Primary Acute Myeloid Leukemia Patient 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.

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.

CD25 Targeting Eliminates Regulatory T Cells and CD25+ Blasts in Acute Myeloid Leukemia

Background Acute Myeloid leukemia (AML) is a heterogeneous disease that requires novel targeted treatment options tailored to the patients' specific microenvironment and blast phenotype. CD25 Mab (also referred to as RG6292 and RO7296682) is an afucosylated, IL-2 non-blocking human IgG1 antibody, shown to efficiently deplete immunosuppressive regulatory T cells (Tregs) in humans and solid tumour models (Kolben 2021) whilst allowing binding of IL-2 to effector T cells and the induction of anti-tumour adaptive immune responses (Solomon, Amann et al. 2020). CD25 Mab binds to CD25+ target cells and its crystallisable fragment (Fc) to Fc receptors (FcR) expressed on the surface of effector cells, such as FcgRIIIa on Natural Killer (NK) cells, monocytes and macrophages. It mediates killing of target cells through antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). CD25 Mab is currently being investigated in a phase I dose escalation monotherapy study (NCT04158583) or in combination with atezolizumab (NCT04642365). In AML patients, a higher frequency of immunosuppressive bone marrow Tregs was observed as compared to that of healthy donors and their abundance correlated with poorer outcome (Szczepanski, Szajnik et al. 2009, Han, Dong et al. 2018, Dong, Han et al. 2020). In addition, CD25 is also expressed on a subset of AML cells and may be restricted to leukemic stem cells (LSC) or cells with a progenitor phenotype (Angelini, Ottone et al. 2015, Bertolini, Kageyama et al. 2018, Aref, Azmy et al. 2020). Considering the critical role of LSC in the propagation of the disease and the high relapse rate in AML, it would be important to understand if CD25 targeting could deepen responses and help prevent relapse in patients. In this study, we hypothesized that CD25 Mab may have a dual mode of action whereby it depletes suppressive Tregs and has a direct cytotoxic effect on CD25+ AML cells. Methods We conducted an in-depth characterization of bone marrow and/or blood samples of 37 AML patients and healthy donors by high dimensional flow cytometry (up to 39 cellular markers) and RNA sequencing using computational analysis. Moreover, we performed ex vivo ADCC assays using allogeneic NK cells isolated from healthy donors and AML patient material to test the cytotoxic potential of CD25 Mab on Tregs and CD25+ AML cells, as compared to isotype control antibody. Results We found that the abundance of Tregs and CD25+ AML cells correlated strongly with that of the blood in 11 patients with time-matched samples, indicating that blood samples could be used for the identification of predictive biomarkers. Moreover, we observed a strong enrichment in the prevalence of CD25 expressing AML cells in patients bearing internal tandem duplication mutations in the FLT3 gene (FLT3-ITD). Furthermore, all four patients treated with a hypomethylating agent in combination with venetoclax displayed detectable levels of CD25+ AML cells. We adopted a patient-centric approach (n=14 patients) to study AML clusters with CD25 expression, due to the high inter-patient heterogeneity. Interestingly, CD25+ clusters most commonly expressed CD34, an LSC marker indicating a preferential expression in immature AML cells. Finally, ex vivo treatment of primary AML patient samples with CD25 Mab led to the specific killing of CD25+ AML cells and Tregs by allogeneic NK cells, as compared to isotype control treatment. Taken together, these results provide a proof of concept of CD25 Mab's dual mode of action in AML patient samples. Conclusions Using high dimensional flow cytometry and computational analysis, we provide a deep characterization of AML patient samples and highlight the heterogeneity in the pattern of expression of common AML targets currently under clinical investigation. We dissected the expression of CD25 on healthy T cells and malignant cells and demonstrated that the blood offers a window into the bone marrow composition and could be used to ascertain Treg and CD25+ AML cells prevalence. Mode of action studies demonstrated that CD25 Mab depletes suppressive Tregs and has a direct cytotoxic effect on the CD25+ AML cells. CD25 targeting represents an attractive target for the treatment of AML, especially in patients where CD25 is expressed on LSC or immature AML cells. Our study warrants further exploration of CD25 Mab as combinatorial treatment with, for instance, FLT3 inhibitors or venetoclax.

NPM1 Mutation Reprograms Leukemic Transcription Network Via Reshaping CTCF-Defined TAD Topology

C-terminal mutation of Nucleophosmin 1 (NPM1C+) was thought to be a primary driving event in acute myeloid leukemia (AML) that reprograms leukemic-associated transcription programs to transform hematopoietic stem and progenitor cells (HSPCs). However, it remains poorly understood how NPM1C+ mutation results in altered myeloid master transcription program and HOX expression profile leading to AML disease and whether NPM1 mutant associated transcription signature can be targeted to prevent pathogenesis of acute myeloid leukemia. Here, we report that NPM1C+ reprograms MIZ-1/MYC regulatory axis by altering NPM1-associated CTCF-driven topologically associated domains (TADs) that switches the balance of MIZ1 interaction with coactivator NPM1/p300 and corepressors MYC/G9A complexes to control cell cycle progression and myeloid lineage-specific PU.1/CEBPa transcription networks leading to impairment of myeloid differentiation and leukemic transformation. To test the hypothesis that mislocalization of the NPM1C+ mutant to cytoplasm may alter CTCF mediated three-dimensional (3D) TADs, we carried out Hi-C to examine whether NPM1C+ mutation altered hematopoietic TAD integrity by comparing mouse bone marrow (BM) Lin-cKit+ (LK) stem- and progenitor-enriched cells purified from WT and NPM1C+ knock-in animals, which develop a myeloproliferative neoplasm/myelodsplasia-like condition with a long latency characterized by enlarged spleens, leukocytosis, and thrombocytopenia. Hematopoietic-specific NPM1C+ knock-in alters TAD topology leading to altered chromatin accessibility and NPM1 mutant associated gene expression signature, which results in disrupted regulation of the cell cycle and myeloid master transcription factors, and eventually myeloid differentiation block. In contract, Retention of NPM1 in OCI-AML3 nucleus, a NPM1C+ mutant driven AML cells, reverses NPM1C+-driven signature TAD formation and gene transcription leading to proper cell cycle control and myeloid differentiation. Mechanistically, we demonstrated that cytoplasmic mislocalization of NPM1C+ mutant exerts two coordinated effects: (a) alteration of CTCF dependent TAD structure, and (b) MYC-MIZ-1 transcription axis. Both of these regulate target genes critical for cell cycle control and myeloid master transcription factors, Pu.1 and CEBPA, mediated myeloid differentiation. Re-activation of Pu.1 or CEBPa within the nucleus by CRISPR-dCas9 mediated endogenous promoter activation restores myeloid differentiation programs by reorganizing TADs critical for myeloid TFs and cell cycle regulators, and prevents NPM1C+-driven gene expression. FinalIy, when we transplanted NPM1OE, activated Pu.1, or activated CEBPA OCI-AML3 cell line or primary NPM1C+ AML patient cells into NSG mice along with control cells, NPM1 overexpression or re-activation of PU.1 or CEBPA in OCI-AML3 cells or NPM1C+ primary AML patient cells prolonged transplanted or PDX mice survival and mitigate AML leukemic blast in transplant or PDX mouse models. In sum, our data reveal that NPM1C+ reshapes CTCF-defined TAD topology to reprogram signature leukemic transcription programs required for cell cycle progression and leukemic transformation. Restoration of myeloid transcription program in nucleus reversed NPM1C+-driven transcription signature and promotes myeloid differentiation leading to mitigation of AML.

Loss of Cell Fate Determinant LLGL1 Impairs Leukemia Development in Acute Myeloid Leukemia

Cell fate determinants and polarity regulators influence the fine balance between self-renewal and differentiation in HSC and leukemic stem cells (LSC). Previously our group provided first evidence that inactivation of the cell fate regulator Llgl1 in murine HSCs leads to alterations in self-renewal, proliferative capacity and differentiation (Heidel et al., 2013). The impact of Llgl1 on malignant transformation and especially acute myeloid leukemia (AML) development had not been investigated in detail. Inactivation of LLGL1 in several human AML cell lines (MOLM13, HEL, MV4;11, HL-60) by CRISPR/Cas9 resulted in decreased proliferation of leukemic cells. Consistently, xenograft studies using LLGL1-depleted human AML cells resulted in delayed disease development compared to non-targeting control (median survival NT-control: 37.5 days vs. LLGL1-KD: 92 days; p=0.0002). Genetic inactivation of LLGL1 in primary AML patient cells resulted in reduced colony formation in vitro and improved survival of recipient mice in a patient-derived xenograft model of AML. To recapitulate these findings in defined model systems of murine AML, we used a conditional Llgl1 knockout mouse model (Klezovitch et al, 2004) and intercrossed it with AML-models harboring different driver mutations: MllAF9 (Cerral et al., 1995) or Flt3ITD/ITD (Lee et al., 2006). Conditional deletion of Llgl1 in MllAF9 knockin mice using an Mx1-Cre-recombinase (Cre+) resulted in significant delay of leukemia development (median survival +/+: 174 days; -/-: 203 days; p=0.0361). To confirm these findings, we investigated a second oncogenic model and induced deletion of Llgl1 in Flt3ITD/ITD knockin mice. Leukemic Flt3ITD/ITD; Llgl1+/+; Cre+ animals (n=12) showed rapid onset of leukemia development, whereas the Flt3ITD/ITD; Llgl1-/-; Cre+ animals (n=25) showed significant delay in leukemia progression (median survival +/+: 38 days; -/-: 51 days; p=0.0005). Llgl1-/- animals show reduced peripheral blast counts (WBC +/+: 332.558 Gpt/l; -/-: 141.083 Gpt/l; p=0.0012) and decreased organ infiltration. Importantly, immunophenotypic analysis of bone marrow (BM) compartments indicated a shift of immature cells towards a more differentiated state. The overall abundance of leukemic Lin-Kit+Sca-1+ (LSK) cells was reduced in Flt3ITD/ITD; Llgl1-/- animals compared to Flt3ITD/ITD; Llgl1+/+ controls (LSK +/+: 13099±3144/106 BMC; -/-: 8117±2810 /106 BMC; p=0.0227). In contrast, we found increased numbers of Lin-Kit+Sca-1+FcgR+CD34+ of leukemic granulocyte-macrophage progenitors (LGMPs: +/+: 8306±1963/106 BMC; -/-: 14755±4783/106 BMC; p=0.0141) upon deletion of Llgl1 in Flt3ITD/ITD animals. These findings indicate that genetic inactivation of Llgl1 results in loss of leukemia stemness and shifts the leukemia stem cell phenotype from HSC-like to GMP-like LSCs. To test for this hypothesis, we performed global transcriptome analysis of sorted LSK cells of Flt3ITD/ITD mice early after genetic deletion of Llgl1. Among the significantly downregulated genes, we found several members of the HoxA-cluster, consistent with the immunophenotypic loss of stemness. Gene-set-enrichment analysis revealed downregulation of leukemia stem cell signatures (Gal et al., 2006) and enrichment of a gene set associated with LGMPs (compared to leukemic-HSC) signatures (Krivtsov et al., 2006). To validate whether loss of Hox-gene expression is relevant for impaired leukemia development, we performed rescue experiments in vivo. LSK cells derived from Flt3ITD/ITD; Llgl1+/+ or Flt3ITD/ITD; Llgl1-/- animals were retrovirally transduced with HoxA9 or empty vector control (eV). When transplanted into sublethally irradiated recipient mice, overexpression of HoxA9 (OE) in Llgl1-/-; Flt3ITD/ITD cells rescued the disease phenotype comparable to Flt3ITD/ITD; Llgl1+/+ cells (median survival +/+;eV: 62.50%; -/-; OE: 55.56%). In contrast, empty vector control Flt3ITD/ITD; Llgl1-/- recipients showed no disease development (median survival -/-; eV: 100%). Taken together our results may provide first evidence for a functional role of Llgl1 in models of AML. In contrast to its function in normal HSCs, deletion of Llgl1 appears to reduce stemness of leukemic stem cells and impair leukemia development in vitro and in vivo. Experiments to assess for the effects of Llgl1 on LSC polarity and cell fate decisions are currently under way.

Data-Driven Molecular Hallmarks of Acute Myeloid Leukemia: Biological, Prognostic and Therapeutic Implications

The cancer hallmark concept defines the biological processes that contribute to cancer development and progression. While the hallmarks represent an important theoretical framework, they reflect generic properties of all cancer types and hence most hallmarks are not measurable nor applicable to guiding diagnosis or therapy. Here, we carried out deep multi-omic profiling (DNA panel sequencing, , transcriptome, DNA methylation and proteome) and functional testing of responses to 525 drugs in 118 primary acute myeloid leukemia (AML). In addition, 47 primary AML patients were used as an independent validation cohort, including measurements by single-cell assays. Using unsupervised multi-omic dimensionality reduction, we defined 11 independent axes of biological variability that we considered here to reflect the data-driven hallmarks (DDHM) of AML. We defined the molecular features that contributed most to each hallmark, hence forming an understanding of the biological nature and pathways of each DDHM We hypothesized that the 11 DDHMs define biologically and clinically important features of AML applicable to diagnostics, therapy and precision medicine. For example, each patient had a unique composition of the DDHMs that reflected the AML biology of that patient case. Each DDHM was associated with distinct AML biology, including NPM1 mutation, transcriptional deregulation, stem cell properties, cell cycle, stromal signals, HOX signaling, and stress response. The DDHMs captured both biology that is already well known in AML, but with multi-omic resolution, and highlighted also novel, previously poorly understood, properties of AML. Each DDHM included a ranked list of drugs that explained the biology reflected in the hallmarks. For instance, the DDHM2 that defined sensitivity to Bcl-2 (Venetoclax) and MEK inhibitors highlighted myeloid cell differentiation patterns. Moreover, DDHM8 was strongly associated with patient prognosis, a higher response to hypomethylating agents and complete remission,. Combining this prognostic DDHM8 with hallmark DDHM1 that explained the omics of the NPM1 mutation landscape, captured the ELN risk classification and other prognostic cell surface markers. This revealed that several previously independently defined biomarkers of prognosis converge in the multi-omic paradigms defined by our DDHMs. In summary, we have defined through multi-omics and functional analysis a set of eleven data-driven hallmarks of AML. These capture some of the most important known prognostic and therapeutic biomarkers in AML but also highlight opportunities for novel biomarker discovery and drug repurposing. This approach provides a new quantitative and measurable framework to understand the biological heterogeneity of AML and exploit it for diagnosis, prognosis and precision therapy.

DIACC2010, Sole-in-Class Selective Inhibitor of Kinesin KIF20A, Has Potent Preclinical Efficacy in Acute Myeloid Leukemia

Context: Despite recent therapeutic advances in Acute Myeloid Leukemia (AML) and the decent proportion of complete remission after induction chemotherapy, relapse rate is still high and long-term survival does not exceed 20%. The therapeutic arsenal of AML could benefit from differentiated strategies. Mitotic kinesins are essential regulators of cancer cell replication and migration. The microtubule associated motor protein KIF20A (also called MKlp2), a member of the kinesin-6 family, plays an essential role during cytokinesis and is also involved in the fission of RAB6-positive vesicles from Golgi membrane, therefore contributing to intracellular vesicular trafficking. KIF20A plays a critical role in the development and progression of many cancers, and its high expression is associated with disease progression and poor survival outcome. More specifically, immature hematopoietic cells, exhibit high KIF20A expression, whereas mature peripheral blood cells do not. Herein we describe the preclinical anti-leukemic efficacy of DIACC2010, sole-in-class selective KIF20A inhibitor. Methods: We used cell lines (U-937, KG-1, CEM, KASUMI, SKM-1, MOLM-14, HL-60, MV-4-11, THP1) provided by ATCC and a Cell line-Derived Xenograft models to demonstrate the anti-leukemic activity od DIAC2010. Briefly, NOD.Cg-Prkdc scid/J (NSG) mice were obtained from Charles River France and maintained under specific pathogen-free conditions. Animals were injected via tail vein with 0.2 106 viable human leukemia cells. Mice were treated with 0.9% NaCl or Diacc2010 (200 mg/kg IV; once a week). Daily monitoring of mice for symptoms of disease determined the time of killing for animals with signs of distress. In addition, primary AML samples were obtained after informed consent. Cells were transplanted to NSG mice, collected after the 3rd transplantation and drug tests were performed ex vivo. All animal experiments were performed in compliance with the laws and in agreement with the French Guidelines for animal handling and approved by animal ethics committees (Agreement no. APAFIS#6743). Golgi imaging was performed using anti-GM130 antibody. Cell cycle, Caspase-Glo 3/7 and Annexin V/PI tests were performed according to the manufacturer's protocols. Results: DIACC2010 demonstrated potent and consistent cytotoxic activity in vitro against a panel of 9 human AML cell lines, with median IC50 of 40 nM (range 10-77 nM). In the same experimental conditions, cytarabine (CYTA) had median IC50 of 207 nM (range 4-1580 nM). DIACC2010 was subsequently evaluated in vivo in xenograft models of AML cell lines. DIACC2010 significantly improved overall survival as compared to CYTA in all models, with dose-dependent efficacy as shown in Figure A ( MOLM14 cells. Next, DIACC2010 cytotoxic activity was evaluated in vitro on 8 primary AML patient samples, IC50 ranged from 57 nM to 728 nM across the tested samples. DIACC2010 antitumoral activity is currently evaluated in Patients Derived Xenograft model. In parallel, no in vitro toxicity of DIACC2010 was observed on human normal cells such as peripheral blood mononuclear cells (PBMC) and primary hepatocytes (IC50 >50 µM). On a mechanistic standpoint, DIACC2010-exposed AML cells displayed characteristic Golgi scattering, observed within 1 hour of treatment and majoring at 6 hours confirming target engagement (Figure B). In parallel, we observed a strong block in cell cycle in G2/M after 6 hours of treatment (45% of cells versus 21% in the control condition). This was associated with a high caspase activation that occurred mainly after 24 hours of treatment. This cascade led to cell death assessed by annexin V/PI between 24 and 48 hours after treatment initiation. Nevertheless, caspase inhibitors ZVAD-fmk and Emricasan partially rescued DIACC2010 cell death, although these inhibitors significantly rescued caspase activation induced by Venetoclax, a BCL-2 inhibitor triggering intrinsic apoptosis commonly used in AML treatment. We ruled out necroptosis and ferroptosis as cell death pathways triggered by DIACC2010 using pharmacological inhibitors. These data strongly suggests that DIACC2010 induces a non-redundant cell death pathway that could be advantageously used to kill resistant AML cells. Conclusion: Altogether, these results confirm the relevance of KIF20A-directed therapeutic approaches and support the development of DIACC2010 for the treatment of AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Activating a Non-Genetic MYC-Regulating Pathway Sensitizes Acute Myeloid Leukemia to Bromodomain Inhibition

MYC (c-Myc) plays a central role in the oncogenic progression and is commonly overexpressed or amplified in more than 50% of human cancers, including acute myeloid leukemia (AML), which is an aggressive disease with over 70% of patients who cannot survive more than five years. Elevated MYC expression is associated with therapeutic resistance and predicts a poorer outcome in AML patients. While numerous lines of evidence have validated MYC as a therapeutic target of AML and many other cancer types, MYC protein itself has been a challenge for therapeutic targeting. Recently, MYC gene transcription has been linked to the bromodomain and extra-terminal domain (BET) family proteins. BET inhibitors (BETi) inhibit MYC expression and have shown promising antitumor activities in preclinical models and are being evaluated in clinical trials. Despite the therapeutic promise, BETi-associated drug resistance and dose-limiting side effects hinder the clinical applications. Therefore, there is a pressing need to understand the mechanisms controlling drug response and resistance to BET inhibitors. To understand the mechanisms that regulate BETi sensitivity, we leveraged a functional CRISPR screen focusing on about 1,700 human transcription factors. We discovered the aryl hydrocarbon receptor (AHR) as a novel regulator of BETi sensitivity in AML. Our results have further identified that a novel AHR-ELMSAN1 axis is critically involved in regulating MYC and BETi sensitivity. Specifically, we show that constitutive or ligand activation of AHR sensitizes leukemia cells to BETi. AHR signaling represses MYC and synergizes with BETi for maximum inhibition of MYC transcription. Ectopic expression of MYC could rescue the anti-leukemia effects induced by combined AHR activation and BET blockade. Mechanistically, our unbiased bioinformatic analysis unexpectedly identified that AHR functions through activating a non-canonical target, ELMSAN1, a less-studied epigenetic factor known as a member of the MiDAC histone deacetylase complex. Depletion of ELMSAN1 leads to upregulation of MYC and blunts AHR signaling-mediated MYC repression and BETi sensitization. Importantly, we demonstrate that AHR agonist and BETi synergistically inhibit MYC and leukemia cell growth in CD34+ primary AML patient samples in vitro and patient-derived xenografts in vivo. In summary, our data suggest that AHR-ELMSAN1 is a novel pathway for MYC repression, activation of which could sensitize AML to BET-targeting agents. As neither AHR nor ELMSAN1 is found mutated in AML, our research may open a new door to new MYC targeting paradigms by activating novel non-genetic MYC repressors.

Efficacy of venetoclax combined azacitidine in newly diagnosed acute myeloid leukemia unfit for standard chemotherapy: a single center experience

Objective: To investigate the effectiveness and safety of the VA regimen, which combines venetoclax with azacitidine in the treatment of patients with newly diagnosed acute myeloid leukemia (AML) who are not suitable candidates for conventional chemotherapy. Methods: In the Department of Hematology at the Second Hospital of Hebei Medical University, 66 AML patients who received venetoclax and azacitidine treatment from May 2020 to March 2022 were the subject of a retrospective study. The complete remission (CR) rate, cCR rate, ORR rate, MRD negative rate, the incidence of adverse events,1-year EFS, and OS were retrospectively analyzed. Patients subgroups with varying ages, ECOG scores, primary and secondary, risk stratifications, and gene mutation were compared for differences in efficacy and survival. Results: The median follow-up was 4.25 (0.9-19.9) months, and the median number of treatment courses was 2 (1-8) cycles. After the first cycle, the cCR rate was 78.8% , and the MRD negative rate was 51.9% . After prolonged treatment, the cCR rate was 81.8% and MRD negative rate was 66.7% . The median EFS and OS, respectively, were13.2 and 15.3 months. Secondary AML showed inferior efficacy and prognosis. IDH1/2 or NPM1 mutation groups had a significantly higher rate of CR than the control group (P<0.05) . The CR rate and MRD negative rate of patients with rebound thrombocytosis were significantly higher than those without rebound thrombocytosis (P<0.05) . Those who had epigenetic modification mutations (DNMT3, ASXL1, TET2) were more likely to benefit from ongoing therapy. The most common grade 3 and 4 adverse reactions were neutropenia, thrombocytopenia, and anemia. Conclusions: In real-world patients with newly diagnosed AML who are not candidates for standard chemotherapy, the VA regimen produces rapid deep remission. Primary AML patients, rebound thrombocytosis, IDH1/2, and NPM1 gene mutations are favorable factors for treatment benefit, and adverse reactions were tolerable.

Phosphoproteomics of primary AML patient samples reveals rationale for AKT combination therapy and p53 context to overcome selinexor resistance.

SummaryAcute myeloid leukemia (AML) is a heterogeneous disease with variable patient responses to therapy. Selinexor, an inhibitor of nuclear export, has shown promising clinical activity for AML. To identify the molecular context for monotherapy sensitivity as well as rational drug combinations, we profile selinexor signaling responses using phosphoproteomics in primary AML patient samples and cell lines. Functional phosphosite scoring reveals that p53 function is required for selinexor sensitivity consistent with enhanced efficacy of selinexor in combination with the MDM2 inhibitor nutlin-3a. Moreover, combining selinexor with the AKT inhibitor MK-2206 overcomes dysregulated AKT-FOXO3 signaling in resistant cells, resulting in synergistic anti-proliferative effects. Using high-throughput spatial proteomics to profile subcellular compartments, we measure global proteome and phospho-proteome dynamics, providing direct evidence of nuclear translocation of FOXO3 upon combination treatment. Our data demonstrate the potential of phosphoproteomics and functional phosphorylation site scoring to successfully pinpoint key targetable signaling hubs for rational drug combinations.

Abstract 3338: Concurrent treatment with Pim kinase inhibitor enhances efficacy of FLT3 inhibitors in AML with FLT3-ITD through GSK-3βactivation and GSK-3β-mediated enhanced proteasomal degradation of c-Myc and Mcl-1

Abstract BACKGROUND: fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in acute myeloid leukemia (AML) cells in 30% of patients, with poor patient outcomes. FLT3 inhibitors are in clinical use, but with incomplete responses and onset of resistance. New targeting strategies are needed. The oncogenic serine/threonine kinase proviral integration site for Moloney murine leukemia virus (Pim-1) is upregulated downstream of FLT3-ITD, directly stimulates cell growth and inhibits apoptosis, and also phosphorylates and stabilizes FLT3 in a positive feedback loop. We previously showed that concurrent treatment of FLT3-ITD cells with Pim kinase and FLT3 inhibitors enhanced apoptosis induction through post-translational downregulation of the master transcription factor c-Myc and the anti-apoptotic protein Mcl-1. The serine/threonine kinase GSK-3β regulates c-Myc and Mcl-1 protein via phosphorylation at T58 and S159, respectively; we hypothesized downregulation of both proteins by combination treatment through phosphorylation by GSK-3β. METHODS: FLT3-ITD cells lines (Ba/F3-ITD, MV4-11, and MOLM-14) and primary FLT3-ITD AML patient blasts were cultured with the pan-Pim kinase inhibitor AZD1208, the FLT3 inhibitors gilteritinib or quizartinib and the GSK-3β inhibitor TCG-24. Protein expression was measured by immunoblotting. To measure protein half-lives, cells were pretreated with cycloheximide with and without the proteasome inhibitor MG-132. Ba/F3-ITD cells were infected with Myc-T58A plasmid, preventing c-Myc T58 phosphorylation, or Mcl-1 S159A plasmid, preventing Mcl-1 S159 phosphorylation, or empty vector controls. Apoptosis was measured by Annexin V labeling, measured by flow cytometry. RESULTS: Combined treatment of FLT3-ITD-expressing cell lines and AML patient blasts with Pim and FLT3 inhibitors caused rapid downregulation of c-Myc, and then Mcl-1, protein expression, relative to single drugs. Combined Pim and FLT3 inhibitor treatment caused marked decrease in half-lives of both proteins, which was rescued by proteasome inhibition, as the mechanism of protein downregulation. Combined Pim and FLT3 inhibitor treatment rapidly activated (dephosphorylated) GSK-3β and culture with GSK-3β inhibitor prevented induction of c-Myc and Mcl-1 downregulation, increased c-Myc and Mcl-1 protein turnover by concurrent Pim and FLT3 inhibitor treatment. Finally, combination treatment of Ba/F3-ITD cells infected with T58A c-Myc or S159A Mcl-1 plasmids did not downregulate these proteins or increase their turnover, with reduced apoptosis induction. CONCLUSION: Pim inhibitors enhance efficacy of FLT3 inhibitors via GSK-3β activation and GSK-3β-mediated enhanced proteasomal degradation of c-Myc and Mcl-1, highlighting GSK-3β as a key target in cells with FLT3-ITD. Citation Format: Jonelle Lee, Mario Scarpa, Aditi Chatterjee, Mooath Mustafa Ali, Prerna Singh, Shivani Kapoor, Rossana Trotta, Maria Baer. Concurrent treatment with Pim kinase inhibitor enhances efficacy of FLT3 inhibitors in AML with FLT3-ITD through GSK-3βactivation and GSK-3β-mediated enhanced proteasomal degradation of c-Myc and Mcl-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3338.

Integrated multiomic approach for identification of novel immunotherapeutic targets in AML

BackgroundImmunotherapy of acute myeloid leukemia has experienced considerable advances, however novel target antigens continue to be sought after. To this end, unbiased approaches for surface protein detection are limited and integration with other data types, such as gene expression and somatic mutational burden, are poorly utilized. The Cell Surface Capture technology provides an unbiased, discovery-driven approach to map the surface proteins on cells of interest. Yet, direct utilization of primary patient samples has been limited by the considerable number of viable cells needed.MethodsHere, we optimized the Cell Surface Capture protocol to enable direct interrogation of primary patient samples and applied our optimized protocol to a set of samples from patients with acute myeloid leukemia (AML) to generate the AML surfaceome. We then further curated this AML surfaceome to exclude antigens expressed on healthy tissues and integrated mutational burden data from hematologic cancers to further enrich for targets which are likely to be essential to leukemia biology. Finally, we validated our findings in a separate cohort of AML patient samples.ResultsOur protocol modifications allowed us to double the yield in identified proteins and increased the specificity from 54 to 80.4% compared to previous approaches. Using primary AML patient samples, we were able to identify a total of 621 surface proteins comprising the AML surfaceome. We integrated this data with gene expression and mutational burden data to curate a set of robust putative target antigens. Seventy-six proteins were selected as potential candidates for further investigation of which we validated the most promising novel candidate markers, and identified CD148, ITGA4 and Integrin beta-7 as promising targets in AML. Integrin beta-7 showed the most promising combination of expression in patient AML samples, and low or absent expression on healthy hematopoietic tissue.ConclusionTaken together, we demonstrate the feasibility of a highly optimized surfaceome detection method to interrogate the entire AML surfaceome directly from primary patient samples and integrate this data with gene expression and mutational burden data to achieve a robust, multiomic target identification platform. This approach has the potential to accelerate the unbiased target identification for immunotherapy of AML.

BTX-1188, a first-in-class dual degrader of GSPT1 and IKZF1/3, for treatment of acute myeloid leukemia (AML) and solid tumors.

7025 Background: BTX-1188 is a first-in-class oral molecular glue that degrades GSPT1 and IKZF1/3 and is currently in phase 1 clinical trials for treatment of hematologic and solid malignancies. Targeted protein degradation of cereblon neosubstrates is clinically validated in the treatment (tx) of hematologic malignancies (Lu 2014, Zou 2020). Methods: Cell viability in BTX-1188-treated cells and patient samples was measured by CellTiter-Glo 2.0 assay (Promega). Substrate degradation and apoptosis profiles were analyzed by immunoblots of protein lysates from cells treated with DMSO or BTX-1188. Vehicle, or 30 or 40 mg/kg IP BTX-1188, was used in athymic nude mice AML xenograft models. Results: BTX-1188 is a rapid, deep, and potent degrader of GSPT1 and IKZF1/3 and inhibitor of Myc in several cancer cell lines (Table). Proteomics and immunoblot analysis of AML cell line, MV-4-11, shows significant degradation of GSPT1 and IKZF1 after 2 h tx with 100 nM BTX-1188 ( P&lt;1x10-5) and 6 h tx with 3 nM BTX-1188 (&gt;90% of GSPT1), respectively, indicating rapid and potent neosubstrate degradation. BTX-1188 also durably degrades GSPT1 where tx with 30 nM for 6 h followed by washout maintains significantly lower levels of GSPT1 and sustained apoptosis for up to 24 h. Owing to IKZF1/3 degradation, BTX-1188 has immunomodulatory properties as seen by inhibition of proinflammatory cytokines (IL-1β, IL-6, TNFα) and induction of IL-2 by LPS and αCD3-stimulated PBMCs, respectively. This approach is expected to improve clinical outcomes and reduce toxicities associated with pure GSPT1 degradation (CC-90009), thus expanding the therapeutic window of BTX-1188. Functionally, BTX-1188 is cytotoxic in various cancer cell lines such as Myc-driven lines (IC50 range: 0.5-10 nM) and primary human AML patient samples (IC50 range: 0.4-1.5 nM), including relapsed/refractory-, cytarabine- and venetoclax-resistant samples. The durability of GSPT1 degradation and sustained apoptosis in response to BTX-1188 tx is further reflected in in vivo efficacy models where daily or intermittent dosing of BTX-1188 results in potent and sustained antitumor activity. Conclusions: These preclinical data show that BTX-1188 is a promising drug candidate for AML and other tumor types. Its immunomodulatory properties owing to IKZF1/3 degradation may prevent systemic inflammatory dose-limiting toxicities associated with pure GSPT1 degradation (Uy 2019). BTX-1188 has entered phase 1 clinical studies for advanced solid tumors and AML. DC50 (nM) or inhibitory concentration (nM) of BTX-1188 at 6 h of Tx. [Table: see text]

Emvododstat, a Potent Dihydroorotate Dehydrogenase Inhibitor, Is Effective in Preclinical Models of Acute Myeloid Leukemia.

Blocking the pyrimidine nucleotide de novo synthesis pathway by inhibiting dihydroorotate dehydrogenase (DHODH) results in the cell cycle arrest and/or differentiation of rapidly proliferating cells including activated lymphocytes, cancer cells, or virally infected cells. Emvododstat (PTC299) is an orally bioavailable small molecule that inhibits DHODH. We evaluated the potential for emvododstat to inhibit the progression of acute myeloid leukemia (AML) using several in vitro and in vivo models of the disease. Broad potent activity was demonstrated against multiple AML cell lines, AML blasts cultured ex vivo from patient blood samples, and AML tumor models including patient-derived xenograft models. Emvododstat induced differentiation, cytotoxicity, or both in primary AML patient blasts cultured ex vivo with 8 of 10 samples showing sensitivity. AML cells with diverse driver mutations were sensitive, suggesting the potential of emvododstat for broad therapeutic application. AML cell lines that are not sensitive to emvododstat are likely to be more reliant on the salvage pathway than on de novo synthesis of pyrimidine nucleotides. Pharmacokinetic experiments in rhesus monkeys demonstrated that emvododstat levels rose rapidly after oral administration, peaking about 2 hours post-dosing. This was associated with an increase in the levels of dihydroorotate (DHO), the substrate for DHODH, within 2 hours of dosing indicating that DHODH inhibition is rapid. DHO levels declined as drug levels declined, consistent with the reversibility of DHODH inhibition by emvododstat. These preclinical findings provide a rationale for clinical evaluation of emvododstat in an ongoing Phase 1 study of patients with relapsed/refractory acute leukemias.

3221 – INVESTIGATING THE ROLE OF UBIQUITIN E3 LIGASE, FBXO21, IN THE REGULATION OF CYTOKINE SIGNALING WITHIN ACUTE MYELOID LEUKEMIA

Hematopoiesis is tightly regulated through various molecular mechanisms, including the ubiquitin proteasome system (UPS). Dysregulation can lead to malignancies, including acute myeloid leukemia (AML). AML is characterized by an abnormal proliferation of immature myeloid cells. F-box ubiquitin E3 ligase, FBXO21, is differentially expressed in both normal and malignant hematopoiesis. FBXO21 has low expression in AML, but high expression correlates to decreased survival in AML patients. At the protein level, AML patients with high blast counts have increased expression of FBXO21, suggesting FBXO21 may play a role in leukemic stem cells (LSCs) to promote progression and survival of AML. This paradoxical relationship between FBXO21 expression and patient survival highlights a major gap in knowledge and the necessity of further studies to understand the role of FBXO21 in AML. We have found that silencing of FBXO21 in AML cell lines and primary AML patient samples leads to increased apoptosis, differentiation, and decreased proliferation. Furthermore, silencing of FBXO21 leads to up-regulation of inflammatory cytokines/chemokines and modulation of MAP kinase pathways, which are known to be involved in the activation of many cytokine signaling pathways. We have identified up-regulation of CXCL10 at both the RNA and protein level, which is known to initiate T-cell recruitment and promote anti-tumor activity within the AML microenvironment. These findings lead us to hypothesize that FBXO21 regulates the maintenance and progression of AML through MAP kinase mediated cytokine signaling pathways. Regulation of cytokines and chemokines play a key role in the progression and maintenance of AML, suggesting FBXO21 may impact disease progression and survival.

The clinical characteristics and prognosis of Chinese acute myeloid leukemia patients with CSF3R mutations.

The colony-stimulating factor 3 receptor (CSF3R) controls the proliferation of myeloid progenitors and differentiation into neutrophils. However, the clinical features and prognostic significance of CSF3R mutations in primary acute myeloid leukemia (AML) patients are still unclear. 158 newly diagnosed AML patients were retrospectively evaluated in our study. Amplicon-based next-generation sequencing (NGS) and multiplex-nested reverse-transcription polymerase chain reaction (RT-PCR) were used to investigate the 34 genes and 43 fusion genes associated with leukemia. In addition, clinical features, mutation incidence, and survival outcomes were compared between patients with CSF3R mutation and patients with wild-type CSF3R. In our study, CSF3R mutations were found in 7.6% (12/158) cases. The membrane-proximal amino acid substitution T618I (58.3%) was the most frequent mutation. CSF3R mutations were associated with higher WBC counts (P=.035). CEBPA mutation, TET2 mutation, and RUNX1-RUNX1T1 translocation were the most common co-mutations of CSF3R. The CSF3R gene was mutually exclusive with signal transduction genes (P=.029), while positively associated with TET2 mutations (P=.014). CSF3R mutations had no effect on CR1 (P=.935), R (P=.625) and OS (P=.1172). Patients with CSF3R mutations had a worse DFS (P=.0352) than those with wild-type CSF3R. Multivariate survival analysis showed that CSF3R mutation was an independent risk factor for DFS of primary AML patients (HR=2.048, 95%CI: 1.006-4.170, P=.048). AML patients with CSF3R mutations had unique clinical features and gene co-mutation spectrum. CSF3R mutation was an independent risk factor for DFS and could be a potential prognostic marker and therapeutic target for Chinese primary AML patients.

Pembrolizumab in Combination with Antigen-Specific Cytotoxic T Lymphocytes Enhances Killing of Acute Myeloid Leukemia

BackgroundPembrolizumab, an antibody that blocks programmed cell death protein 1 (PD-1), has been FDA approved for several solid tumors and hematologic malignancies. Currently, pembrolizumab is under investigation for acute myeloid leukemia (AML) in combination with hypomethylating agents. It is established that AML is highly responsive to immunotherapy, as seen with the anti-leukemic effect of allogeneic hematopoietic stem cell transplantation (alloHSCT). However, response to cytotoxic T lymphocytes (CTLs) that target leukemia-associated antigens (LAAs) has been less reliable in eradicating disease. This insufficient response to LAA-specific CTLs is likely partially accounted for by the immune dysregulation seen in AML. Because of promising murine data that blockade of the PD1/PD-L1 pathway enhances the graft versus leukemia effect of alloHSCT, we investigated if adding pembrolizumab to CTLs that target the two LAAs CG1 and PR1 will enhance CTL antileukemia activities. CG1 and PR1are two HLA-A2 restricted nonameric peptides that we validated as promising AML targets derived from cathepsin G (i.e. CG1), and proteinase 3 (P3) and neutrophil elastase (NE) (i.e. PR1). We hypothesized that pembrolizumab added to CG1-CTLs and PR1-CTLs, will enhance their anti-leukemic effects with minimal off target toxicities.MethodsUsing a standard calcein AM in vitro cytotoxicity assay, we co-cultured AML targets, including U937 HLA-A2 + (U937-A2) AML cell line and primary patient HLA-A2 + AML samples, with CG1-CTL and PR1-CTL. AML cells were loaded with calcein AM and then incubated with CG1- and PR1-CTLs at increasing effector to target ratios. Pembrolizumab or isotype antibody were added to the cultures. After 4 hours, calcein AM was measured to determine cell viability. T2 cells pulsed with PR1 or CG1, were used as a positive control and non-pulsed T2 cells were used as negative control cells. For in vivo experiments, we used a human established AML xenograft mouse treatment model to determine the effect of pembrolizumab when added to CG1- or PR1- CTL in vivo. CG1-CTL and PR1-CTLs were expanded from the same donor and used as the effector cells. NOD/SCID gamma (NSG) mice (4-6-week-old females) were engrafted with AML samples by tail vein injection. After confirming engraftment, CG1- and/or PR1-CTL were administered to mice and pembrolizumab or isotype antibody [100ug/mouse] were given three times over two weeks. Mice were monitored for clinical GVHD and AML three times/week. Mice were sacrificed at approximately two weeks following treatment. To assess for GVHD, mouse tissues including spleen, liver, kidney, BM, intestine, brain, heart, and lung were harvested and fixed in 10% formalin. The fixed tissue samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin prior to histologic examination. Bone marrow (BM) was processed using standard methodology and analyzed for residual AML by flow cytometry.Results:In vitro our data demonstrate that U937-A2 and two primary patient HLA-A2 AML samples, have enhanced cell lysis when treated with pembrolizumab (CTL+ pembrolizumab) in comparison with isotype (Iso) + CTL, pembrolizumab only, iso only or CTL only groups (Figure 1). In vivo data show a decrease in U937-A2 disease burden and primary patient AML after treatment with CG1and/or PR1-CTL (CTL-treated) . This decrease was enhanced when pembrolizumab was added to the CTL-treated mice (CTL+ pembrolizumab) in comparison with mice treated with pembrolizumab only (pembrolizumab-treated), or CTL only (CTL-treated) (Figure 2). Pathology data to assess toxicity in mice treated with CTL +/- pembrolizumab showed an enhanced pulmonary (perivascular and parabronchial) lymphocytic infiltration in mice treated with the combination of CTL and pembrolizumab. The other organs investigated, showed no change when combination therapy was used.Conclusion:We have validated in vitro and in vivo the enhanced killing of AML (cell lines and primary patient samples) by CG1-CTL and/or PR1-CTL after addition of pembrolizumab. Toxicity data show mild enhancement of lymphocytic infiltrate in the lungs after addition of pembrolizumab to CTL. Our data suggest that the strategy of combining LAA-specific CTL with immune checkpoint blockade could prove beneficial in the setting of adoptive T cell therapy and allogeneic stem cell transplantation for AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tolerability and Single Agent Anti-Neoplastic Activity of the CD3xCD123 Bispecific Antibody APVO436 in Patients with Relapsed/Refractory AML or MDS

Tolerability and Single Agent Anti-Neoplastic Activity of the CD3xCD123 Bispecific Antibody APVO436 in Patients with Relapsed/Refractory AML or MDS

Pim Kinase Inhibitor Enhances FLT3 Inhibitor Efficacy through GSK-3β Activation and GSK-3β-Mediated Proteasomal Degradation of c-Myc

BACKGROUNDInternal tandem duplication in the fms-like tyrosine kinase 3 receptor tyrosine kinase (FLT3-ITD) is present in acute myeloid leukemia (AML) in 30% of patients, associated with poor treatment outcomes due to rapid relapse. FLT3 inhibitors are used in the clinic, but with incomplete efficacy and development of resistance. Further treatment options are needed. The serine/threonine kinase proviral integration site for Moloney murine leukemia virus (Pim-1) is upregulated downstream of FLT3-ITD; it directly stimulates cell growth and inhibits apoptosis, and also phosphorylates and stabilizes FLT3 in a positive feedback loop in cells with FLT3-ITD. Dual targeting of Pim-1 and FLT3 is a promising treatment strategy. The c-Myc transcription factor contributes to dysregulation of cell growth and apoptosis in cancers, including AML. In addition to transcriptional regulation, c-Myc is regulated post-translationally by T58 phosphorylation by the serine/threonine kinase glycogen synthase kinase-3- β (GSK-3β). Here we show that concurrent treatment of cells with FLT3-ITD with Pim and FLT3 inhibitors activates GSK-3β, which phosphorylates and post-translationally downregulates c-Myc.METHODSBa/F3-ITD and MV4-11 cells, with FLT3-ITD, and FLT3-ITD AML patient blasts were cultured with the pan-Pim inhibitor AZD1208 (1 μM) and/or the FLT3 inhibitors gilteritinib or quizartinib (15 nM, 1 nM), with and without the GSK-3β inhibitor TCG-24 (20 μM). c-Myc, p-GSK3-α/β (S21/9) and GSK3-α/β protein expression was measured by immunoblotting. c-Myc mRNA was measured by qPCR. Cells were also cultured with cycloheximide (100 μg/mL) with and without the proteasome inhibitor MG-132 (20 μM) to measure protein half-life and proteasomal degradation. To study the role of c-Myc overexpression and activation, Ba/F3-ITD cells were infected with retroviral estrogen receptor (ER)-Myc plasmid, causing c-Myc nuclear translocation when activated by 4-hydroxytamoxifen (4-OHT; 300 nM). To study the role of c-Myc phosphorylation at T58, Ba/F3-ITD cells were infected with MycT58A plasmid, preventing c-Myc phosphorylation at T58. Apoptosis was detected by Annexin V and propidium iodide staining, measured by flow cytometry.RESULTSTreatment with Pim inhibitor AZD1208 and FLT3 inhibitor gilteritinib or quizartinib combination rapidly downregulated c-Myc protein expression in Ba/F3-ITD and MV4-11 cells, with FLT3-ITD, and in primary FLT3-ITD AML patient blasts, compared to quizartinib or gilteritinib alone. Pim inhibitor and FLT3 inhibitor combination treatment did not decrease c-Myc mRNA levels, but markedly decreased c-Myc protein half-life, from 36 mins without drugs and 24 mins with gilteritinib to 18 mins with combination. Half-life did not decrease when cells were pre-treated with the proteasome inhibitor MG-132, consistent with post-translational downregulation through proteasomal degradation. Apoptosis induction by Pim inhibitor and FLT3 inhibitor combination decreased by more than 50% in Ba/F3-ITD cells infected with ER-Myc plasmid and treated with 4-OHT, demonstrating the major role of c-Myc downregulation in apoptosis induction by combination treatment. GSK-3b is inactivated by phosphorylation, and combination treatment rapidly decreased p-GSK-3b levels, while total GSK-3b levels were unchanged, indicating activation of GSK-3b. Treatment of cells with FLT3-ITD with the GSK-3b inhibitor TCG-24 in addition to Pim and FLT3 inhibitors abrogated c-Myc protein downregulation, demonstrating that Pim and FLT3 inhibitor combination downregulates c-Myc through activation of GSK-3b. Finally, Pim and FLT3 inhibitor combination treatment did not downregulate c-Myc in Ba/F3-ITD cells transfected with c-Myc T58A, preventing c-Myc phosphorylation at T58, showing that c-Myc phosphorylation at T58 is necessary for its downregulation by combination treatment.CONCLUSIONSConcurrent treatment of cells with FLT3-ITD with Pim kinase inhibitor enhances the efficacy of FLT3 inhibitors through activation of GSK-3β and GSK-3β-mediated phosphorylation of c-Myc at T58, with resulting c-Myc downregulation through increased proteasomal degradation. This work and previous work in our laboratory on PP2A activating drugs and FLT3 inhibitor combination (Mol Cancer Ther 20:676, 2021) support GSK-3β activation as a mechanism for enhancing efficacy of FLT3 inhibitors in AML with FLT3-ITD. DisclosuresNo relevant conflicts of interest to declare.

Efficient Human Acute Myeloid Leukemia Targeting By Universal Chimeric Antigen Receptor T-Cells Via Combinatorial Use of Linkers

Chimeric antigen receptor (CAR) T-cells are genetically engineered T-cells with potent biocidal activity against respective target-expressing cells. Recently, CAR T-cells have been successfully used clinically to eradicate B cell-derived malignancies by targeting B-cell lineage specific surface antigens (e.g. CD19, BCMA). However, several limitations of current single-antigen targeting CAR T-cell therapies are becoming evident: a) low target tumor-antigen expression might lead to low CAR T-cell targeting efficacy; b) single antigen-targeting might lead to rapid selection of tumor cells with low or loss of antigen expression; c) single antigen-targeting does frequently not generate a high tumor-selectivity as tumor-antigens are frequently also expressed on healthy tissues; d) production of single-antigen CAR T-cells is time- and resource-consuming but results in effectors that target only one antigen; e) on-target off-tumor as well as off-target side-effects are difficult to control without terminally eliminating CAR T-cells in the recipient. While a-c) might be overcome by combinatorial tumor-antigen targeting, d-e) might be addressed by production of Universal CAR T-cells that recognize a specific tag on selective bridging molecules with short half-life.To address some of these limitations in principle, we have here developed universal CAR T-cells targeting fluorescein, as well as fluorescein-labeled antibody-constructs directed against several cell surface antigens, that would serve as versatile, combinatorial selective linkers and, upon target binding, also CAR T-cells activators. We then tested the system on human acute myeloid leukemia (AML) cell lines and primary patient AML blasts. Specifically, we engineered CAR T-cells, termed FluA-CAR T-cells, to display the anti-fluorescein engineered lipocalin FluA, which mediated recognition of randomly or site-specifically fluorescein-labeled antibodies in IgG or short half-life diabody (Db) format, directed against the frequently AML expressed antigens CD33, CD117 and CD371. Site-specific chemical modification methods and cysteine-tagged Db mediated the strongest AML killing results in vitro over a broad range of antibody concentrations.We then hypothesized that FluA-CAR T-cells, targeting AML cells via combinatorial use of linker molecules adapted to specific AML antigen-expression profiles, would allow to most efficiently eliminate AML cells in a dose- and timely-regulated fashion. To this end, we tested single and combinatorial use of fluoresceinated anti-CD117 Db and anti-CD371 Db on MOLM13 AML cell lines, engineered to be either CD117 highCD371 neg, CD117 negCD371 high, or CD117 highCD371 high. Indeed, combination of anti-CD117 and anti-CD371 Db-linkers, resulted in significantly improved MOLM13 cell lysis compared to equimolar concentrations of single agents in vitro (example figure). We then tested the same approach, targeting CD117 +CD371 + primary AML cells from two different patients, using either patient-derived or allogeneic FluA CAR T-cells. Again, the combinatorial use of linkers generated a significantly higher AML cell lysis than the use of single Db linkers.We thus here provide proof-of-concept for the generation of highly potent universal targeting FluA CAR T-cells from healthy donors and AML patients. By choosing suitable CAR-adaptors with respect to their conjugation chemistry and size, it is possible to tightly regulate CAR-T cell activity against CD33, CD117 and CD371 expressing AML cells and likely any tumor cell expressed antigen. Short half-life small molecule linkers will allow to control FluA CAR T-cell on-off activity and combinatorial use of linkers will allow to maximize anti-tumor activity and to minimize on-target off-tumor toxicity. [Display omitted] DisclosuresMyburgh: University of Zurich: Patents & Royalties: CD117xCD3 TEA. Neri: Philogen S.p.A.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Multiple patents on vascular targeting; ETH Zurich: Patents & Royalties: CD117xCD3 TEA. Manz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company; University of Zurich: Patents & Royalties: CD117xCD3 TEA.