Primary Acute Myeloid Leukemia Cells Research Articles

C/EBPα Confers Dependence to Fatty Acid Biosynthetic Pathways and Vulnerability to Lipid Oxidative Stress in FLT3-Mutant Leukemia

Despite therapeutic breakthroughs, drug resistance and relapse remain the major barrier for the treatment of patients with acute myeloid leukemia (AML). Therefore, understanding and targeting therapy-resistant AML cells responsible for relapse ("persisters") represent an urgent need in the development of new treatments. In this context, we demonstrated that the hyperactivation of mitochondrial oxidative metabolism plays a crucial role in drug resistance of AML. This high oxidative phosphorylation (OxPHOS) phenotype associated with enhanced mitochondrial BCL2 dependency and ROS detoxification is the consequence of a mitochondrial adaptation driven by inflammatory stress responses in a cAMP-PKA-ATF4-dependent manner. Targeting elevated oxidative phosphorylation (OxPHOS) activity with direct and indirect mitochondrial inhibitors sensitizes resistant cells to chemotherapy cytarabine (AraC) in AML. Furthermore, we have recently shown that the mitochondrial OxPHOS state supports IDH mutant cell proliferation and chemoresistance in a C/EBPa-dependent manner. Importantly, multi-omics analyses uncovered a coordinated activation of CEBPA and Fms-like tyrosine kinase 3 (FLT3) to control lipid metabolism in FLT3-mutant AML cells from primary AML patient specimens, cell lines, and PDX models. Mechanistically, C/EBPα regulated FASN-SCD axis to promote uptake, biosynthesis and desaturation of fatty acids (FA). We further demonstrated that FLT3 or C/EBPα inactivation decreased mono-unsaturated FAs incorporation to membrane phospholipids through SCD downregulation in leukemic cells. Consequently, SCD inhibition enhanced susceptibility to lipid redox stress and accumulation of poly-unsaturated FA increased lipid peroxidation and vulnerability to redox stress. Accordingly, this C/EBPα-dependent adaptation of FA homeostasis was exploited by combining FLT3 and glutathione peroxidase 4 (GPX4) inhibition to trigger lipid oxidative stress, enhancing ferroptotic death of FLT3-mutant AML cells ex vivo and in vivo. Altogether, this study reveals a non-canonical C/EBPα function in lipid homeostasis and adaptation to redox stress, and a previously unreported vulnerability of FLT3-mutant AML with promising therapeutic application. Altogether, our works highlight the role of metabolic adaptations to support the plasticity and drug resistance in AML.

Pre-Clinical Investigation of a Novel Small Molecule Inhibitor Targeting YBX1 in AML

Recently, our group identified the pleiotropic DNA and RNA binding protein YBX1 as a specific dependency and therapeutic target in acute myeloid leukemia (AML) that is essential for stabilizing oncogenic transcripts and thereby maintains oncogenic protein expression (Perner et al., 2021). Selective pharmacologic targeting of YBX1 in AML, however, has not been achieved so far. Here, we investigate the functional consequences of a novel small molecule inhibitor (SU056) of YBX1 in AML. We examined the viability of several human AML cell lines (MOLM13, HL60, EOL1, THP1, Kasumi1, ML2, Mv4;11, OCI-AML3, NOMO1) upon SU056 treatment and found significant reduction of cell growth in a concentration dependent manner. Inhibition of cell growth could be mainly attributed to impaired proliferative capacity at nanomolar concentrations. Pharmacologic inhibition of YBX1 resulted in impaired colony formation (mean colony number DMSO: 231.3±13.88 vs. SU056 500nM: 154.7±8.29; p<0.0001), cell cycle arrest (cells in S phase: DMSO: 37.3±2.4 vs. SU056 500nM: 27.2±1.67; p=0.0127) and induction of myeloid differentiation. Consistently, in a xenograft model of human AML, SU056 treatment resulted in prolonged disease latency (MOLM13 diluent: 62 days OS vs. SU056: not reached, p=0.0013; OCI-AML3 diluent: 79 days OS vs. not reached, p=0.0001) and significant reduction of disease penetrance (MOLM13 diluent: 100% vs. SU056: 24,9%; OCI-AML3 diluent: 100% vs. SU056: 9,1%). Consistently, percentage of SU056 treated human leukemic cells in the bone marrow of recipient mice was decreased for both, MOLM13 (mean diluent: 41,3% vs. SU056: 7,6%) and OCI-AML3 (mean diluent: 28,9% vs. SU056: 2,9%). Likewise, pharmacologic inhibition of YBX1 reduced colony formation and total cell numbers of primary human AML cells in a dose-dependent manner. To be clinically relevant, such treatment must have a suitable therapeutic index between human hematopoietic stem- and progenitor cells (HSPCs) and their malignant counterpart. This is particularly important, considering the role of YBX1 in mRNA splicing and processing. Of note, pharmacologic inhibition of YBX1 did not perturb colony formation of normal HSPCs derived from apheresis donors. These findings were confirmed by investigating primary human leukemic cells in a patient-derived xenograft (PDX) model of AML. We aimed to assess whether loss in leukemia cell competition induced by pharmacologic targeting of YBX1 is sufficient for normal hematopoietic cells to outcompete leukemic cells in vivo. 1x106 normal human HSPCs were transplanted along with 2x104 leukemic cells to assess for efficacy of SU056-treatment versus diluent control. In vivo treatment with SU056 (20mg/kg, i.p.) for 3 weeks resulted in a significant decrease in the AML blast number in the PB (mean blast %: diluent: 38.5%; SU056: 2.25%; p<0.0001). This resulted in increased survival of SU056 treated recipients (OS diluent: 83 days vs. SU056: not reached). To confirm the proposed mechanism by which SU056 impairs leukemia growth we performed global transcriptome and proteome analysis following treatment with SU056 (500nM, 96h). Proteome profiling revealed a total of 2641 deregulated proteins. Gene-ontology analysis indicated deregulation of YBX1 targets and proteins associated with cell cycle, DNA repair, mRNA splicing/processing, and transcriptional regulation. Of note, overlap between transcriptional regulation (as determined by mRNA-Seq) and changes in proteome abundance revealed less than 5% target genes/proteins, indicating that SU056 may in fact interfere with binding of oncogenic mRNAs to YBX1 and thereby may reduce translation of oncogenic transcripts. In order to define functional consequences of pharmacologic YBX1 inhibition and explore potential vulnerabilities of drug combinations, we performed a genome-wide CRISPR/Cas9 screen in human AML cells treated with SU056 versus diluent control. Validation experiments are currently ongoing. Taken together, our data confirm the therapeutic potential to target YBX1 using the novel small molecule SU056. Given its broad applicability across genetic subtypes of AML and lack of toxicity on normal HSPCs, SU056 as a single agent or in combination with established targeted therapies may efficiently help to target and outcompete leukemic cells. Experiments to assess for potential synergy with already established targeted therapies will be presented.

Functional Role of Histone Variant macroH2A1 (H2AFY) in Acute Myeloid Leukemia (AML)

Histone variants can replace replication-coupled histones in the nucleosome. Among core histones, the H2A family exhibits highest sequence divergence resulting in the largest number of variants. Dysregulated expression of macrohistone (macroH2A) variants has been reported in different cancers. Their role in normal hematopoiesis and hematopoietic cancers remained so far poorly understood. We found high expression of macrohistone H2AFY (macroH2A1) in hematopoietic stem and progenitor cells (HSPCs) and primary AML as compared to differentiated hematopoietic cells. To assess for the functional dependency of human AML cells on macroH2A1, we performed in vitro CRISPR-Cas9 negative selection competition assays. Genetic inactivation of H2AFY in 4/9 AML cell lines (Molm-13, MV4;11, THP-1, OCI-AML3) resulted in outcompetition of H2AFY-deleted cells against non-deleted competitors. To confirm these findings in vivo, we performed xenotransplantation experiments. Genetic inactivation of H2AFY in human AML cells resulted in delayed leukemia development in recipient mice (median survival for NT-control: 35.5 days vs. H2AFY-KO: 44.0 days; p=0.002). To validate the functional impact of macroH2A1 on leukemic stem cells (LSCs), we generated a novel conditional knockout mouse model for H2AFY [B6(Cg)-H2afytm1Flh/J (H2afy flox/flox)]. Here, isolated lineage-depleted H2afy+/+, Mx1-Cre+ and H2afyfl/fl, Mx1-Cre+ bone marrow cells (Lin-BMCs) were retrovirally infected with MLL-AF9-GFP (MA9) and transplanted into sub-lethally irradiated recipient mice. Following establishment of leukemia, we transplanted 1x105 leukemic cells and inactivated H2afy in the leukemic cells by pIpC injection on day 7, 9 and 11 post-transplantation. Genetic deletion of H2afy resulted in delayed disease progression (median survival +/+: 83.5 days vs. -/-: 162.0 days, p=0.0013) and reduction of disease penetrance to 61%. Organ infiltration by leukemic cells led to disease development in a limited number of H2afy-/- recipient mice. Isolated H2afy-/- leukemic cells showed a decreased in proliferation and an increased in differentiation. Importantly, 37% of H2afy-/- recipient mice that developed AML showed only partial excision of H2afy, indicating selection of partially excised clones. Long-term survivors without leukemia development showed no molecular evidence for counterselection. Irrespective of the time of disease development and survival, immunophenotypic analysis of (Lin-cKit+Sca-1-FcγR+CD34+GFP+) LGMPs revealed decreased numbers of LSCs following H2afy-deletion (+/+: 2631/106 BMC; -/-: 727/106 BMC; p=0.0002). To assess for the functional consequences of H2afy-deletion on the LSC pool, we performed limiting dilution assays. When transplanting 1x105, 1x104, 1x103 and 1x102 H2afy+/+ or -/- leukemic cells into sub-lethally irradiated recipients, LGMP frequency in H2afy-deficient cells was significantly reduced (1/1 vs. 1/29928; p=1.25e-19). The reduction of LGMP frequency correlated with reduced plating efficiency and increased differentiation. Consistently, primary human AML cells showed impaired colony forming capacity following H2afy deletion (mean colony number NT-control: 60.0±17.1 vs. H2afy-KD: 30.1±8.9; p=0.0023). To assess for the impact of H2afy deletion on normal HSPCs, we performed competitive repopulation assays. Immunophenotypic abundance of HSPC in H2afy+/+ vs. -/- animals revealed comparable numbers in all relevant subpopulations. Serial competitive transplantation of H2afy+/+ and -/- BMC into primary and secondary recipient animals showed no competitive disadvantage or lack of self-renewal capacity of H2afy-/- cells. Likewise, colony formation of healthy human donor CD34+ cells was not impaired following depletion of H2AFY by RNAi. Transcriptome analysis of LGMP populations indicated increased of myeloid cell development and loss of LSC signatures in H2afy-deficient LGMPs. Likewise, ATAC-seq of H2afy+/+ and -/- LGMP population revealed profound changes in the chromatin structure and a strong correlation between chromatin accessibility and mRNA expression. Together, our results provide first evidence for a critical function of H2afy (macroH2A1) in models of AML while being dispensable for normal HSPC function. Experiments to assess for the mechanistic consequences of H2afy deletion on chromatin are currently under way.

Ectopic Viral Integration Site-1 Promotes NRAS/ERK Pathway through Epigenetic Silencing of microRNA-124 in AML

BACKGROUNDː Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by genetic mutations that promote proliferation of myeloid progenitors and prevent their differentiation. Over-expression of Ectopic Viral Integration site-1(EVI-1) is related to the poor prognosis in myeloid leukemia, but the underlying mechanism remains unclear. METHODSː Using qRT-PCR and western blotting, we quantified expressions of EVI-1, NRAS and ERK/p-ERK in leukemia cell lines and PBMCs. Using WTS-8 and cell cycle analysis, we further investigated whether downregulation of EVI-1 by siRNA can inhibit cell proliferation. Microscopic observation of peripheral blood cells from EVI-1 transgenic zebrafish and WT control were analyzed by Wright Giemsa staining. Using miR-seq, qPCR, dual-luciferase reporter and coimmunoprecipitation assays, we revealed the relationship between EVI-1, miR-124 and NRAS. RESULTSː EVI-1 was highly expressed in both primary AML and leukemia cell lines (THP-1 and K562). In a transgenic zebrafish model, EVI-1 mediated higher mortality and induced immature hematopoietic cells in the blood circulation, suggesting its oncogenic role. Furthermore, our results suggested that EVI-1 upregulated NRAS expression, thereby activating the RAS/ERK pathway through epigenetic silencing of a potent NRAS suppressor, miR-124. In this study, we found that EVI1 physically interacts with Dnmt3a to form a protein complex that targets and binds to regulatory elements of miR-124. CONCLUSIONSː Overall, the current findings demonstrate that EVI-1 overexpression converges on the regulation of miR-124 promoter methylation and activation of the RAS/ERK pathway in AML carcinogenesis, and suggest EVI-1 and/or miR-124 as therapeutic targets for this dismal disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting Myeloid Leukemia Using Novel TCR-like Chimeric Antigen Receptor-T Cells Against Cathepsin G

Cathepsin G (CG) is a serine protease that is normally contained within primary granules of neutrophils and is expressed during the promyelocyte stage of myeloid cell development. We reported CG to be highly expressed in acute myeloid leukemia (AML) blasts compared with normal myeloid progenitors and leukocytes. We also showed CG to be a promising immunotherapeutic target in AML, having eluted a CG-derived peptide, FLLPTGAEA (hereafter referred to as CG1), from the surface of primary HLA-A*0201 (HLA-A2+) AML blasts and AML cell lines. Additionally, we showed CG expression in acute lymphoblastic leukemia (ALL) through endogenous expression by some ALL subsets and by cross-presentation from the marrow microenvironment. We also showed that CG1-specific cytotoxic T-lymphocytes (CTL) killed HLA-A2+ primary AML blasts and cell lines, but not normal myeloid progenitor cells. Furthermore, we detected CG1-CTL in peripheral blood mononuclear cells (PBMC) from AML patients following allogenic stem cell transplant. Here we report on the engineering, preclinical efficacy, and safety of CG1-targeting chimeric antigen receptor (CAR) T cells, which employ a novel T cell receptor-like (TCR-L) CAR construct that binds cell surface CG1/HLA-A2. Methods Time-of-flight mass spectrometry was used to quantify CG1 expression on the cell surface. The antibody targeting CG1/HLA-A2 was developed by immunizing Harbour Mice with CG1/HLA-A2 complex, FACS-sorting of single B cells, screening B cell antibody-containing supernatant for binding to CG1/HLA-A2, and fusion cloning of VH and VL sequences from positively screened B cells. OCTET BLI assays were used to determine the binding of different alanine substituted CG1 monomers to lead antibodies. The affinity of each monomer was calculated based on BLI data. T2 binding assays were used to validate the binding of antibody monomers to cell surface CG1/HLA-A2 complexes. Immunohistochemistry (IHC) using human fresh and frozen PBMC and marrow was used to show the specificity of anti-CG1/HLA-A2 antibodies. CG1 ScFV was cloned into pORBIT-LENTI-IRES-EGFR-III vector co-expressing either CD28 (CG1-CD28-CAR) or 4-1BB (CG1-41BB-CAR). HLA-A2+ normal donor (ND) T- cells were activated with anti-CD3 and anti-CD28 antibodies before lentiviral transduction with CG1 CAR constructs. Transduced cells were expanded and then characterized in vitro to determine CAR T quality. In vitro flow cytometry cytotoxicity assays were used to confirm in vitro killing of leukemia by CG1-CAR T cells. NSG mice engrafted with AML cell lines and AML patient-derived xenografts (PDX) were used to demonstrate the in vivo efficacy/pharmacokinetics/pharmacodynamics of CG1-CAR T cells from different donors. Results We eluted CG1 peptide from the cell surface in 7 of 8 HLA-A2+ myeloid leukemia cell lines. CG1 was absent on the cell surface of HLA-A2+ non-myeloid solid tumor and lymphoid cell lines, as well as in normal HLA-A2+ neutrophils and PBMC. Screening of different antibody clones for binding to CG1 peptide with different alanine amino acid substitutions identified top 5 antibody candidates with high affinity (single to sub-digit nM) for CG1-HLA-A2. T2 binding assays demonstrated high binding specificity of these five CG1 antibodies to CG1 monomers. The lead antibodies for CAR-T development were selected based on high antibody binding affinities (0.14-5 nM), specificity for CG1/HLA-A2, and no binding to ND marrow and PBMC. In vitro cytotoxicity assays confirmed in vitro killing of HLA-A2-transduced U937 (U937-A2) cells by CG1-CAR T cells. Both CG1-CD28- and the CG1-41BB-CAR constructs demonstrated high levels of killing. Lastly, in vivo AML mouse models using ML-2 and U937-A2 cell lines, and AML PDX validated the in vivo activity of CG1-CD28- and CG1-41BB-CAR T cells (Figure). Conclusion Targeting CG1 using immunotherapy appears to be a valid approach in the setting of AML. We first showed that CG was highly expressed and presented on AML blasts. We developed TCRL-scFv that targets CG1/HLA-A2 on the cell surface and generated two different CAR constructs based on the scFv antibody sequence. We then showed potent anti-leukemia activity and specificity of CG1-CAR T cells against human leukemia in vitro and in vivo. Together, these data provide the pre-clinical proof of concept work that lays the foundation for further development of CG1-CAR T cells as a potential immunotherapy for patients with AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Novel Bispecific Innate Cell Engager (ICE®) AFM28 Efficiently Directs Allogeneic NK Cells to CD123+ Leukemic Stem- and Progenitor Cells in AML

Introduction: Acute myeloid leukemia (AML) is a common hematologic malignancy characterized by expansion of undifferentiated myeloid cells in the bone marrow (BM) and peripheral blood. Although AML can be treated with curative intent, residual leukemic stem cells (LSCs) are frequently reservoirs for relapse, which is associated with a poor prognosis. Moreover, especially elderly patients are often not eligible for intensive treatment regimen. Therefore, there is a high need for novel treatment strategies, which are capable of eliminating LSCs in order to induce long-term remissions, suppress measurable residual disease (MRD) and prevent relapse. A promising emerging approach is the adoptive transfer of allogeneic natural killer (NK) cells, which was shown to be clinically active and could induce complete remissions in relapsed/refractory (R/R) AML. Innate Cell Engagers (ICE®) may further augment the activity of NK cell therapies, in particular against LSCs and hold the potential to increase frequency and durability of responses. The tetravalent bispecific CD123/CD16A ICE®, AFM28, directs the cytotoxic activity of CD16A-expressing NK cells towards CD123-expressing (CD123+) AML blasts and LSCs. In this study, we evaluated the efficacy of AFM28 in pre-clinical models of AML. Methods: Antibody-dependent cell-mediated cytotoxicity (ADCC) data were obtained by a 4h calcein release assay using AML cell lines and primary human NK cells (E:T ratio 2.5:1) in the presence of titrated antibodies. CD64 and CD123 expression levels on AML cell lines were evaluated by flow cytometry. Intracellular FACS was used to detect STAT5/pSTAT5. Proliferation of TF-1 cells in the presence of IL-3 or GM-CSF was measured via CellTiter Glo Assay (Promega). Using primary BM samples of n=6 AML patients and n=4 healthy donors, ADCC assays and colony-forming unit (CFU) assays were performed. BM-MNC (ADCC assay) or enriched CD34+ cells (CFU assay) and allogeneic NK cells (E:T ratio 1:1) were treated with AFM28 or an Fc-enhanced anti-CD123 antibody comparator at different concentrations of AFM28 (0-500 or 0/10/100/1000 pM, respectively) for 24h. Subsequently, ADCC assays were analyzed by flow cytometry and CFU assays were evaluated after 7-14 days. Systemic anti-tumor activity of AFM28 in vivo was assessed using in-life imaging (BLI) in a T cell-depleted huFcgR-C57BL6 transgenic mouse model against intravenously administered luciferase-transfected murine AML cells expressing human CD123 as tumor antigen. Results: Pre-clinical in vitro models using a panel of AML cell lines showed efficacious ADCC against CD123+ tumor cells by allogeneic NK cells in the presence of AFM28. ADCC induction by AFM28 was independent of leukemic cell mutational profiles and even targeted cells with low levels of CD123 surface expression. Of note, expression of the high-affinity IgG receptor CD64 on AML cell lines completely abolished ADCC induction by an Fc-enhanced anti-CD123 antibody whereas AFM28 also showed high ADCC efficacy against all tested CD64+ cell lines. In addition, AFM28 exerted an NK cell-independent inhibitory effect on the IL-3-induced phosphorylation of STAT5 and abolished proliferation of CD123+ TF-1 cells, indicating an antagonistic effect on IL-3R signaling. In ex vivo models using primary AML patient cells, AFM28 demonstrated strong ADCC mediated by allogeneic NK cells. CFU assays of patient-derived AML BM samples performed subsequent to incubation with allogeneic NK cells with and without AFM28 showed significantly reduced numbers of outgrowing colonies in AFM28 treated samples, indicating that AML LSCs and progenitor cells were eliminated. In the in vivo efficacy study with huFcgR-C57BL6 mice, treatment with AFM28 at 15 mg/kg b.w. led to complete inhibition of tumor growth in 5/5 animals throughout a treatment period of 42 days. In contrast, all untreated control mice (6/6) developed systemic disease. Conclusion: AFM28 induced highly potent and selective lysis of CD123+ leukemic cells, including LSCs and progenitor cells, by allogeneic NK cells in vitro and in vivo. The capacity to eradicate LSCs holds the promise for durable responses and the potential for long-term remissions in patients with R/R AML. AFM28 is currently being prepared for a first-in-human clinical investigation as monotherapy and in combination with allogeneic NK cells.

VIP943 Is a Novel CD123 Antibody Drug Conjugate with in Vitro and In Vivo Efficacy in Acute Myeloid Leukemia (AML) Models

Background: Current treatment options (cytarabine with anthracyclines) for patients with acute myeloid leukemia (AML) are often associated with severe and barely tolerable toxicities. In patients who tolerate induction therapy, the complete remission rate is 60%-80%; however, 50% of patients relapse due to the presence of leukemic stem cells (LSC). Antibody-drug conjugates (ADCs) are putatively designed to deliver potent drugs directly to cancer cells while sparing normal tissue; however, first-generation ADCs are associated with severe bone marrow and liver toxicity. VIP943 is a CD123-targeting ADC which uses a kinesin spindle protein inhibitor (KSPi) as a new payload class and a unique legumain linker designed to overcome liabilities of currently approved ADC therapies. The payload is delivered with a drug-antibody ratio of 6. It consists of the KSPi warhead which is modified with a CellTrapperTM moiety so it accumulates within the CD123-targeted tumor cell and cannot enter normal cells. Non-human primate toxicology studies demonstrated a highly favorable and differentiated safety profile for VIP943 (Kirchhoff et al, Cancers 2020). Here we characterize VIP943 in various patient-derived AML models. Methods: For co-localization experiments, MOLM-13 AML cell line was stained with labelled anti-CD123 antibody or isotype control and CytoPainter LysoGreen indicator reagent and analyzed by imaging flow cytometry. AML primary patient mononuclear cells from blood and bone marrow were treated with VIP943 for 24, 48, and 72 hours. Flow cytometry immunophenotyping for AML blasts and CD34+/CD38- LSCs was used to analyze these populations. An in vivo patient-derived AML PDX mouse model was treated with VIP943 (5 mg/kg IV every 7 days) or in combination with 5-azacytidine (2.5 mg/kg SC days 1-5 x 3) and venetoclax (50 mg/kg PO days 1-5 x 3). Cytokine release syndrome was analyzed comparing VIP943 to isotype control ADC, anti-CD123 antibody, and positive comparator antibodies by in vitro cytokine release assays (CRA) in two different assay formats using whole blood and isolated mononuclear cells from 10 healthy donors. Flow cytometric lymphocyte and leukemic immunophenotyping was performed from blood of non-human primates. A leukemia cell line panel drug screen with VIP943 was evaluated and baseline genomic features were correlated with response. Results: Co-localization studies confirm specific internalization and efficient intracellular trafficking to the lysosome of VIP943. In vitro treatment with VIP943 (66 nM, continuous exposure) delivers blast reductions of 37% after a 24h incubation with VIP943, 45% after 48h incubation and 83% after an 72h incubation. In the CD34+/CD38- cell population, a reduction of 71% was achieved. In a patient-derived AML model, VIP943 with 5-azacytidine and venetoclax led to complete tumor growth inhibition after 21 days showing superiority of this triplet therapy (complete regression: 8/9 mice) with high tolerability (maximum body weight loss -4.8 %). The immunotoxicological evaluation in both CRA formats and with the tested antibody concentrations (100µg, 10µg, 1µg) exerts no induction of cytokines by VIP943 compared to controls. Immunophenotyping demonstrates no impact of VIP943 treatment on lymphocytic subpopulations, whereas the CD123-positive basophils are reduced after 24h followed by an almost complete recovery. VIP943 IC50 ranges from 0.1nM-300nM in a leukemia cell line panel screen and features associated with sensitivity are investigated. Conclusions: VIP943 is a next-generation ADC with a differentiated safety profile from currently approved ADCs, including lack of in vitro cytokine release. In vitro and in vivo studies using patient-derived AML cells show VIP943 has favorable monotherapy and combination efficacy including targeting of leukemic stem cells, which drive relapse. These findings warrant evaluating VIP943 in clinical trials.

CD84 Is a Therapeutically Targetable Driver of Leukemogenesis Via Disruption of Energy Supply in Acute Myeloid Leukemia

CD84 is a member of the signaling lymphocyte activation molecule (SLAM) family; it forms homophilic dimers by self-association. CD84 is reported as an important survival receptor in chronic lymphocytic leukemia and its activation upregulates PD-L1 expression on leukemia cells. We and others recently reported that CD84 is highly expressed on myeloid derived suppressor cells, but its function in myeloid malignancies has yet to be investigated. Using publicly available microarray data (GSE13509), we found that CD84 is overexpressed on acute myeloid leukemia (AML) cells compared to normal hematopoietic counterparts (AML, n=542 vs. normal, n=73, p<0.0001). and that its upregulation is associated with poor prognosis in AML patients (GSE10358; p=0.0105). Knocking down CD84 expression in AML cell lines (THP1 and HEL) by shRNAs caused a substantial inhibition of cell viability and extended THP1-luciferase xenografted mice survival in vivo (median survival: shCtrl, 38 days vs shCD84, 53 days, p=0.0015, n=5). Moreover, we observed that CD84 depletion in AML primary patient cells dramatically induced cell apoptosis (shCtrl: 13.0±1.84% vs shCD84: 29.33±1.3%; n=3; p=0.02) and inhibited colony formation (shCtrl: 68.44±11.65 vs shCD84: 15.56±6.32; n=3; p=0.01). In light of highly expressed CD84 in MLL-r AML and inv(16) AML, we employed MLL-AF9 and CBFB-MYH11(CM) AML mouse models, and found that CD84 knockdown robustly dampened cell growth and decreased colony-forming units in vitro. In both models, bone marrow (BM) transplantation shown that CD84 deficiency significantly decreased leukemic engraftment in BM (MLL-AF9: shCtrl: 95.92±0.61% vs shCD84: 76.42±4.11%, p=0.0016, n=5/group; inv(16): shCtrl: 39.02±2.98% vs shCD84: 13.66±1.24%, p<0.0001, n=5/group), spleen, and peripheral blood (PB). In AML patient derived xenografts (PDX), we observed AML burden was markedly reduced in BM (shCtrl:51.22±10.29% vs shCD84: 2.304±0.16%; n=5/group, p=0.0014) and spleen (shCtrl: 8.790±2.989% vs shCD84: 0.8420±0.13%; n=5/group, p=0.0255) in the CD84 knockdown group. Mechanistically, CD84 deletion depressed mTOR signaling, induced mitochondrial stress, and disrupted energy supply chains based on RNA-seq analysis. We demonstrated CD84 deletion caused mitochondrial dysfunction as indicated by decreasing oxidation phosphorylation (shCtrl vs shCD84, p<0.0001) and fatty acid oxidation (shCtrl vs shCD84, p=0.0004). Next, we determined that CD84 deletion caused disruption of mitochondrial matrix morphology and loss of mitochondrial cristae in AML. We also observed a dramatic loss of mitochondrial membrane potential upon CD84 deletion. We generated a novel murine-human chimeric anti-human CD84 monoclonal antibody (mAb) with high affinity and specificity. In the presence of NK cells, anti-CD84 mAb induced strong antibody-dependent cell-mediated cytotoxicity in CD84-expressing AML lines (n=2) and primary samples (n=2), but not in healthy donor CD34+ cells (n=2). Next, the luciferase-expressing THP1 was transplanted into NSG mice. Mice with comparable tumor burden were randomized into 2 groups: control human IgG or anti-CD84 mAb (5mg/kg, i.v, bid, n=6/group), every week together with 3x106 human PB mononuclear cells (PBMCs) as effector cells (for 4 weeks). Anti-CD84 mAb significantly suppressed leukemia development and extended median survival (IgG: 30 days vs anti-CD84 mAb: undefined, p=0.0018). However, without PBMCs, human IgG or anti-CD84 mAb did not induce significant differences in tumor burden or median survival (n=5/group; p=0.5082). Finally, we developed two AML PDXs in NSG mice. Following engraftment >1% in PB, mice were divided into 2 groups (n=6/group). We maintained the same treatment strategy as applied above. In both AML PDXs, the BM engraftment of CD45+CD33+ cells markedly decreased in anti-CD84 mAb treated mice (AML #1: IgG: 94.25± 0.41% vs anti-CD84 mAb: 71.52±3.96%, p= 0.0002; AML #2: IgG: 88.74±0.7% vs anti-CD84 mAb: 53.98±5.61%, p=0.0003). Anti-CD84mAb treatments in secondary transplantation of BM harvested from the first treatment groups resulted in further significant reduction in BM engraftment (IgG: 86.48± 0.67% vs anti-CD84 mAb: 29.74±6.02%, n=5, p<0.0001), and extended survival (IgG, 28 days vs anti-CD84 mAbs, 36 days, p=0.0026, n=5) following third BM transplantation. The results indicated our pharmacological targeting of CD84 inhibits leukemogenesis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mitochondrial Regulation of Ferroptosis in Acute Myeloid Leukemia

Background: Investigations into targeting apoptosis in acute myeloid leukemia (AML) have resulted in breakthrough therapies utilizing Bcl-2 inhibition by venetoclax; however, acquired resistance poses major clinical challenges. Non-apoptotic regulated cell death (RCD) pathways have recently drawn attention especially in solid tumors, but studies targeting these pathways including ferroptosis in leukemia are limited. While ferroptosis is well-defined by iron- and lipid peroxidation-dependency, accumulating evidence suggests that there are also context-specific mechanisms. Since leukemia cells exhibit increased oxidative stress with particular dependency of cell survival on mitochondria, we hypothesized that AML exhibits a unique vulnerability to ferroptosis involving mitochondria. We here study targeting ferroptosis pathway in AML and its mitochondrial (mito-) regulation to develop mechanism-based combination with mito-targeting agents. Results: We first analyzed DepMap datasets to determine the essentiality of ferroptosis-regulating genes in cancer cell lines. Among major anti-ferroptosis genes, GPX4 exhibits high essentiality prominently in leukemia cells, suggesting its potential as a therapeutic target. AML patient survival data from TCGA revealed that high GPX4 expression is associated with shorter survival, indicating prognostic impact. GPX4 is the most downstream molecule to block ferroptosis by reducing lipid hydroperoxide. Although GPX4 is essential for mouse embryonic development, acquired depletion is reported to have no significant effect on hematopoietic stem cells, suggesting tolerability of GPX4-targeted therapies for normal hematopoiesis. We demonstrate that GPX4 inhibition with doxycycline-inducible shRNA or the specific inhibitor ML210 induces ferroptosis in leukemia cells, evidenced by iron-dependent lipid peroxidation and blockade of cell death by the iron chelator deferoxamine. Of note, the classical ferroptosis inducers (xCT inhibitors) sulfasalazine and sorafenib did not induce ferroptosis in AML cells, suggesting a specific relevance of GPX4 in AML ferroptosis. Consistent with solid tumor cells, AML cells show early (6h post-treatment) morphological alterations in mitochondria characterized by shrinkage and increased membrane density upon GPX4 inhibition (Figure 1). Importantly, and in contrast to most solid tumor studies, mitochondria-targeted antioxidants MitoQ and MitoTEMPO completely blocked ferroptosis, suggesting that AML ferroptosis is regulated predominantly by mitochondrial rather than cytoplasmic redox metabolism. To further determine the role of mitochondria in ferroptosis, we utilized HL60 rho0 cells that exhibit abrogated mito-respiration due to lack of mito-DNA. Surprisingly, HL60 rho0 cells were nearly 10 times more sensitive to ML210 than HL60 parental cells, in clear contrast to sarcoma studies demonstrating similar or lower sensitivity to ferroptosis of mito-DNA deficient cells. Consistently, degradation of the respiratory complex with hyperactivation of mito-protease ClpP by imipridone ONC212 sensitized cells to GPX4 inhibition in HL60 parental cells but not in rho0 cells, further supporting the role of mito-respiration in the protection of cells against ferroptosis. As venetoclax (Ven) is one of the most widely used mitochondria-targeting agents in AML that also inhibits mito-respiration, we tested the combination of GPX4 inhibitor ML210 and Ven. This combinatorial treatment exerts synergistic anti-leukemia effects in AML cell lines and primary cells from AML patients including those with Ven resistance (Figure 2). Unexpectedly, and as a novel mechanism of GPX4 inhibition-mediated ferroptosis, we found that ML210 induces BAX/BAK-independent cytochrome C release from mitochondria, which is blocked by MitoQ. Combination with Ven further enhanced cytochrome C release, as one of potential mechanisms of the observed synergistic cell death. Conclusion: Our data suggest therapeutic potential of ferroptosis induction in AML by targeting GPX4 with the mechanistic involvement of mito-respiration. The combination of GPX4 and BCL-2 inhibition is synergistic and capable of overcoming Ven resistance. Studies are in progress to elucidate the molecular link between mito-respiration/redox and cytochrome C release and to investigate ferroptosis induction in vivo. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Circulating Microbial Metabolite Drives the Clonal Expansion of Pre-Leukemic Cells

Inflammatory mediators, including cytokines and microbial ligands, are implicated in the development of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). A consequence of aging and various autoimmune and inflammatory conditions is loss of intestinal barrier integrity and translocation of microbial species and metabolites into circulation. More recent findings revealed that patients with inflammatory bowel disease (IBD) harbor mutant hematopoietic clones and often present with clinical manifestations of MDS/AML. Moreover, intestinal barrier dysfunction is commonly reported at diagnosis in patients with MDS and AML. These observations led us to speculate that microbial-derived signals may contribute to the expansion of pre-leukemic hematopoietic stem and progenitor cells (HSPCs) and development of myeloid malignancies. To determine if loss of intestinal barrier integrity contributes to the expansion of pre-leukemic DNMT3a-mutant HSPCs, we generated a model of IBD by inducing gut epithelial injury using dextran sulfate sodium (DSS). Upon gut epithelial injury, we observed a significant expansion of DNMT3a-mutant HSPCs, but not of wild-type (WT) HSPCs, in vivo. Unexpectedly, DNMT3a-mutant HSPCs do not exhibit a competitive advantage over WT cells in the absence of intestinal barrier dysfunction, such as following DSS or high-dose radiation. Fecal material from DSS-treated WT mice was sufficient to induce expansion of DNMT3a-mutant HSPCs and this expansion was prevented with broad-spectrum antibiotics. These findings indicate that the competitive advantage of DNMT3a-mutant HSPCs following gut epithelial injury was due to alterations in the microbiome. In parallel sequencing studies, we identified a significant enrichment of gram-negative bacterial phyla in stool samples from the IBD mouse model, IBD patients, and treatment-naive AML patients. All gram-negative bacteria uniquely generate a microbial intermediate, ADP-heptose, as part of the lipopolysaccharide biosynthesis pathway. ADP-heptose can enter the blood circulation and freely translocate into cells and activate the ALPK1-TIFA complex ("TIFAsome"). Alpha-protein kinase 1 (ALPK1) and tumor necrosis factor receptor-associated factor (TRAF)-interacting protein with an FHA domain (TIFA) are overexpressed in primary human MDS and AML HSPCs as compared to healthy donors and their expression is correlated with poor prognosis. Gram negative bacteria and corresponding ADP-heptose levels were also significantly increased in the plasma of individuals with intestinal barrier injury, such with IBD, MDS, or upon aging, as compared to young healthy individuals. The circulating levels of ADP-heptose from aged individuals and IBD or MDS patients were sufficient to induce the TIFAsome in leukemic cells. Moreover, administration of disease relevant levels of ADP-heptose resulted in expansion and enhanced function of mouse DNMT3a-mutant HSPCs and of human AML cell lines and primary MDS cells in vivo. Expansion of DNMT3a-mutant HSPCs and human MDS/AML cells was directly related to overexpression of ALPK1, as WT HSPCs or ALPK1-deficient AML cells did not respond to ADP-heptose. To gain deeper mechanistic insight, we examined global transcriptional and epigenetic changes in purified WT and DNTM3a-mutant HSCs treated with ADP-heptose. DNMT3a-mutant HSCs treated with ADP-heptose resulted in epigenetic changes and increased expression of gene programs related to non-canonical NF-kB, ERK/MAPK, cell cycle activation, cell survival, and stem cell function. Indeed, ADP-heptose caused increased cycling and self-renewal of DNMT3a-mutant HSPCs, but not WT HSPCs, via the TIFAsome in vitro and in vivo. Based on the enriched pathways in DNMT3a-mutant HSCs treated with ADP-heptose, we screened small molecule inhibitors of TIFAsome activation. We found that targeting UBE2N abrogated ADP-heptose-mediated signaling via the TIFAsome and expansion of patient-derived AML xenografts. In summary, we report that conditions leading to intestinal barrier dysfunction and circulating ADP-heptose from gram-negative bacteria induce expansion of pre-leukemic and MDS/AML cells via ALPK1-TIFA. Lastly, we provide a potential therapeutic strategy to prevent the competitive advantage of pre-leukemic cells prior to overt malignant transformation.

Mesenchymal Stromal Cells Protect Acute Myeloid Leukemia Cells from Venetoclax-Induced Apoptosis through Contact-Dependent Mechanisms

Acute myeloid leukemia (AML) patients have a poor prognosis with standard treatments due to a high relapse rate. Recently, Venetoclax (VEN), the first B-cell lymphoma 2 (BCL-2) selective inhibitor to enter the clinic, has shown a breaking-through effect in combination with demethylating agents in older patients with treatment-naïve AML. However, resistance mechanisms were described, hampering the complete remission of VEN-treated patients. It has been proposed that the bone marrow microenvironment in general and Mesenchymal stromal cells (MSCs), in particular, favor a protective niche that sustains leukemic cell viability and plays a crucial role indetermining drug sensitivity in several hematological disorders, including AML. A previous study demonstrated that VEN, as well as other drugs commonly used in AML therapy, showed a reduced effect in the presence of an MSC-conditioned medium, suggesting that MSC-dependent release of inflammatory signals and other soluble factors could be involved in drug resistance. Our study aims to further investigate the mechanisms of MSC-mediated VEN resistance in AML. We found that primary AML cells isolated from patients at diagnosis and treated with VEN showed different apoptotic rates, indicating an AML cell-specific resistance/sensitivity to the drug. In co-culture experiments, primary MSCs, isolated from healthy donors or AML patients, protected VEN-sensitive AML cells from apoptosis, confirming the critical role of MSCs in drug resistance. To deeply investigate this issue, we performed co-culture experiments using the AML cell line MV4-11, which we demonstrated as sensitive to VEN in a dose-dependent manner. We found that MSCs significantly reduced MV4-11 apoptosis after VEN exposure. The addiction of transwells to the co-culture system abrogated the MSC-protective effect on VEN-induced apoptosis, suggesting the establishment in our setting of a complex interplay between AML cells and MSCs, not only mediated by soluble factors as already demonstrated but also involving direct cell-contact. Furthermore, when we cultured MV4-11 cells in direct contact with MSCs, we were able to distinguish, among leukemic cells, a cell population that remained floating in the supernatant and a cell population that kept adherent to MSCs and could be collected by trypsin detachment only. Interestingly, MSCs protected the adherent but not the floating cells from VEN-induced apoptosis, suggesting a central role of a close adhesion in the MSC-driven resistance to VEN. To gain insight into this mechanism, we sorted adherent vs. floating cells and analyzed and compared Gene Expression Profiling (GEP). Interestingly, the principal component analysis suggested that the floating cell GEP was more similar to the GEP of MV4-11 cells cultured without MSCs compared to adherent cell GEP. We found 159 differential expressed genes (DEGs) in adherent vs. floating MV4-11 cells (FC| ≥ 2 and P ≤ 0.05, according to Transcriptome Analysis Console software guidelines). Enrichment analysis revealed that DEGs were involved in cell adhesion migration and proliferation pathways. Among DEGs, we found that CD90/Thy-1, an adhesion molecule whose expression correlated with unfavorable karyotypes and shorter survival in AML, was significantly up-regulated in adherent MV4-11 cells. Flow cytometry analysis confirmed that the adherent compared to the floating MV4-11 cells, showed a higher expression of CD90 as well as CXCR4 as expected. Our study demonstrated that MSCs protect AML cells from VEN-induced apoptosis through a complex mechanism involving tight adhesion-mediated pathways. Further clarification of these interactions can shed light on putative targets to counteract MSC-mediated drug resistance to VEN treatment.

The Transcription Factor PU.1 Controls Alternative Splicing of Cflip By Binding to SRSF2/10 Splice Factors in AML Therapy Responses

cFLIP (CFLAR) is an inactive homolog of caspase 8 that functions downstream of death-receptor mediated apoptosis. CFLAR gene is alternatively spliced and exists in three isoforms: a long isoform (cFLIPL), which partially inhibits CASP8 activity, and two short forms (cFLIPR and cFLIPS) that entirely block caspase 8 activity. Moreover, cFLIPL is crucial for macrophage differentiation. Therefore, the ratio between cFLIPL and cFLIPS/R may regulate myeloid differentiation. The transcription factor PU.1 (SPI1) is necessary for myeloid differentiation as well as myeloid cell survival. PU.1 is expressed at low levels in hematopoietic progenitors and increasing levels of PU.1 promote macrophage and granulocyte differentiation. Additionally, aberrant low PU.1 expression contributes to an immature myeloid phenotype, e.g. seen in acute myeloid leukemia (AML). Interestingly, two studies indicated that high PU.1 protein levels were associated with alternative splicing in a murine erythroleukemia model by either direct binding to splice factors or RNA. Based on these observations we hypothesized that PU.1 controls alternative splicing of cFLIP during myeloid differentiation. In line with above hypothesis, differentiating primary CD34+ hematopoietic progenitors into granulocytes and monocytes or AML cells into granulocytes shifted cFLIP splicing in favor of cFLIPL paralleled by increased PU.1 mRNA and protein levels. Next, we assessed the direct impact of PU.1 protein on splicing during granulocytic differentiation by knocking down PU.1 in NB4 acute promyelocytic leukemia (APL) and MOLM13 AML cells. Silencing PU.1 in NB4 and MOLM13 cells decreased cell death and differentiation during all-trans retinoic acid (ATRA)-mediated granulocytic differentiation. Moreover, knocking down PU.1 reduced cFLIPL/S protein and mRNA ratios during ATRA treatment. Interestingly, increasing the cFLIPL/S ratio by knocking down specifically cFLIPS or overexpressing cFLIPL reduced MOLM13 cell viability during ATRA treatment. Since the canonical function of CFLAR isoforms is the inhibition of extrinsic apoptosis, we next tested if increased cFLIPS levels found in AML cells depleted for PU.1 would show reduced sensitivity to TRAIL treatment. Indeed, TRAIL-induced extrinsic apoptosis via TRAIL receptor was significantly reduced in PU.1 depleted MOLM-13 AML cells. To investigate if PU.1 is directly involved in CFLAR splicing, we analyzed the CFLAR pre-mRNA sequence encompassing the alternative splicing site for PU.1 and splice factor binding motifs. We identified one putative PU.1 and SRSF2 as well as SRSF10 splice factor binding sites in the CFLAR pre-mRNA sequence. Using RNA immunoprecipitation, PU.1 pull-down and proximity ligation assays, we showed that all three proteins bind the CFLAR mRNA and physically interact with each other. Lastly, we asked if PU.1 additionally influences cFLIP alternative splicing via regulating splice factor transcription. Knocking down PU.1 in MOLM13 cells resulted in significantly reduced SRSF2 and SRSF10 mRNA and protein levels. We confirmed PU.1 binding to their promoter using ChIP assays. Accordingly, these splice factors showed altered mRNA expression when comparing mature granulocytes to primary AML cells. Since silencing SRSF2 and SRSF10 switched the CFLARL/S isoform ratio towards the long isoform, we suggest that PU.1-mediated induction of these splice factores represents a negative feedback loop. In conclusion, we provide evidence that PU.1 directly regulates alternative splicing of CFLAR during ATRA-mediated neutrophil differentiation and TRAIL-induced cell death in human AML cells. We propose a model where PU.1 binds to CFLAR pre-mRNA and blocks SRSF2 and SRSF10-mediated splicing ultimatively leading to exon 7 skipping and predominant expression of cFLIPL.

MicroRNA profiling of paediatric AML with FLT-ITD or MLL-rearrangements: Expression signatures and invitro modulation of miR-221-3p and miR-222-3p with BRD4/HATs inhibitors.

Novel therapeutic strategies are needed for paediatric patients affected by Acute Myeloid Leukaemia (AML), particularly for those at high-risk for relapse. MicroRNAs (miRs) have been extensively studied as biomarkers in cancer and haematological disorders, and their expression has been correlated to the presence of recurrent molecular abnormalities, expression of oncogenes, as well as to prognosis/clinical outcome. In the present study, expression signatures of different miRs related both to presence of myeloid/lymphoid or mixed-lineage leukaemia 1 and Fms like tyrosine kinase 3 internal tandem duplications rearrangements and to the clinical outcome of paediatric patients with AML were identified. Notably, miR-221-3p and miR-222-3p resulted as a possible relapse-risk related miR. Thus, miR-221-3p and miR-222-3p expression modulation was investigated by using a Bromodomain‑containing protein 4 (BRD4) inhibitor (JQ1) and a natural compound that acts as histone acetyl transferase inhibitor (curcumin), alone or in association, in order to decrease acetylation of histone tails and potentiate the effect of BRD4 inhibition. JQ1 modulates miR-221-3p and miR-222-3p expression in AML with a synergic effect when associated with curcumin. Moreover, changes were observed in the expression of CDKN1B, a known target of miR-221-3p and miR-222-3p, increase in apoptosis and downregulation of miR-221-3p and miR-222-3p expression in CD34+ AML primary cells. Altogether, these findings suggested that several miRs expression signatures at diagnosis may be used for risk stratification and as relapse prediction biomarkers in paediatric AML outlining that epigenetic drugs, could represent a novel therapeutic strategy for high-risk paediatric patients with AML. For these epigenetic drugs, additional research for enhancing activity, bioavailability and safety is needed.

SpinTip: A Simple, Robust, and Versatile Preanalytical Method for Microscale Suspension Cell Proteomics.

Sample loss and contamination are critical preanalytical pitfalls in microscale proteomic applications of nonadhering cells. Common assays and workflows are not easily adoptable to microscale sample sizes of suspension cells due to inadvertent sample loss. This impedes preanalytical experimental manipulation of limited suspension cell samples for microscale proteomics applications, such as encountered for primary human materials. Here, we describe and test a simple manual batch technique for single-step 100-fold concentration of scarce numbers of diluted suspension cells (down to 5000 cells) by volume reduction, facilitating microscale experiments with suspension cells. Pipette tips with heat-sealed orifices (SpinTips) are manufactured within 1 min and serve as versatile microcentrifugation vessels from which supernatant can be aspirated with minimal cell loss. A residual volume of approximately 3 μL can be achieved without visualization of the cell pellet. The results show that SpinTips enable the concentration, medium exchange, washing, and culture of highly limited amounts of suspension cells for functional manipulation and microscale proteomics and are readily incorporated into standard workflows. The application is illustrated by profiling ex vivo responses of primary acute myeloid leukemia (AML) cells from one AML patient to daunorubicin (DNR) to a depth of 3462 quantified proteins with excellent repeatability.

Co-targeting of HDAC, PI3K, and Bcl-2 results in metabolic and transcriptional reprogramming and decreased mitochondrial function in acute myeloid leukemia

Despite the recently approved new therapies, the clinical outcomes of acute myeloid leukemia (AML) patients remain disappointing, highlighting the need for novel therapies. Our lab has previously demonstrated the promising outlook for CUDC-907, a dual inhibitor of PI3K and HDAC, in combination with venetoclax (VEN), against AML both in vitro and in vivo at least partially through suppression of c-Myc. In this study, we further elucidated the mechanism of action of the combination in preclinical models of AML. We demonstrated that the combination significantly reduced primary AML cell engraftment in immunocompromised mice. RNA sequencing and metabolomics analyses revealed that the combination reduced the levels for mRNAs of key TCA cycle genes and metabolites in the TCA cycle, respectively. This was accompanied by a reduced oxygen consumption rate (OCR), demonstrating that the combination suppressed oxidative phosphorylation (OXPHOS). Metabolomics analyses revealed that a large number of metabolites upregulated in AraC-resistant AML cells could be downregulated by the combination. CUDC-907 synergized with VEN in inducing apoptosis in the AraC-resistant AML cells. In conclusion, the CUDC-907 and VEN combination induces metabolic and transcriptomic reprograming and suppression of OXPHOS in AML, which provides additional mechanisms underlying the synergy between the two agents.

Antileukemic activity of YPN-005, a CDK7 inhibitor, inducing apoptosis through c-MYC and FLT3 suppression in acute myeloid leukemia

Acute myeloid leukemia (AML) is an aggressive blood cancer with a high rate of relapse associated with adverse survival outcomes, especially in elderly patients. An aberrant expression of cyclin dependent kinase 7 (CDK7) is associated with poor outcomes and CDK7 inhibition has showed antitumor activities in various cancers. We investigated the efficacy of YPN-005, a CDK7 inhibitor in AML cell lines, xenograft mouse model, and primary AML cells. YPN-005 effectively inhibited the proliferation of AML cells by inducing apoptosis and reducing phosphorylation of RNA polymerase II. The c-MYC expression decreased with treatment of YPN-005, and the effect of YPN-005 was negatively correlated with c-MYC expression. YPN-005 also showed antileukemic activities in primary AML cells, especially those harboring FMS-like tyrosine kinase 3–internal tandem duplication (FLT3-ITD) mutation and in in vivo mouse model. Phosphorylated FLT3/Signal transducer and activator of transcription 5 (STAT5) was decreased and FLT3/STAT5 was downregulated with YPN-005 treatment. Our data suggest that YPN-005 has a role in treating AML by suppressing c-MYC and FLT3.

AML-250 Mitochondrial Respiration Regulates GPX4 Inhibition-Induced Ferroptosis in Acute Myeloid Leukemia

Ferroptosis, a form of non-apoptotic cell death regulated by iron-dependent lipid peroxidation, has drawn extensive attention as a potential anti-cancer strategy. However, it remains to be explored in hematologic malignancies. We here investigate the molecular mechanisms of ferroptosis in acute myeloid leukemia (AML) and its therapeutic potential of co-targeting mitochondrial respiration. Analyses of publicly available databases (TCGA, DepMap) showed that AML patients with higher mRNA expression of major anti-ferroptotic genes had significantly shorter survival and that leukemia cells are one of the cell types that are highly dependent on GPX4 among other cancers. This suggests the potential prognostic impact and relevance in therapeutics of this pathway in AML. Indeed, pharmacological inhibition or genetic knockdown of a well-established anti-ferroptosis regulator GPX4 induced profound ferroptosis, evidenced by dependency on iron and lipid peroxidation. Importantly, the induced cell death was agonistic of TP53 mutational status. As a potentially AML-specific mechanism that is unusual in solid tumor models, we found that GPX4 inhibition-induced ferroptosis was efficiently blocked by the mitochondria-targeted ubiquinone MitoQ in AML cells. Ubiquinone is an endogenous antioxidant protecting cells from lipid peroxidation, and in the mitochondria, the respiratory chain is essential for the recycling of ubiquinone. Thus, we hypothesized that mitochondria protect AML cells from ferroptosis by activating the ubiquinone cycle. Consistently, mitochondrial DNA-depleted HL60 Rho0 cells, which lack mitochondrial respiration, were more sensitive to ML210 compared to the parental cells. To translate this finding to a pharmacological approach, we utilized the imipridone ONC201, which hyperactivates mitochondrial protease ClpP to degrade mitochondrial proteins, including the respiratory chain complex, and exerts cancer-selective lethality (Ishizawa et al. Cancer Cell 2019). Indeed, the combination of ONC201 and ML210 resulted in synergistic anti-leukemia effects in primary AML cells. The combinatorial effect was also validated by utilizing genetically engineered AML cells (genetic knockdown of GPX4 or overexpression of hyperactivated mutant ClpP). GPX4 inhibition induces ferroptosis involving mitochondrial redox machinery in AML. Combinatorial targeting of mitochondrial respiration with GPX4 inhibition exerts synergistic anti-leukemia effects. Further studies are in progress to assess the molecular mechanisms and the in-vivo efficacy of the combinatorial treatments.

AML-266 Optimization of a Drug Screening Platform in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a group of hematological diseases with a poor prognosis. Functional ex-vivo drug screening of primary AML cells could identify patient-specific vulnerabilities and improve the treatment of AML patients. To determine the chemosensitivity of primary cells from AML patients, we previously developed a multiparametric niche-like drug screening platform (NEXT platform, Reinaldo Dal Bello, ASH, 2020). Optimization of an ex-vivo niche-like drug screening platform on primary AML samples to prospectively recommend second-line treatment for AML patients. Primary cells from 49 consecutive patients with newly diagnosed AML were cultured for 72 hours in niche-like conditions (conditioned media, mesenchymal stroma, hypoxia) and exposed to 36 molecules at 8 concentrations. Subsequently, total leukemic cells, blasts, lymphocytes, differentiated cells, and leukemic stem cell counts were assessed using multiparametric high-throughput flow cytometry (iQue3 Intellycyt). Drug activity was assessed using a drug sensitivity score (DSS, Yadov et al., Scientific Reports, 2014) after normalization to DMSO controls. A drug was considered inactive (DSS=0), moderately effective (0<DSS<50), or very effective (DSS≥50). We validated the use of frozen cells that provided a similar number of live cells (3492±2808, n=32) as fresh samples (2,398±2,567, n=5, P=0.29). We upscaled the assay to a 384-well plate to increase the number of tested drugs, with a minimum of 7.5 million cells required for testing the 36 drugs. Across the 49 patients, the median number of drugs with strong or moderate activity was 4 (range 0 to 13). In 42 (86%) patients, at least one drug had moderate activity and 22 (49%) had strong activity. We identified 6 top drugs that are moderately effective or very effective in more than 40% of patients: selinexor, ponatinib, palbociclib, sunitinib, and S63845. During this study, we validated and upscaled our NEXT platform on 49 patients. The prospective application of our niche-like drug screening platform is now being investigated in relapsed/refractory AML patients accrued to the multicenter study ALFA-PPP (NCT04777916).

Expression of the immune checkpoint modulator OX40 indicates poor survival in acute myeloid leukemia

Despite therapeutic advances, mortality of Acute Myeloid Leukemia (AML) is still high. Currently, the determination of prognosis which guides treatment decisions mainly relies on genetic markers. Besides molecular mechanisms, the ability of malignant cells to evade immune surveillance influences the disease outcome and, among others, the expression of checkpoints modulators contributes to this. In AML, functional expression of the checkpoint molecule OX40 was reported, but the prognostic relevance of OX40 and its ligand OX40L axis has so far not been investigated. Here we described expression and prognostic relevance of the checkpoint modulators OX40 and OX40L, analyzed on primary AML cells obtained from 92 therapy naïve patients. Substantial expression of OX40 and OX40L on AML blasts was detected in 29% and 32% of the investigated subjects, respectively, without correlation between the expression of the receptor and its ligand. Whereas OX40L expression was not associated with different survival, patients with high expression levels of the receptor (OX40high) on AML blasts survived significantly shorter than OX40low patients (p = 0.009, HR 0.46, 95% CI 0.24–0.86), which identifies OX40 as novel prognostic marker and a potential therapeutic target in AML patients.

Quercetin induces autophagy-associated death in HL-60 cells through CaMKKβ/AMPK/mTOR signal pathway.

Acute myeloid leukemia (AML) is one of the most common malignancies of the hematopoietic progenitor cell in adults. Quercetin has gained recognition over the years because of its anti-cancer effect with minimal toxicity. Herein, we aim to investigate the anti-leukemia mechanism of quercetin and to decipher the signaling pathway of quercetin in HL-60 leukemic cells. We observed that quercetin induces apoptosis and autophagic cell death, in which both pathways play an important role in suppressing the viability of leukemia cells. Phosphorylated AMPK (p-AMPK) protein expressions are lower in primary AML cells, HL-60 cells, KG-1 and THP-1 cells than in peripheral blood monocular cells. After quercetin treatment, the expression of p-AMPK is increased while the expression of p-mTOR is decreased in a dose-dependent manner. Mechanistically, compound C, an AMPK phosphorylation inhibitor, upregulates the phosphorylation of mTOR and inhibits autophagy and apoptosis in quercetin-induced HL-60 cells, while silencing of CaMKKβ inhibits the quercetin-induced phosphorylation of AMPK, resulting in increased mTOR phosphorylation. Furthermore, silencing of CaMKKβ inhibits the autophagy in HL-60 cells. Taken together, our data delineate that quercetin plays its anti-leukemia role by inhibiting cell viability and inducing apoptosis and autophagy in leukemia cells. Quercetin inhibits the phosphorylation of mTOR by regulating the activity of AMPK, thus playing a role in the regulation of autophagy and apoptosis. CaMKKβ is a potential upstream molecule for AMPK/mTOR signaling pathway, through which quercetin induces autophagy in HL-60 cells.