a key role in mitochondrial function of AML cells. mitoSTAT3 inhibition leads to mitochondrial dysfunction, a decrease in mitochondrial mass and eventually cell death. We show that Stattic is an effective inhibitor of mitoSTAT3 in AML, leading to cell death and decreased leukemia stem cell function. Further studies are needed to assess the mechanistic function of STAT3 in the mitochondria.

Targeting G3BP2 Sensitizes Acute Myeloid Leukemia to Venetoclax Treatment Via ELF1-MCL1 Axis

Venetoclax-based combination therapies have emerged as promising treatments for elderly acute myeloid leukemia (AML) patients. However, venetoclax resistance renders the treatment ineffective, mainly due to the upregulation of MCL1. Thus, new venetoclax-based combination therapy is under development to circumvent venetoclax resistance. The Ras-GTPase-activating protein SH3-domain binding protein 2 (G3BP2) is upgraded in several solid tumors. Targeting G3BP2 re-sensitized tumor cells to chemotherapy. To date, whether targeting G3BP2 sensitizes venetoclax treatment in AML and its role in leukemogenesis has not been defined. To this end, the primary AML cells and AML cells were collected and cultured with compound C108 (G3BP2 inhibitor) and venetoclax alone or both. The result showed that co-culture with compound C108 and venetoclax decreased the cell viability in primary AML and AML cells, indicating the potent synergy of G3BP2 inhibitor and venetoclax. The Quantitative real-time PCR (RT-qPCR) showed that G3BP2 was overexpressed in AML patients and was associated with poor prognosis. Knockdown (KD) of G3BP2 decreased proliferation and induced apoptosis in AML cells. In addition, G3BP2 inhibition impeded the tumorigenicity in the subcutaneous AML xenograft tumor model. These data demonstrated that G3BP2 sensitized AML to venetoclax treatment and is critical in AML development. The RNA-seq analysis was performed to investigate the role of G3BP2 in venetoclax resistance and leukemogenesis. The results showed that differentially expressed genes (DEG) were enriched in apoptotic processes when G3BP2 were downregulated. The immunoblotting results indicated that G3BP2 knockdown decreased the expression of MCL1 and BCL2, and vice versa. Moreover, we found that the transcription factor ELF1 was positively correlated with G3BP2 by the immunoblotting and RNA-seq analysis. The result was further validated by the Pearson correlation analysis based on the online database. The expression of ELF1 was decreased when G3BP2 were downregulated. Thus, we concluded that G3BP2 is an upstream gene of ELF1 and MCL1. To further investigate the relationship among G3BP2, ELF1, and MCL1, the RNA binding protein immunoprecipitation assay (RIP) and mRNA stability analysis was performed. The results suggested that G3BP2 interacted with the ELF1 mRNA to enhance its stability. We employed bioinformatics analysis via the JASPAR database and RPISeq tools and found ELF1 may facilitate the transcription of MCL1, which was validated in the following study. The expression of MCL1 was restored in the G3BP2 KD cells when ELF1 was overexpressed. We subsequently performed CHIP-qPCR, dual-luciferase reporter gene assays with truncated MCL1 promoter fragments. A 576bp of the binding region upstream of MCL1 promoter was defined, indicating that ELF1 regulated MCL1 expression by modulating its transcription activity. Taken together, our data suggested that targeting G3BP2 decreases MCL1 expression by disturbing ELF1 mRNA stability, thus increasing the sensitivity to venetoclax treatment. We finally established the intra-venous tumorigenesis xenograft model to verify the role of G3BP2 downregulation in venetoclax-treated leukemia mice. The NCG immunodeficient mice were transplanted with U937 cells and administrated with venetoclax daily from 7-21 days post transplantation (Figure 1A). The data showed that G3BP2 KD in combination with venetoclax reduced clinical scores and prolonged the overall survival of AML mice (combination vs. venetoclax: p&amp;lt;0.01; combination vs. G3BP2 KD: p&amp;lt;0.05, Figure 1B). The reduced leukemic burden was observed when targeting G3BP2 in combination with venetoclax. Meanwhile, we verified the decreased expression of ELF1 and MCL1 in mice targeting G3BP2 in combination with venetoclax by immunofluorescence (IF) and flow cytometry analysis. These data supported the synergic effect of G3BP2 inhibition and venetoclax treatment in AML. Our findings emphasize the critical role of G3BP2 in conferring resistance to venetoclax in AML by stabilizing ELF1 mRNA to regulate MCL1 expression. Targeting G3BP2 represents an appealing therapeutic strategy for patients with venetoclax-resistant AML.

CD33-TIM3 Dual CAR T Cells: Enhancing Specificity While Maintaining Efficacy Against AML

Background: The myeloid associated antigen CD33 is overexpressed in over 88% of Acute myeloid leukemia (AML) patients, making it a suitable target antigen for CAR T cell therapy across different genetic subtypes. Despite the development of clinical trial data, there remains a concern about on-target off-leukemia toxicity. To enhance specificity, we generated dual targeting CAR T cells that target CD33 and/or T-cell immunoglobulin and mucin-domain containing-3 (TIM3). The latter is known to be expressed on LSCs, but not on healthy hematopoietic precursor or stem cells. Notably, TIM3 is recognized for its inhibitory immunomodulatory role further enhancing the suitability of this target antigen. The objective of this research was to explore various CD33 and TIM3 dual-targeting CAR T cells approaches that enhance AML specificity and maintain high anti-AML activity. Methods: The hybridoma technology was used to generate antibodies against TIM3 immunized mice. Antibodies were screened for TIM3 specificity via ELISA and FACS. The anti-TIM3 single-chain variable fragment (scFv) DNA sequence was sequenced from hybridoma cells. Furthermore, the binding ability to TIM3 antigen (PDB ID: 5F71) was evaluated using AlphaFold. The CD33 scFv was derived from Gemtuzumab ozogamicin (clone: hP67.6). ScFv sequences and co-stimulation domains (CD28 or 4-1BB) were cloned into a pMP71 vector. Retrovirus for transduction was produced using the 293Vec-GALV and RD114 retroviral production system. In vitro co-culture assays of CAR T cells and target cells were performed to study the efficacy of CAR T cells. The cytotoxicity against wild-type and TIM3 transduced AML cell lines (THP-1 and OCI-AML3) was assessed by multiparameter flow cytometry (MPFC). The secretion of effector cytokines (IFN-γ, TNF, IL-2) was analyzed via CBA assays. In addition, avidity between CAR T cells and target cells was determined by Z-Movie analyzer. The different CAR constructs were screened for on-target off-tumor toxicity in colony forming unit assays (CFU) using isolated CD34 + hematopoietic stem and progenitor cells (HSPCs) from healthy doners after 14 days. In order to compare CAR constructs regarding long-term efficacy in antigen restimulation assays, CAR T cells were co-cultured with irradiated TIM3 transduced OCI-AML3 every 4 days at an E: T ratio of 1:1 for 24 days. Moreover, CAR T-cell proliferation, checkpoint marker expression and T-cell subset differentiation were analyzed via MPFC. Results: All dual CAR T cells (compound, split, tandem, pooled) were generated with high and robust transduction efficacy (Figure 1A). TIM3-dependent fratricide was not observed during CAR T cell manufacturing. Compared to single antigen targeting CAR T cells, dual CAR T cells showed enhanced cytotoxicity against AML cell lines and primary AML cells (Figure 1B). Moreover, we also observed a strong increase in the secretion of proinflammatory cytokines (IFN-γ and IL-2) and higher avidity of dual CAR T cells. Notably, split CAR T cells demonstrated greater specificity in cocultures directed against mono vs. dual-target antigen expression cell lines. In accordance with these observations, split CAR T cells did not exert on-target off-leukemia toxicity against healthy HSC in CFU assays. In the antigen restimulation assay, we observed that compound CAR T cells exhibited diminished expanding capacity and heightened expression of exhaustion markers in comparison to the other CAR T constructs. Conclusion: We developed multiple CD33 and TIM3 dual CAR T cells using both “AND” and “OR” gating strategies in this study. Our findings revealed that dual CAR T cells provided higher avidity and cytotoxicity compared to single targeting CAR T cells against dual antigen expressing target cells in vitro. Importantly, only the split CAR T constructs demonstrated specific killing of CD33 +TIM3 + cell lines and primary AML cells while sparing HSPCs. Prospectively, compound, tandem, pooled CD33 TIM3 specific CAR T cells might be helpful in a bridge to transplant setting, whereas split CAR T cells might provide a higher safety profile, thereby allowing a transplant independent approach. To advance our concepts, we are currently conducting in vivo experiments in an NSG mouse model.

Primary U2AF1 S34F Mutated Hematopoietic Cells Are Sensitive to Nonsense-Mediated RNA Decay Disruption In Vivo

Expression of mutant splicing factors alters RNA splicing in myeloid malignancies, including increased production of nonsense transcripts that rely on nonsense-mediated RNA decay (NMD) for clearance. Cell lines expressing spliceosome mutants are more sensitive to NMD inhibition than wild-type cells, and the differential sensitivity is partially dependent on R-loop formation. However, the impact of NMD disruption on the viability of primary hematopoietic cells with splicing factor mutations has not been tested in vivo. We engineered mouse MLL-AF9 acute myeloid leukemia (AML) cells to inducibly express a wild type ( U2AF1 WT) or mutant ( U2AF1 S34F) transgene of the U2AF1 splicing factor, and tested the effects of inhibiting the NMD pathway protein kinase, SMG1, in vivo. We injected U2AF1 WT and U2AF1 S34F mouse AML cells (B6, CD45.2) into congenic, irradiated secondary recipient mice (B6, CD45.1), induced transgene expression, and then treated the mice for 2 weeks with either a SMG1 inhibitor (SMG1i) at 60 mg/kg/day or vehicle. SMG1i treatment increased the overall survival (OS) of mice bearing U2AF1S34F AML cells (median OS of 53 days compared to 42 days for mice treated with vehicle; p =0.0003), but did not impact the OS of mice with U2AF1WT AML cells (median OS of 33 days for SMG1i-treated mice compared to median OS of 32 days for vehicle-treated mice, p=0.360). We extended these findings to a second cohort of mice, where we transplanted one primary U2AF1 S34F banked mouse AML sample into 48 secondary recipient mice. To allow direct comparison of isogenic cells with or without U2AF1 S34F expression, half the mice were treated with doxycycline to induce U2AF1 S34F expression, and half of the mice did not receive doxycycline. SMG1i treatment increased the OS of mice expressing the U2AF1S34F mutant allele (median OS of 66 days, compared to median OS of 54 days for mice treated with vehicle; p&amp;lt;0.0001), but had no impact on survival of mice bearing AML cells where U2AF1 S34F was not expressed (median OS of 50 days, compared to median OS of 52 days for mice treated with vehicle; p=0.95). To explore the underlying mechanism for the sensitivity of spliceosome mutant cells to NMD inhibition, we first examined the effects of SMG1i treatment on cell cycle progression and cell death in primary cells. We observed that primary U2AF1 S34F-expressing AML cells treated with SMG1i ex vivo (24 hours) had an increased fraction of cells in G2/M phase compared to U2AF1 WT-treated cells (mean 48% vs. 11% at 100nM, respectively, p&amp;lt;0.001). In addition, the fraction of Annexin V + cells was increased in U2AF1 S34F-expressing cells treated with SMG1i ex vivo compared to U2AF1 WT treated cells (mean fold-change at 500nM relative to DMSO 4.0 vs. 2.3, respectively, p=0.027). We then isolated primary mouse AML cells after 7 days of in vivo SMG1i treatment and analyzed R-loops (S9.6 antibody) and DNA damage (gH2AX) by immunofluorescence. SMG1i-treated U2AF1 S34F cells had a marked increase in R-loop levels compared to U2AF1 WT-treated cells (mean S9.6 nuclear intensity, 306.9 vs 182.6, respectively, p&amp;lt;0.0001). In addition, H2AX phosphorylation was increased in SMG1i-treated U2AF1 S34F compared to U2AF1 WT cells (mean gH2AX nuclear intensity, 136.7 vs 87.8, respectively p&amp;lt;0.0001).Thus, disrupting NMD in splicing factor mutant AML cells alters cell cycle progression and causes R-loop accumulation, DNA damage, and apoptotic cell death. The NMD pathway has been implicated in the regulation of the unfolded protein response (UPR) that controls protein homeostasis and impacts cell viability. We generated K562 cell lines expressing doxycycline-inducible U2AF1 S34F or U2AF1 WT constructs and confirmed increased sensitivity of U2AF1 S34F cells treated with SMG1i(IC50 128.1nM vs 578.5nM at 72 hours, respectively, p&amp;lt;0.0001). Next, we performed RNA-sequencing on U2AF1 WT and U2AF1 S34F cells that were treated with vehicle or SMG1i for 24 hours (n=3) and observed upregulation of endoplasmic reticulum and UPR pathway genes in SMG1i-treated U2AF1 S34F compared to U2AF1 WT cells (GSEA; FDR&amp;lt;0.01). Validation of these findings using primary mouse AML cells is ongoing, including the analysis of key regulators and protein biomarkers of the UPR pathway. The vulnerability of primary hematopoietic cancer cells with spliceosome mutations to NMD inhibition suggests the possibility for therapeutic targeting of NMD to treat myeloid malignancies with aberrant splicing.

Effect and Mechanism of NL-101 Combined with Bcl-2 Inhibitor Venetoclax in Acute Myeloid Leukemia

Background Acute myeloid leukemia (AML) is a kind of malignant clonal disease originating from hematopoietic stem cells with strong heterogeneity,with the highly recurrence rate and mortality rate.Although the application of hematopoietic stem cell transplantation and new targeted drugs has improved the prognosis of some patients in recent years, chemotherapy is still the most important treatment for AML. 50-80% of AML patients eventually relapsed after CR, with a low remission rate after relapse.There are reports that BCL-2 overexpression is a clinical feature of human hematologic malignancies that exacerbates the malignant state and drives apoptosis resistance phenotypes.Therefore, how to reduce treatment-related toxic and side effects and overcome tumor cell drug resistance is a hot and difficult topic in current AML treatment. Venetoclax (ABT-199) is an oral, selective Bcl-2 inhibitor. It showed strong anti-leukemia activity in preclinical stage, but its clinical efficacy as a single drug for AML was limited.Nl-101 (also known as EDE-S101)is a new small molecule compound formed by covalently linking the DNA damage core group of Bendamustine hydrochloride with the inhibition core group of histone deacetylase of the histone inhibitor Volinostat (also known as SAHA).Our previous study found that NL-101 single drug can reduce tumor load and prolong survival of AML mice in vivo.In this study, we used Venetoclax in combination with NL-101 to understand its efficacy and safety in vitro and in vivo, and to explore the synergies and mechanisms of action between the two drugs, providing new effective options for the treatment of AML patients. Purpose To explore the efficacy of Bcl-2 inhibitor Venetoclax (ABT-199) and a novel small molecule compound NL-101 in the treatment of acute myeloid leukemia（AML） and the related mechanisms. Experimental Design AML cell lines and primary patient samples were collected and used for cell proliferation assay. MTS colorimetric assay was used to determine the effect of Venetoclax alone or in combination with NL-101 on the growth of AML cells，the combination index (CI) was calculated to evaluate the synergistic effect of the two drugs. Cell death induced by Venetoclax alone or in combination with NL-101 was analyzed by Annexin V-FITC/ Propyl -iodide staining and flow cytometry. Western blot was used to analyze the protein expression levels of MAPK/ERK, PI3K/AK/GSK pathway members and BCL-2 family members.The MV4-11-derived xenograft mouse model was used to assess in vivo efficacy of the combination of venetoclax and NL-101. Results Venetoclax alone and in combination with NL-101 can inhibit the growth of AML cell lines and primary cells to a certain extent, and the combination of drugs has a synergistic effect. Venetoclax combined with NL-101 effectively synergistically induced apoptosis in AML cell lines and primary patient samples. Venetoclax combined with NL-101 can down-regulate the expression of pro-survival proteins in the Bcl-2 family, such as McL-1, Bcl-2, and Bcl-XL, and up-regulate the expression of pro-apoptotic proteins, such as BIM, BID, and BAX/BAK. Phosphorylated ERK, AKT, and JNK expressions were eliminated by venetoclax in combination with NL-101. In vivo results suggest that Venetoclax combined with NL-101 has the potential to treat AML. Conclusions Venetoclax combined with NL-101 can synergistically inhibit the growth and promote apoptosis of AML cell lines and primary AML cells. In vivo experiments demonstrated that Venetoclax combined with NL-101 can synergistically reduce tumor load and prolong survival time in AML mice. These results suggest that Venetoclax in combination with NL-101 is effective in the treatment of acute myeloid leukemia.

Ferritinophagy Is a Druggable Vulnerability of Quiescent Leukemic Stem Cells

Acute myeloid leukemia (AML) poses significant challenges in terms of prognosis and treatment options. Leukemic stem cells (LSCs) contribute to therapy resistance, relapse and adverse outcomes. This study aimed to explore one of the major features of LSCs (e.g. quiescence) and its underlying molecular mechanisms, as well as identify potential targets for therapeutic intervention. Using in vivo cell labeling in patient-derived xenograft (PDX) assays, we identified a quiescent cell subpopulation with high LSC potential. Transcriptomics experiments conducted separately on quiescent and cycling cell subsets revealed gene signatures characteristic of quiescent (Q_AML_UP) and cycling cells (C_AML_UP). Using single-cell RNA sequencing, we identified clusters of quiescent leukemic cell that were highly enriched in gene signatures related to LSCs (Figure 1A). Through a machine learning approach, we identified a gene set highly specific of quiescent cells (named the QS35 gene signature) that emerged as a significant prognostic factor for poor outcomes in independent cohorts of AML patients (Figure 1B). Next, we aimed to identify new vulnerabilities of LSCs by investigating the quiescent subset of leukemic cells. Transcriptomics and proteomics approaches on quiescent vs cycling cells revealed enhanced autophagy within the quiescent subset. Additionally, the transferrin receptor TFRC and ferritin, two key regulators of iron metabolism, exhibited lower expression in the quiescent subpopulations. As intracellular iron availability is tightly regulated by ferritinophagy, a specific form of autophagy leading to the degradation of ferritin, we investigated the coordinated regulation of autophagy and iron metabolism in the quiescent LSC subset. Intriguingly, the intracellular iron pool was lower in quiescent LSCs, particularly in the CD34+CD38- compartment compared to more mature leukemic cells. Moreover, autophagy inhibition selectively induced cytotoxicity in the CD34+CD38- compartment and reduced the frequency of long-term culture-initiating cells (LTC-IC), and these effects were rescued by exogenous iron supply, underscoring the critical role of autophagy in iron bioavailability within the quiescent LSC subpopulation. To target ferritinophagy in this compartment, we depleted NCOA4, the main carrier in ferritinophagy responsible for delivering ferritin-bound iron to the autophagosome, using shRNAs in primary AML samples. Interestingly, NCOA4 was found more expressed in quiescent compared to cycling AML cells. In ex vivo assays, NCOA4 downregulation significantly reduced the frequency of LTC-IC and inhibited the clonogenic capacities of leukemic cells. Furthermore, NCOA4 invalidation abrogated leukemia initiation and self-renewal in serial transplantation experiments in vivo, particularly within the quiescent LSC population (Figure 2A). Additionally, treatment with a novel small molecule inhibitor named compound 9a that disrupts the NCOA4-ferritin interaction, depleted intracellular iron in primary AML cells, reduced their clonogenic potential and LTC-IC frequency ex vivo, and induced cell death specifically within the quiescent CD34+CD38- subset. Importantly, the viability of LSCs treated with compound 9a was rescued by the addition of iron, demonstrating that the cytotoxic effects of this compound were dependent on iron depletion. Notably, compound 9a exhibited minimal toxicity to normal hematopoietic cells ex vivo. In PDX models, in vivo short-term treatment with compound 9a decreased tumor burden and induced cell death within the CD34+CD38- LSC population (Figure 2B). Mechanistically, genetic of chemical inhibition of NCOA4 led to the accumulation of dysfunctional mitochondria, and to the generation of reactive oxygen species especially within the LSCs population. Furthermore, inhibition of NCOA4 by compound 9a decreased mitophagy, strongly suggesting that NCOA4 coordinates ferritinophagy and mitophagy pathways to increase intracellular iron, thereby specifically promoting the survival of LSCs. These findings highlight the critical vulnerability of quiescent LSCs to ferritinophagy. Thus, NCOA4 inhibition represents an innovative therapeutic approach for AML patients.

Label-Free Identification of Biochemical Footprints of Key Mutations in Acute Myeloid Leukemia Using Raman Spectroscopy

Introduction: With increasing understanding of the complexity and heterogeneity of acute myeloid leukemia (AML), the diagnostic process becomes more complicated and time-consuming. Therefore, there is a need to supplement AML diagnostics with a quick method to identify key, druggable genetic lesions that serve as a target for small molecule drugs. To address this need, we used Raman spectroscopy (RS)-based imaging methods. RS uses non-elastic scattering of laser light on cell biomolecules. When coupled to computational image segmentation and analysis, composition of the analyzed fragment of the cytoplasm can be characterized. In this way, by mapping the focal surface of the cell, we obtain a multidimensional image containing information about the distribution of its biochemical components. In our study, we utilized RS microscopy to investigate how single mutations affect the Raman spectral images of leukemic cells. The primary objective was to determine whether this technique could be utilized to identify mutations in key genes related to leukemia development. Methods: Genetic models of AML were generated by introducing wild-type and mutated forms of three frequently mutated and/or druggable genes (FLT3, IDH1/2, and MLL) into myeloid cell lines (THP1, HEL, HL60). The presence of the desired phenotype was confirmed using flow cytometry, immunoblotting, high-throughput kinase activity assay (Pamgene), and confocal microscopy. For RS experiments, the cells were fixed, and subjected to Raman imaging measurements using the WITec Alpha 300 microscope. Reference or control samples consisted of cells with wild-type genes (WT). The recorded hyperspectral data was subjected to multidimensional chemometric analysis, including k-means cluster analysis (KMCA), principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). To validate the findings from the cell models, primary AML cells from 30 patients diagnosed with FLT-3 ITD, MLL or IDH1/2 mutations were examined with RS. Peripheral blood mononuclear cells (PBMC) isolated from healthy donors blood served as reference. Results: The spectral differences between cells with wild-type FLT3 and FLT3/ITD were subtle, yet significant. The results revealed distinct variances in lipid (1440, 1240, 1660, and 2850 cm -1) and hemoprotein content (740-760 cm -1). Additionally, we successfully distinguished cells harboring IDH1/2 and MLL mutations from those with the wild-type genes. In these cases Raman bands corresponding to protein and nucleic acids composition were changed in comparison to control cells. Raman features in PCA loadings were also related to proteins and nucleic acids. Using the PLS-DA method and simple AI model, we built successful algorithm for distinguishing transgenic models carrying mutation from wild-type cells. PCA analysis also enabled us to distinguish AML primary cells from PBMC. Analysis weather primary cells carrying the selected mutations can be distinguished from non-mutated AML cells is concordant with our findings on models, however, it requires further analysis in larger group of patients to increase statistical power of these observations. Conclusions: Collectively, these findings suggest that Raman spectroscopy coupled with appropriate chemometric analysis may serve as rapid, label-free tool for identification of key druggable mutations in AML. Acknowledgements: The studies were performed as a part of the “ Label-free and rapid optical imaging, detection and sorting of leukemia cells” project carried out within the Team-Net programme of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund.

LSD1 Inhibition Synergizes with Venetoclax in Acute Myeloid Leukemia By Targeting Cellular Metabolism

Introduction: Acute myeloid leukemia (AML) is a hematological neoplasm with poor clinical outcomes. The introduction of the BCL2 inhibitor venetoclax in combination with hypomethylating agent has improved response rates in older patients with AML by targeting the metabolism of leukemic stem cells (LSCs). Resistance, however, is unfortunately still common. Thus, there remains a need to enhance the clinical efficacy of venetoclax by combining it with novel agents. In diverse AML models, lysine-specific demethylase 1 (LSD1) inhibition promotes cellular differentiation and reduces LSCs. We therefore asked if LSD1 inhibition could enhance the efficacy of venetoclax in the treatment of AML. Methods: We have studied the effect of single-agent and co-treatment with venetoclax and LSD1 inhibitor bomedemstat on cell proliferation in human AML cell lines (N=3), Hoxa9/ Meis1(H9M)- and MN1-transformed murine myeloid progenitor lines as well as primary AML patient samples (N=5). We have analyzed the effect of co-treatment with these two compounds on functional cellular bioenergetics in MOLM-13 cells using Seahorse extracellular flux analyzer and followed up by performing NMR spectrometry to study changes in metabolite abundance. Further, we have studied the efficacy of this drug combination in vivo in patient-derived xenograft (PDX) AML models (N=2). Results: Human AML cell lines showed a differential cytocidal activity at 96 hours (h) to venetoclax with MOLM-13 (IC 50 0.04 µM) being most sensitive and OCI-AML3 most resistant (IC 50 9.81 µM). A similar range (between 0.15 µM and 7.3 µM) was also observed in the primary AML samples. For bomedemstat, we observed a time-dependent response where the IC 50 decreased over time, in particular, in the most sensitive model systems. In murine H9M cells, the IC 50 decreased from 30.43 µM at 48 h to 0.034 µM at 96 h and further down to 0.0068 µM at 168 h. Combining bomedemstat and venetoclax, the IC 50 for venetoclax decreased; and synergism was observed in MOLM-13 cells, the most sensitive AML model, but no synergism was observed in venetoclax-resistant cells. Synergism was also observed in MN1 cells as well as 3 of the 5 primary AML samples studied (Table 1). To investigate the mechanism of the observed synergism, we explored the effect of these treatments on metabolic pathways using the Seahorse extracellular flux analyzer. LSD1 inhibition had no effect on oxidative phosphorylation (measured as oxygen consumption rate (OCR)). However, glycolysis, measured as proton efflux rate (PER), was significantly reduced from 102.88 ± 11.03 pmol/min to 54.17 ± 6.18 pmol/min (P &amp;lt;0.01) after 96 h of treatment with 1μM bomedemstat. With the combined treatments, we observed a reduction in both OCR and PER. This synergistic effect was further investigated by studying metabolite abundance using NMR spectrometry where co-treatment showed a reduction in metabolite abundance across a broad spectrum of metabolic pathways (Fig. 1). To determine if the observed combined effect of bomedemstat and venetoclax could enhance activity against AML cells in vivo, we employed a PDX model with two primary AML samples. In the first model system (monosomy 7), co-treatment resulted in a significant reduction in human CD45-positive cells as compared to vehicle (P &amp;lt;0.001) and single-agent treatments with venetoclax (P= 0.011) and bomedemstat (P &amp;lt;0.001). Similarly, in the second PDX model ( NPM1-mutated, FLT3-ITD positive), the co-treatment exhibited a significant reduction in leukemic burden in bone marrow as compared to vehicle (P=0.003) and single-agent treatments with venetoclax (P= 0.024) and bomedemstat (P &amp;lt; 0.001) (Fig. 1). Conclusion: The combination of bomedemstat and venetoclax had synergistic cytocidal effects on AML cell line and primary AML cells in vitro. It significantly reduced the leukemic burden in PDX AML models and synergistically downregulated cellular energy metabolism. These findings suggest that combining venetoclax with LSD1 inhibition holds promise as a combination treatment in AML and warrants further clinical investigation.