Monocytic Acute Myeloid Leukemia Cells Research Articles

Abstract 2372: ATG-102, a novel LILRB4 x CD3 T cell engager, targeting two nonoverlapping epitopes of LILRB4, for the treatment of monocytic AML

Abstract Background: Acute myeloid leukemia (AML) is the most common acute leukemia in adults, and the treatment of AML, especially monocytic AML subtypes still has a poor outcome. T cell engagers (TCE) have been well used for the treatment of hematological malignancies, such as B cell leukemia and multiple myeloma. However, lack of ideal target antigens that only express on AML and leukemic stem cells (LSCs) but not on normal hematopoietic stem cells (HSCs) hampers the development of TCE therapy for AML. Leukocyte immunoglobulin-like receptor B4 (LILRB4), an inhibitory receptor belonging to the LILR family, is highly and restrictedly expressed on monocytic M4/M5 AML subtypes and LSCs, but not on normal HSCs which makes it an ideal target for TCE. Here we report a novel LILRB4 x CD3 bispecific T cell engager based on AnTenGagerTM platform, ATG-102. It targets two distinct epitopes of LILRB4, inducing potent T cell-dependent cellular cytotoxicity (TDCC) with low CRS, resulting in a potent anti-tumor efficacy in vitro and in vivo. Method: ATG-102 was constructed by introducing a high affinity anti-CD3 single chain fragment variable (scFv) to the hinge region of one of the heavy chains of a LILRB4 monoclonal antibody. A scFv targeting another epitope of LILRB4 was connected to the C terminal of the same heavy chain by GSSS linker, enabling a trivalent, dual epitope recognition of LILRB4. ATG-102 was evaluated in a series of preclinical studies for binding epitope and affinity, T cell activation, T cell dependent cytotoxicity (TDCC), and cytokine release. The in vivo antitumor efficacy of ATG-102 was evaluated in a humanized PBMCs and THP-1-luciferase cells engrafted NCG mice model. Results: ATG-102 binds to LILRB4 positive cells with a single-digit nM affinity. It binds to two distinct and nonoverlapping epitopes of LILRB4. ATG-102 showed limited binding capability to CD3+ cells before LILRB4 crosslinking. However, with the presence of LILRB4 positive THP-1 cell, ATG-102 strongly activated primary T cells, upregulating early and later markers of T cell activation, CD69 and CD25, respectively. ATG-102 induced strong TDCC against LILRB4-high expressing THP-1 cells, and it induced more potent TDCC against LILRB4-low expressing MOLM-13 cells than a benchmark antibody. While compared with benchmark, ATG-102 induced significantly lower release of IL-6, which is one of the major cytokines involved in cytokine release syndrome (CRS). In addition, ATG-102 demonstrated more potent in vivo anti-tumor efficacy compared with the benchmark antibody at 1 mg/kg and 0.1 mg/kg dose level in PBMC-humanized NCG mice bearing THP-1-luciferase cells. Conclusion: ATG-102 demonstrates trivalent, dual epitope recognition of LILRB4, conditionally redirects and activates T cells to recognize and kill monocytic AML cells. It demonstrates potent in vitro and in vivo anti-tumor efficacy with low risk of CRS. Citation Format: Ao Sun, Mengshi Gao, Rong Guo, Huiling Liu, Zaoshun Hu, Enlin Zheng, Hui Yuwen, Peng Chen, Jay Mei, Bo Shan, Bing Hou. ATG-102, a novel LILRB4 x CD3 T cell engager, targeting two nonoverlapping epitopes of LILRB4, for the treatment of monocytic AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2372.

SETDB1 Suppresses Interferon Responses and NK Cell-Mediated Immunosurveillance Specifically in Monocytic AML

Acute myeloid leukemia (AML) with monocytic differentiation (monocytic AML) responds poorly to current treatments, including venetoclax-based therapy. To identify novel therapeutic targets in monocytic AML, we conducted in vivo and in vitro CRISPR/Cas9 library screens using a pooled sgRNA library targeting epigenetic regulators with a mouse monocytic AML model driven by SETBP1 and ASXL1 mutations (cSAM: combined expression of SETBP1 and ASXL1 Mutations). We ranked all the epigenetic genes according to the in vivo specific essentiality, and selected several candidates which were more depleted in mice rather than in cell cultures. The top-ranked gene was Setdb1, which encodes a histone H3 lysine 9 (H3K9) methyltransferase. In addition, several H3K9 trimethylation (H3K9me3) regulators, ATF7IP and TRIM33, were also more important in vivo than in vitro in our screens. ATF7IP is a cofactor of SETDB1 to enhance its enzymatic activity. TRIM33 is also involved in H3K9me3 modification. These data suggest the critical role of the SETDB1-ATF7IP-TRIM33 H3K9 methyltransferase complex in the development of AML in vivo. To validate the results of our screenings and to investigate the role of immune cells in our transplantation assay, we next transplanted control and Setdb1-knockout (KO) cSAM cells into three types of mice with different immune systems: the immunocompetent C57BL/6 mice, the immunodeficient NSG mice lacking T, B and natural killer (NK) cells, and the C57BL/6J- Rag2-/- mice lacking mature T and B cells but possessing NK cells. Although Setdb1 depletion did not inhibit the growth of cSAM cells in NSG mice, it showed the strong growth-inhibitory effect in C57BL/6 mice and Rag2-/- mice (Fig 1A). Since both C57BL/6 mice and Rag2-/- mice have NK cells, these data suggest that SETDB1 promotes leukemogenesis by suppressing NK cell-mediated cytotoxicity in vivo. Mechanistically, loss of Setdb1 induced upregulation of NKG2D ligands, interferon-stimulated genes (ISGs), and endogenous retrovirus (ERV) sequences through H3K9me3 reduction, thereby rendering cSAM cells more susceptible to NK cell-mediated cytotoxicity. Setdb1 depletion also induced myeloid maturation of cSAM cells towards macrophages and downregulation of major histocompatibility complex class I (MHC-I), which contributed to the increased susceptibility of Setdb1-depleted cSAM cells to NK cell-mediated cytotoxicity. Next, we investigated whether SETDB1 inactivation also sensitizes human AML cells to NK cells using the co-culture assay with an IL-2 dependent NK cell line NK-92. Loss of SETDB1 rendered monocytic AML cell lines, NOMO-1, THP-1, OCI-AML3, U937, and NB4, more susceptible to NK-92 cell-mediated cytotoxicity. SETDB1 depletion also induced overactivation of interferon signaling and intrinsic apoptosis in these monocytic AML cells. In contrast, non-monocytic AML cells, such as K562, TF-1, and Kasumi-1, were not sensitive to SETDB1 depletion. SETDB1 depletion neither increased the susceptibility to NK cell-mediated cytotoxicity nor induced apoptosis in non-monocytic AML cells. Subsequent analyses revealed that SETDB1 depletion reduced H3K9me3 at the monocyte-specific enhancer regions with ERV sequences to upregulate NK cell-activating ligands and ISGs. Because the “ERV enhancers” are active only in monocyte lineage, SETDB1 is essential specifically in monocytic AML. Finally, we identified MNDA and its mouse counterpart Ifi203 as biomarkers to predict the sensitivity of each AML to SETDB1 depletion. MNDA is one of the ISGs and is involved in myeloid cell differentiation. Human monocytic AML cells that are dependent on SETDB1 expressed higher MNDA at both RNA and protein levels compared to other subtypes of AML. We also found that Ifi203-high mouse AML cells transformed by MLL-AF9 were sensitive to Setdb1 depletion, whereas Setdb1 was dispensable in Ifi203-low mouse AML cells transformed by RUNX1-RUNX1T1. In summary, our findings reveal the critical and selective role of SETDB1 in monocytic AML. SETDB1 mediates H3K9me3 at the monocyte-specific ERV enhancers to repress the expression of NK cell-activating ligands and ISGs, thereby suppressing immunogenicity and apoptosis in monocytic AML (Fig 1B). SETDB1 as well as its binding partners could be promising therapeutic targets to treat monocytic AMLs that are relatively resistant to the current standard therapies.

A Novel CD64 CAR-T Therapy for the Treatment of Monocytic Acute Myeloid Leukemia

Introduction: Acute Myeloid Leukemia (AML) is a difficult to treat, life threatening malignancy with overall survival remaining extremely poor, approximately 15% at 5 years. Recent advances in therapeutic approaches for AML have significantly improved outcomes, most notably venetoclax and azacitidine which induces a remission in approximately 70% of patients. Despite this, the majority of patients ultimately relapse and succumb to their disease. We and others have previously shown that more monocytic (M5 by FAB classification) AML cells demonstrate increased resistance to venetoclax therapy and that relapse after venetoclax and azacitidine treatment shows a monocytic phenotype in approximately 30% of patients (Pei, et. al. Cancer Discovery, 2020). These patients experience decreased overall survival compared to patients with non-M5 AML. Furthermore, we have demonstrated that AML with a dominant monocytic phenotype can arise from a unique subclass of leukemia stem cells (LSCs) that we have termed monocytic-LSCs (m-LSCs) due to their immunophenotype, including expression of CD64. m-LSCs are highly resistant to venetoclax-based therapies (Pei, et. al., Cancer Discovery, 2023). As venetoclax is increasingly utilized in AML treatment regimens, it is essential to identify methods of targeting this m-LSC subtype to prevent and treat relapsed and refractory disease. Methods: To more effectively target monocytic AML, we have developed a novel chimeric antigen receptor (CAR) T cell therapy targeting CD64, an antigen commonly expressed in monocytic cells and strongly expressed in the m-LSC population. We generated two second-generation CAR constructs targeting CD64, one with CD28 and the other with 41BB costimulatory domains. We expressed these CARs in healthy donor human T cells via lentiviral transduction and tested their function in vitro and in vivo. Results: We demonstrate that CD64 CAR-T cells effectively treat monocytic AML using pre-clinical models. CD64 CAR-T cells eliminate CD64+ leukemia cell lines in vitro in a dose dependent manner with an effector to target (E:T) ratio of 1:4 and higher eliminating 100% of Molm14 leukemia cells at 18 hours compared to 0-10% in mock T-cell controls. We also show that CD64 CAR-T cells eliminate established Molm14 leukemia in vivo, with 100% of CAR-T treated mice surviving over 50 days with no evidence of leukemia versus less than 30 days survival with high tumor burden in mock treated mice (p<0.01, Figure 1). We find that CD64 CAR-T cells with CD28 verses 41BB costimulatory domains are similarly efficacious against monocytic AML, though we find more prolonged persistence of CD64-41BBz CAR-T cells in mice with long term leukemia free survival. Furthermore, we demonstrate that CD64 CAR-T cells kill CD64+ primary patient derived AML cells in ex vivo co-culture, resulting in a 60-80% reduction in viable AML cells at 18 hours across 1:1 to 1:16 E:T ratios compared to mock T cell controls. We further show that CD64 CAR-T cells target m-LSCs and reduce the engraftment potential of primary AML cells in mice with a nearly 9-fold reduction in leukemia burden in CAR-T treated versus control mice. Finally, preliminary experiments with AML patient derived xenograft models further support the in vivo efficacy of CD64 CAR-T cell treatment. Conclusions: Our data provide promising preclinical evidence for the efficacy of a novel CD64 CAR-T therapy in the treatment of monocytic AML. We propose that using this regimen to augment AML therapy is a potentially powerful means to eradicate m-LSCs, which are inherently resistant to venetoclax, and thereby increase remission duration. This concept serves as the basis for an upcoming clinical trial of CD64-directed CAR-T cells for CD64 expressing AML. Figure 1: CD64 CAR-T cells eliminate AML in in vivo murine leukemia models. A) NSG mice leukemia models of GFP expressing Molm14, a CD64+ human leukemia cell line, experience leukemia clearance within 3 days after treatment with CD64-28z CAR-T cells, as shown by decreased bioluminescent signal in images of CAR-T treated mice. Mock transduced human T-cells from the same human donor serve as a control. B) Survival curves demonstrate 100% survival of CAR-T treated mice over the experiment (p<0.01).

Abstract PR02: RSK1 targeting impedes oncogenic driver and inflammatory cytokine signaling to attenuate myeloid neoplasms

Abstract Myeloproliferative neoplasms (MPNs), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML) exist among a spectrum of myeloid malignancies, governed by critical genetic dependencies, and propagated by a ripe pro-inflammatory niche. We previously identified elevated RPS6KA1 (RSK1) expression in myeloid malignancies associated with poor overall survival. Here, we demonstrate that RSK1 plays a fundamental role in mediating inflammation and serves as a central signaling node driving leukemogenesis. We further evaluate a novel therapeutic regimen utilizing a first-in-class RSK1 inhibitor, PMD-026, currently evaluated in Phase 1/1b clinical trials for breast cancer, for use in myeloid malignancies. Plasma analysis of 144 MPN patients and RNA-seq of CD14+ monocytes from 54 MPN patients revealed enrichment of inflammatory cytokines and hyperactive NFκB signaling. RSK1 inhibition by shRNA or PMD-026 in THP-1 monocytic AML cells potently suppressed this NFκB node, decreased TNF and NFKB1 mRNA, and reduced luminescence in cells expressing a NFκB-luciferase reporter. Immunoblotting and cytokine mass cytometry revealed suppression of RelA/p65 phosphorylation with reduction of TNF, IL-6, IL-8, CCL3, and CCL4 in MPN and CMML CD14+ monocytes. We also observed that RSK1 inhibition attenuated NFκB activation by TNF, LPS, and Pam3CSK4, while also suppressing monocyte differentiation to M1 macrophages. In the MPL W515L myelofibrosis (MF) model, mice treated with PMD-026 had significant reduction in TNF, IL-6, IL-1b, CCL3, and CCL5 by multiplex cytokine profiling and diminished disease burden (reduction of leukocytosis, splenomegaly, and bone marrow fibrosis). Finally, amelioration of disease was observed across MF and sAML patient-derived xenograft (PDX) models with PMD-026 treatment. We extended our findings to FLT3-ITD de novo AML models, in which RSK1 inhibition led to lethality in FLT3-ITD cells and PMD-026 potently suppressed leukemic engraftment in a MV4-11 mouse xenograft. Both genetic and pharmacological inhibition of RSK1 reduced FLT3 phosphorylation and revealed a novel, bi-directional regulatory network. Moreover, we found that RSK1 perturbation also attenuated FLT3 expression and altered protein stability, which was not observed with FLT3 inhibitor quizartinib. Mechanistically, RSK1 modulated FLT3 dynamics through deubiquitinase USP1, which was downregulated upon RSK1 perturbation. USP1 perturbation led to cell death and phenocopied FLT3 suppression observed with RSK1 inhibition. We further identified elevated USP1 expression associated with worse survival outcomes across the TCGA LAML, BeatAML, and Leucegene cohorts, thus indicating the potential of USP1 as a novel biomarker. We uncover a role for targeting RSK1 as a two-pronged approach in reducing disease burden across myeloid malignancies via: 1) suppressing driver signaling, and 2) dampening the pro-inflammatory milieu. These findings highlight a critical dependency along the RSK1-USP1-FLT3 axis that is potentially exploitable with RSK1 inhibitors such as PMD-026. Citation Format: Tim Kong, Angelo Laranjeira, Stephen Oh. RSK1 targeting impedes oncogenic driver and inflammatory cytokine signaling to attenuate myeloid neoplasms [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr PR02.

Abstract LB217: A novel bispecific LILRB4/CD3 antibody with potent killing of monocytic acute myeloid leukemia cells

Abstract Background: Acute myeloid leukemia (AML) remains one of the highest unmet needs among human cancers. LILRB4 is specifically expressed on M4 myelomonocytic and M5 monocytic AML cells with a lack of expression on normal hematopoietic stem cells and progenitors, making LILRB4 an attractive target for a T-cell redirecting bispecific antibody to treat AML. Using a proprietary bispecific antibody platform, we engineered and optimized a LILRB4/CD3 bispecific antibody demonstrating potent and specific killing of monocytic AML cells in vitro and in vivo. The strong chemistry, manufacturing and control (CMC) attributes and cross-reactivity to non-human primate LILRB4 and CD3 favor this bispecific for rapid advancement to the clinic. Experimental Procedures: Binding EC50 values for LILRB4/CD3 bispecific antibodies were determined by flow cytometry analysis. The selectivity of the anti-LILRB4 modality to other LILRA/B proteins was assessed by ELISA. The potency of T-cell mediated killing of LILRB4-expressing AML cells was determined by co-culturing human peripheral T cells and monocytic AML THP-1 cells. A human peripheral mononuclear cell (PBMC) assay evaluated potency against LILRB4-positive primary cells (CD14+ monocytes), using CD19+ B cells as a negative control. Tumor bioluminescence kinetics was performed in NSG mice administrated intravenously with human T cells and THP-1-luciferase cells to measure T cell-directed killing of AML cells in vivo. Single intravenous dose of 5 mg/kg was used for PK evaluation in human FcRn transgenic mice. Results: Two top candidates were selected using biophysical criteria: 1) A monovalent anti-LILRB4 arm together with a monovalent anti-CD3 (1+1); 2) A bivalent anti-LILRB4 arm together with a monovalent anti-CD3 (2+1). The binding EC50 values of the 2+1 or the 1+1 variants to LILRB4 in the THP-1 cells were 0.7 nM and 1.3 nM, respectively. ELISA showed the anti-LILRB4 arm to be highly selective for LILRB4. Both 2+1 and 1+1 variants demonstrated potent killing of the THP-1 cells with EC50 values of 0.5 pM and 4.3 pM, respectively. In the PBMC assay, both variants induced killing of monocytes while sparing B cells, supporting the specificity in inducing T-cell directed killing of the LILRB4-expressing primary cells. The anti-CD3 arm was designed to have a low affinity for CD3 to mitigate the risk of cytokine release while ensuring sufficient T cell activation. Both variants showed potent tumor-growth inhibition at doses as low as 0.2 mg/kg in the NSG mice with the 2+1 molecule showing superiority in vivo. The PK profile of the 2+1 bispecific in human FcRn-transgenic mice was similar to what is expected of human IgG1 with linear clearance. Conclusions: A novel bispecific LILRB4/CD3 (2+1) has been identified for development to treat AML based on its potency and specificity in killing monocytic AML cells in both in vitro and in vivo preclinical studies. Citation Format: Tao Huang, Kyu Hong, Krista McCutcheon, Jing-Tyan Ma, Ying Zhu, Mi Deng, Cheng Cheng Zhang, Paul Woodard, Hong Xiang, Xiao Min Schebye, X. Charlene Liao. A novel bispecific LILRB4/CD3 antibody with potent killing of monocytic acute myeloid leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB217.

Survival and Outcomes after Relapse in Newly Diagnosed Acute Myeloid Leukemia (AML) Patients Treated with Venetoclax-Based Chemotherapy

Background Venetoclax (bcl-2 inhibitor) was first approved in 2018 for use in combination with hypomethylating agents (HMA) or low-dose cytarabine (LDAC) for treatment of newly diagnosed acute myeloid leukemia (AML) in older patients, or those who have comorbidities that preclude use of intensive induction chemotherapy. However, AML is also known to evolve and undergo clonal selection when treated with venetoclax, leading to treatment failure. Mechanisms for adaptive resistance include kinase activation (e.g. FLT3-ITD), clones that acquire biallelic silencing of TP53, and monocytic AML cells that upregulate MCL1 to evade BCL2 blockade. Other issues with venetoclax-based regimens include prolonged cytopenias and increased risk of infection. Our aim was to investigate survival after relapse, subsequent responses to second- line agents, and pattern of infections in patients treated with first-line venetoclax-based combination therapies. Methods This was a retrospective study of patients who received venetoclax-based regimens for untreated AML or myelodysplastic syndrome with excess blasts (MDS-EB) at LAC+USC Medical Center and Norris Cancer Center in Los Angeles, CA from January 1, 2018 to June 30, 2022. The electronic medical record was reviewed for demographics, disease characteristics, clinical history, transplant status, and survival. Results 61 patients received frontline venetoclax-based regimens for untreated AML or MDS-EB with a mean age of 65 (range 22-84); 36% were female, 44% non-Hispanic white and 31% were Hispanic/Latino. 41% had AML-MRC (myelodysplasia-related changes), 36% had De Novo AML, 16% had therapy-related AML, and 7% had MDS-EB. The majority (70%) were adverse risk group per ELN 2017 and 46% had complex cytogenetics. 58 patients had available myeloid mutation panel at time of diagnosis. The most common mutations (median allele frequencies) were: TP53=33% (0.45), ASXL1=17% (0.37), NPM1=16% (0.39), IDH2=9% (0.42), IDH1=7% (0.29), FLT3-ITD=5% (0.51). 70% received venetoclax with decitabine, 21% received venetoclax with azacitidine, and 2% received venetoclax with LDAC. Median number of cycles was 3 (1-16). 10 patients underwent allogeneic stem cell transplant. Out of 61 patients, 12 (20%) had primary refractory disease and 23 (38%) relapsed on frontline venetoclax. 49% of these relapsed/refractory patients received salvage chemotherapy. The median time to relapse and number of cycles prior to relapse was 6.3 months (1.6-17.4) and 4 (1-12) respectively. There was no correlation between cytogenetics and genetic variables and risk of relapse. Only 3 (19%) out of 16 patients and 3 (27%) out of 11 patients who received second- and third-line treatment achieved CRi respectively. Myeloid mutation panel was available for 12 patients at time of relapse. Most common mutations were FLT3-ITD/TKD (n=5), TP53 (n=3) and NPM1 (n=3). Six patients acquired new mutations at the time of relapse: FLT3-ITD (n=2), TP53 (n=1), WT1 (n=2) and ETV6 (n=1). Median time from relapse to death was 3 months (1-23). Median overall survival was 10.2 months. Median survival in responder compared to non-responders after cycle 1 and cycle 2 was 16.8 vs. 5.3 months (p <0.001) and 19.4 months vs. 7.4 months (p=0.031) respectively. Median survival in patients with relapse compared to non-relapse was 12.1 vs 6 months (p=0.187). 40 (66%) patients died; major causes of death were disease progression (50%), bleeding (10%) and infection (25%). 28% patients died in intensive care unit. 9 patients had fungal infections; most common were aspergillosis (n=5) and mucormycosis (n=2). Conclusions Venetoclax-based regimens are effective options for untreated AML in elderly patients. Response to first and second cycle of combination treatment predict survival. Although we observed clonal evolution in relapsed patients, it did not affect their survival. FLT3-ITD was a commonly acquired mutation at time of relapse, thus patients received salvage with targeted agents. Also, as patients relapse, the chance of attaining CR/CRi with subsequent lines of treatment is less than 30% and risk of invasive fungal infection was approximately 15%. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Preclinical Evaluation of CD64 As a Potential Target For CAR-T-cell Therapy For Acute Myeloid Leukemia.

The relapsed and refractory acute myeloid leukemia (AML) patients receiving traditional chemotherapies have poor survival rate. Chimeric antigen receptor (CAR)-modified T cells have demonstrated remarkable effectiveness against some malignancies. However, most of CAR-Ts targeting the candidate proteins on AML cells induce hematopoietic cell suppression. Because of extensive heterogeneity among different types of AML, it is essential to expand the choice of target antigen for the CAR-T treatment of AML. CD64 (FcγRI) is a transmembrane protein with broad expression on various types of AML cells, especially monocytic AML cells, but it is absent on hematopoietic stem cells (HSCs) and most of nonmonocytes. Here, we found that some types of AML patients showed the homogeneous high-level expression of CD64. So, we created a CAR-T targeting CD64 (64bbz) and further verified its high efficiency for eradicating CD64+AML cells. In addition, 64bbz showed no cytotoxicity to HSCs. Overall, we developed a new treatment option for AML by using CD64 CAR-T cells while avoiding ablation of HSCs.

Anti-Leukemic Properties of Histamine in Monocytic Leukemia: Role of NOX2

We aimed at defining effects of histamine dihydrochloride (HDC) on the maturation and expansion of monocytic acute myeloid leukemia (AML) cells in vitro and in vivo with focus on the role of the myeloid NADPH oxidase (NOX2), which is targeted by HDC acting via H2-type histamine receptors (H2R). The human myelomonocytic/monocytic AML cell lines PLB-985 and OCI-AML3 were found to express NOX2 and H2R and responded to HDC with increased expression of differentiation markers and reduced cell cycle proliferation in vitro . These effects were absent in NOX2-/- (NOX2 -KO) PLB-985 cells. Microarray analysis revealed that HDC regulated the expression of genes involved in cell differentiation and cell cycle progression in NOX2+ but not in NOX2 -KO AML cells. In a xenograft mouse model using immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac (NOG) mice, systemic treatment with HDC reduced the expansion of engrafted monocytic AML cells in bone marrow but did not exert anti-leukemic activity against engrafted NOX2 -KO cells. Primary leukemic cells recovered from patients with monocytic forms of AML co-expressed NOX2 and H2R and commonly upregulated the expression of the maturation markers HLA-DR, FPR1 and FPR2 after exposure to HDC. Results from a phase IV trial supported the clinical benefit of treatment with HDC, used in conjunction with low-dose IL-2, in preventing relapse among patients with monocytic forms of AML. These results imply that HDC targets NOX2 to exert anti-leukemic activity against monocytic AML cells and point towards NOX2 as a conceivable target in AML therapy. DisclosuresGrauers Wiktorin:Immune Pharmaceuticals: Patents & Royalties: Pending patent protecting the use of NOX2 inhibitors in cancer. Aurelius:Immune Pharmaceuticals: Patents & Royalties: Pending patent protecting the use of NOX2 inhibitors in cancer. Thoren:Immune Pharmaceuticals: Patents & Royalties: Pending patent protecting the use of NOX2 inhibitors in cancer. Hellstrand:Immune Pharmaceuticals: Patents & Royalties: Filed or pending patents protecting the use of NOX2 inhibitors in cancer. Martner:Immune Pharmaceuticals: Patents & Royalties: Pending patent protecting the use of NOX2 inhibitors in cancer.

ILT3 promotes tumor cell motility and angiogenesis in non-small cell lung cancer.

Immunoglobulin-like transcript (ILT) 3 is an immunosuppressive molecule that negatively regulates myeloid cell activation. ILT3 overexpression in tumor cells induces immune escape of solid tumors and facilitates invasion of monocytic acute myeloid leukemia cells. However, the expression and function of ILT3 in non-small cell lung cancer (NSCLC) cells remain elusive. Herein, we found that ILT3 was enriched in human NSCLC cells, and predicted advanced disease and poor overall survival. ILT3 overexpression enhanced the migration and invasion of NSCLC cells and tubule formation of human umbilical vein endothelial cells by upregulating and interacting with its ligand apolipoprotein E (ApoE) in vitro. Mechanistically, ILT3 recruited SHP2 and SHIP1, and subsequently activated ERK1/2 signaling mediating epithelial-mesenchymal transition (EMT) and increasing vascular endothelial growth factor (VEGF)-A expression in NSCLC cells, which are responsible for tumor cell motility and angiogenesis, respectively. Using murine metastasis models, we further confirmed ILT3 promoted NSCLC metastasis and explored the exact correlation of ILT3 with ApoE, EMT, and VEGF-A in vivo. These results unraveled novel mechanisms for ILT3-induced tumor progression and proposed ILT3 as a potential therapeutic target and prognostic biomarker for NSCLC patients.

LILRB4-targeting Antibody-Drug Conjugates for the Treatment of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the most common and aggressive blood cancer in adults. In particular, significant unmet medical needs exist for effective treatment strategies for acute myelomonocytic leukemia (M4) and acute monocytic leukemia (M5) AML subtypes. Antibody-drug conjugates (ADC) are a promising drug class for AML therapy, as demonstrated by the FDA-approved anti-CD33 ADC, gemtuzumab ozogamicin (Mylotarg). However, CD33 is expressed in normal hematopoietic stem cells, highlighting the critical need to identify AML-specific targets to minimize the risk of potential adverse effects. We have demonstrated that the leukocyte immunoglobulin-like receptor subfamily B4 (LILRB4) is expressed at significantly higher levels on monocytic M4 and M5 AML cells than on normal counterparts. Here, we test whether LILRB4 is a promising ADC target to kill monocytic AML cells while sparing healthy counterparts. To this end, we generated ADCs from a humanized anti-LILRB4 mAb and the antimitotic payload, monomethyl auristatin F. The conjugates constructed were characterized and evaluated for LILRB4-specific cell killing potency, toxicity to progenitor cells, pharmacokinetics, and therapeutic efficacy. Our ADC linker technology platform efficiently generated homogeneous anti-LILRB4 ADCs with defined drug-to-antibody ratios. The homogeneous anti-LILRB4 ADCs demonstrated the capacity for LILRB4-mediated internalization, suitable physicochemical properties, and high cell killing potency against LILRB4-positive AML cells. Importantly, our data indicate that these ADCs spare normal progenitor cells. One of our homogeneous conjugates exerted a remarkable therapeutic effect and no significant toxicity in a xenograft mouse model of disseminated human AML. Our findings highlight the clinical potential of anti-LILRB4 ADCs in monocytic AML therapy.

Monocytic Subclones Confer Resistance to Venetoclax-Based Therapy in Patients with Acute Myeloid Leukemia.

Venetoclax-based therapy can induce responses in approximately 70% of older previously untreated patients with acute myeloid leukemia (AML). However, up-front resistance as well as relapse following initial response demonstrates the need for a deeper understanding of resistance mechanisms. In the present study, we report that responses to venetoclax +azacitidine in patients with AML correlate closely with developmental stage, where phenotypically primitive AML is sensitive, but monocytic AML is more resistant. Mechanistically, resistant monocytic AML has a distinct transcriptomic profile, loses expression of venetoclax target BCL2, and relies on MCL1 to mediate oxidative phosphorylation and survival. This differential sensitivity drives a selective process in patients which favors the outgrowth of monocytic subpopulations at relapse. Based on these findings, we conclude that resistance to venetoclax + azacitidine can arise due to biological properties intrinsic to monocytic differentiation. We propose that optimal AML therapies should be designed so as to independently target AML subclones that may arise at differing stages of pathogenesis. SIGNIFICANCE: Identifying characteristics of patients who respond poorly to venetoclax-based therapy and devising alternative therapeutic strategies for such patients are important topics in AML. We show that venetoclax resistance can arise due to intrinsic molecular/metabolic properties of monocytic AML cells and that such properties can potentially be targeted with alternative strategies.

A Novel Anti-LILRB4 CAR-T Cell for the Treatment of Monocytic AML.

To effectively improve treatment for acute myeloid leukemia (AML), new molecular targets and therapeutic approaches need to be identified. Chimeric antigen receptor (CAR)-modified Tcells targeting tumor-associated antigens have shown promise in the treatment of some malignancies. However, CAR-T cell development for AML has been limited by lack of an antigen with high specificity for AML cells that is not present on normal hematopoietic stem cells, and thus will not result in myelotoxicity. Here we demonstrate that leukocyte immunoglobulin-like receptor-B4 (LILRB4) is a tumor-associated antigen highly expressed on monocytic AML cells. We generated a novel anti-LILRB4 CAR-T cell that displays high antigen affinity and specificity. These CAR-T cells display efficient effector function invitro and invivo against LILRB4+ AML cells. Furthermore, we demonstrate anti-LILRB4 CAR-T cells are not toxic to normal CD34+ umbilical cord blood cells in colony-forming unit assays, nor in a humanized hematopoietic-reconstituted mouse model. Our data demonstrate that anti-LILRB4 CAR-T cells specifically target monocytic AML cells with no toxicity to normal hematopoietic progenitors. This work thus offers a new treatment strategy to improve outcomes for monocytic AML, with the potential for elimination of leukemic disease while minimizing the risk for on-target off-tumor toxicity.

Anti-Leukemic Properties of Histamine in Monocytic Leukemia: The Role of NOX2.

In patients with acute myeloid leukemia (AML), treatment with histamine dihydrochloride (HDC) and low-dose IL-2 (HDC/IL-2) in the post-chemotherapy phase has been shown to reduce the incidence of leukemic relapse. The clinical benefit of HDC/IL-2 is pronounced in monocytic forms of AML, where the leukemic cells express histamine type 2 receptors (H2R) and the NAPDH oxidase-2 (NOX2). HDC ligates to H2Rs to inhibit NOX2-derived formation of reactive oxygen species, but details regarding the anti-leukemic actions of HDC remain to be elucidated. Here, we report that human NOX2+ myelomonocytic/monocytic AML cell lines showed increased expression of maturation markers along with reduced leukemic cell proliferation after exposure to HDC in vitro. These effects of HDC were absent in corresponding leukemic cells genetically depleted of NOX2 (NOX2−/−). We also observed that exposure to HDC altered the expression of genes involved in differentiation and cell cycle progression in AML cells and that these effects required the presence of NOX2. HDC promoted the differentiation also of primary monocytic, but not non-monocytic, AML cells in vitro. In a xenograft model, immunodeficient NOG mice were inoculated with wild-type or NOX2−/− human monocytic AML cells and treated with HDC in vivo. The administration of HDC reduced the in vivo expansion of NOX2+/+, but not of NOX2−/− human monocytic AML cells. We propose that NOX2 may be a conceivable target in the treatment of monocytic AML.

NOX2-derived ROS in myeloid cell differentiation: Implications in cancer

The NOX2 enzyme is highly expressed in normal myeloid cells, as well as in subpopulations of malignant myeloid cells, where it contributes to the generation of reactive oxygen species (ROS). We have utilized mice and leukemic cells genetically depleted of NOX2 and the NOX2-specific inhibitor histamine dihydrochloride (HDC) to further elucidate the role of NOX2-derived ROS in hematopoietic cancer. HDC was found to promote the maturation of human DCs from monocytes and to enhance T cell priming. In addition, treatment of mice with HDC reduced the growth of murine EL-4 lymphomas in parallel with an increment of tumor-infiltrating DCs in NOX2-sufficient mice but not in Nox2-/- mice. HDC was also found to facilitated the maturation and inhibit the proliferation of NOX2-expressing human acute myeloid leukemia (AML) cells. These effects were absent in NOX2-/- AML cells. In microarray analysis, HDC was found to regulate the expression of genes involved in cell differentiation and cell cycle progression in NOX2+ but not in NOX2-/- AML cells. In a xenograft mouse model, systemic treatment with HDC reduced the expansion of engrafted monocytic AML cells in bone marrow but did not exert anti-leukemic activity against engrafted NOX2-/- AML cells.

Misfolded N-CoR is Linked to the Ectopic Reactivation of CD34/Flt3-Based Stem-Cell Phenotype in Promyelocytic and Monocytic Acute Myeloid Leukemia.

Nuclear receptor co-repressor (N-CoR) is the key component of generic co-repressor complex essential for the transcriptional control of genes involved in cellular hemostasis. We have recently reported that N-CoR actively represses Flt3, a key factor of hematopoietic stem cells (HSC) self-renewal and growth, and that de-repression of Flt3 by the misfolded N-CoR plays an important role in the pathogenesis of promyelocytic and monocytic acute myeloid leukemia (AML). The leukemic cells derived from the promyelocytic and monocytic AML are distinctly characterized by the ectopic reactivation of stem cell phenotypes in relatively committed myeloid compartment. However, the molecular mechanism underlying this phenomenon is not known. Here, we report that N-CoR function is essential for the commitment of primitive hematopoietic cells to the cells of myeloid lineage and that loss of N-CoR function due to misfolding is linked to the ectopic reactivation of generic stem cell phenotypes in promyelocytic and monocytic AML. Analysis of N-CoR and Flt3 transcripts in mouse hematopoietic cells revealed a positive correlation between N-CoR level and the commitment of myeloid cells and an inverse correlation between N-CoR and Flt3 levels in primitive as well as committed myeloid cells. Enforced N-CoR expression in mouse HSCs inhibited their growth and self-renewal potentials and promoted maturation toward cells of myeloid lineage, suggesting a role of N-CoR in the commitment of cells of myeloid lineage. In contrast to AML cells with natively folded N-CoR, primary and secondary promyelocytic and monocytic AML cells harboring the misfolded N-CoR were highly positive for Flt3 and myeloid antigen-based HSC marker CD34. Genetic and therapeutic restoration of N-CoR conformation significantly down-regulated the CD34 levels in monocytic AML cells, suggesting an important role of N-CoR in the suppression of CD34-based HSC phenotypes. These findings collectively suggest that N-CoR is crucial for the commitment of primitive hematopoietic cells to cells of myeloid lineage and that misfolded N-CoR may contribute to transformation of committed myeloid cells through the ectopic reactivation of Flt3/CD34-based stem cell phenotypes in promyelocytic and monocytic AML. Moreover, these findings provide novel mechanistic insights into the formation of leukemic stem cells in subsets of AML and identify the misfolded N-CoR as a subtype-specific biomarker of AML.

Cathecholamines Differently Regulate Human AML and Normal Hematopoietic Progenitor Cell Motility Via miR126 and RGS16

Abstract Introduction Motility, proliferation, bone marrow (BM) retention and egress to the circulation of hematopoietic stem and progenitor cell (HSPCs) are key elements in normal hematopoiesis and also in the pathogenesis of acute myeloid leukemia (AML). HSPCs are tightly regulated in the BM niche by various molecules. Among them, CXCL12 and its major receptor CXCR4, expressed by both HSPCs and BM niche components, govern HSPC retention in the BM. Administration of CXCR4 antagonists lead to mobilization of both normal HSPCs and leukemia-inducing stem cells (LICs) from the BM to the circulation. We have previously found that normal human HSPCs functionally express b2-adrenergic receptors (b2-AR) that are up-regulated by myeloid cytokines such as G-CSF. The catecholaminergic neurotransmitters epinephrine and norepinephrine (NE) activate both Wnt and GSK3β signaling pathways via b2-AR, leading to enhanced HSPC proliferation, motility and BM repopulation (Spiegel et al, Nat Immunol 2007; Lapid et al, JCI 2013). These findings indicate HSPC regulation by dynamic interactions of the sympathetic nervous and hematopoietic systems. However, the role of catecholamines in regulation of AML remains elusive. Results We found that several human AML cell lines from different FAB subtypes express b2-AR. NE, a b2-AR activating ligand, increased b2-AR expression and the CXCL12-induced migration of AML primary patients’ cells and cell lines. In addition, NE treatment significantly enhanced CXCL12induced actin polymerization, which drives most of the cellular movements. Looking for a downstream effector of b2-AR, we focused on RGS16, a G-protein signaling regulator, which negatively regulates CXCL12/CXCR4 axis (Berthebaud et al., Blood 2005). Concurrently with the increase in cell migration, NE decreased RGS16 expression (both in protein and mRNA level) in monocytic AML cells. However, no effect on either cell migration or RGS16 expression was observed in non-monocytic AML cells. These data provided evidence for the involvement of catecholamines in the regulation of AML cell migration and showed a correlation between AML FAB subtypes, cell motility and RGS16 expression. One of the regulators of RGS16 levels is miR126, which is highly expressed in AML and normal HSPCs and plays an important role in mobilization and proliferation of normal HSPCs. Indeed, in search for the mechanisms underlying the above observed differences, we found that the enhancing effect of NE on CXCL12-induced migration of monocytic AML cells was accompanied by up-regulation of miR126 expression concurrently with down-regulation of RGS16 expression, whereas in non-monocytic AML cells we observed the opposite effects, suggesting that NE differently regulates AML cells belonging to different FAB subtypes. Upon studying human normal HSPCs, we found that in steady state, normal cells express low levels of β2-AR and NE did not affect either RGS16 expression or CXCL12-induced migration of both mononuclear and CD34+ cells derived from human cord blood and BM. Conclusions Our results demonstrate that while normal and AML cells share common mechanisms that govern their motility, there are unrevealed yet mechanisms, apparently cell-type dependent, which uniquely lead to opposite effects in normal HSPCs, monocytic and non-monocytic AML cells. Altogether, these findings suggest that targeting of miR126 and RGS16 pathways by specific agonists and antagonists may serve as a new approach for selective eradication of LICs. Disclosures: No relevant conflicts of interest to declare.

Monocytic AML cells inactivate antileukemic lymphocytes: role of NADPH oxidase/gp91phox expression and the PARP-1/PAR pathway of apoptosis

AbstractDysfunction of T cells and natural killer (NK) cells has been proposed to determine the course of disease in acute myeloid leukemia (AML), but only limited information is available on the mechanisms of lymphocyte inhibition. We aimed to evaluate to what extent human malignant AML cells use NADPH oxidase-derived reactive oxygen species (ROS) as an immune evasion strategy. We report that a subset of malignant myelomonocytic and monocytic AML cells (French-American-British [FAB] classes M4 and M5, respectively), recovered from blood or BM of untreated AML patients at diagnosis, expressed the NADPH oxidase component gp91phox. Highly purified FAB M4/M5 AML cells produced large amounts of ROS on activation and triggered poly-[ADP-ribose] polymerase-1−dependent apoptosis in adjacent NK cells, CD4+ T cells, and CD8+ T cells. In contrast, immature (FAB class M1) and myeloblastic (FAB class M2) AML cells rarely expressed gp91phox, did not produce ROS, and did not trigger NK or T-cell apoptosis. Microarray data from 207 AML patients confirmed a greater expression of gp91phox mRNA by FAB-M4/M5 AML cells than FAB-M1 cells (P < 10−11) or FAB-M2 cells (P < 10−9). Our data are suggestive of a novel mechanism by which monocytic AML cells evade cell-mediated immunity.

Abstract 5414: Malignant monocytic AML cells trigger apoptosis in anti-leukemic lymphocytes: Role of NADPH oxidase gp91phox expression and PARP-1 activity

Abstract The dysfunction of natural killer (NK) cells and T cells in acute myeloid leukemia (AML) determines the course of disease, but only limited information is available on the mechanisms of lymphocyte inhibition. This study aimed to evaluate to what extent malignant AML cells utilize NADPH-oxidase derived reactive oxygen species (ROS) as an immune evasion strategy. We report that a subset of genetically defined malignant monocytic AML cells (FAB class M4/M5), recovered from blood or bone marrow of untreated AML patients at diagnosis, express gp91phox, a component of the NADPH oxidase. These AML cells produced large amounts of ROS upon activation and triggered PARP 1-dependent apoptosis in adjacent natural killer (NK) cells, CD4+ T cells, and cytotoxic T cells. In contrast, myeloblastic (FAB class M2) or immature (FAB class M1) AML cells did not express gp91phox, did not produce ROS, and did not trigger NK or T cell apoptosis. Our data are suggestive of a novel mechanism by which monocytic AML cells evade lymphocyte immunity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5414. doi:1538-7445.AM2012-5414