Monocytic Acute Myeloid Leukemia Research Articles

Selective eradication of venetoclax-resistant monocytic acute myeloid leukemia with iron oxide nanozymes

The clinical treatment of human acute myeloid leukemia (AML) is rapidly progressing from chemotherapy to targeted therapies led by the BCL-2 inhibitor venetoclax (VEN). Despite its unprecedented success, VEN still encounters clinical resistance. Thus, uncovering the biological vulnerability of VEN-resistant AML disease and identifying effective therapies to treat them are urgently needed. We have previously demonstrated that iron oxide nanozymes (IONE) are capable of overcoming chemoresistance in AML. The current study reports a new activity of IONE in overcoming VEN resistance. Specifically, we revealed an aberrant redox balance with excessive intracellular reactive oxygen species (ROS) in VEN-resistant monocytic AML. Treatment with IONE potently induced ROS-dependent cell death in monocytic AML in both cell lines and primary AML models. In primary AML with developmental heterogeneity containing primitive and monocytic subpopulations, IONE selectively eradicated the VEN-resistant ROS-high monocytic subpopulation, successfully resolving the challenge of developmental heterogeneity faced by VEN. Overall, our study revealed an aberrant redox balance as a therapeutic target for monocytic AML and identified a candidate IONE that could selectively and potently eradicate VEN-resistant monocytic disease.

Monocytic Differentiation of Human Acute Myeloid Leukemia Cells: A Proteomic and Phosphoproteomic Comparison of FAB-M4/M5 Patients with and without Nucleophosmin 1 Mutations.

Even though morphological signs of differentiation have a minimal impact on survival after intensive cytotoxic therapy for acute myeloid leukemia (AML), monocytic AML cell differentiation (i.e., classified as French/American/British (FAB) subtypes M4/M5) is associated with a different responsiveness both to Bcl-2 inhibition (decreased responsiveness) and possibly also bromodomain inhibition (increased responsiveness). FAB-M4/M5 patients are heterogeneous with regard to genetic abnormalities, even though monocytic differentiation is common for patients with Nucleophosmin 1 (NPM1) insertions/mutations; to further study the heterogeneity of FAB-M4/M5 patients we did a proteomic and phosphoproteomic comparison of FAB-M4/M5 patients with (n = 13) and without (n = 12) NPM1 mutations. The proteomic profile of NPM1-mutated FAB-M4/M5 patients was characterized by increased levels of proteins involved in the regulation of endocytosis/vesicle trafficking/organellar communication. In contrast, AML cells without NPM1 mutations were characterized by increased levels of several proteins involved in the regulation of cytoplasmic translation, including a large number of ribosomal proteins. The phosphoproteomic differences between the two groups were less extensive but reflected similar differences. To conclude, even though FAB classification/monocytic differentiation are associated with differences in responsiveness to new targeted therapies (e.g., Bcl-2 inhibition), our results shows that FAB-M4/M5 patients are heterogeneous with regard to important biological characteristics of the leukemic cells.

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.

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

Abstract Background: Myeloid epithelial reproductive tyrosine kinase (MERTK), a transmembrane protein receptor from the TAM (Tyro3, Axl, Mertk) family, is overexpressed in cancer and associated with reduced apoptosis and chemoresistance, making it an attractive therapeutic target. RGX-019-MMAE, a novel humanized IgG1-MMAE antibody-drug conjugate (ADC) (Inspirna, Inc), selectively binds to MERTK with high affinity on tumor cells, resulting in internalization and degradation of the receptor. It then induces cytotoxicity through the release of the payload, MMAE (monomethyl auristatin E), which disrupts mitosis. We hypothesize that targeting MERTK via RGX-019-MMAE exerts an antileukemic effect. Methods: First, to determine the clinical relevance of MERTK, using the Oregon Health and Science University (OHSU) acute myeloid leukemia (AML) dataset, we divided patients into low and high MERTK mRNA expression based on mean expression and compared their overall survival. To identify the AML subtypes with the highest MERTK expression, we analyzed samples from 810 AML patients at MD Anderson using reverse phase protein arrays (RPPA), as well as 8 AML cell lines and the peripheral blood of 9 AML patients using flow cytometry. Then, the cell lines with the highest MERTK expression were treated with varying doses of RGX-019-MMAE or isotype control for 120 hours and assessed for viability with CellTiter-Glo 2.0. Likewise, we administered RGX-019-MMAE to primary cells from AML patients and determined the anti-leukemic effect. Finally, we investigated the synergistic effect of RGX-019-MMAE with venetoclax, a BCL2 inhibitor, or cytarabine, a chemotherapy agent, in vitro. Results: In the AML OHSU dataset, patients with high MERTK mRNA expression had significantly worse overall survival (p=0.02). RPPA revealed that patients with PTPN11 mutation, t (9;11), and monocytic AML subtypes had significantly higher MERTK protein expression. MERTK protein expression varied in AML cell lines and was highest in Kasumi-1 and OCI-AML3. Treatment of these two cell lines with RGX-019-MMAE resulted in significantly more killing of leukemic cells than the isotype control ADC in a dose-dependent manner (p<0.01). Similarly, in primary AML cells with high MERTK expression, RGX-019-MMAE treatment induced significant cell death compared to the control ADC. In addition, Kasumi-1 and OCl-AML3 cells treated with RGX-019-MMAE combined with venetoclax or cytarabine showed a synergistic cytotoxic effect in a dose-dependent manner. Conclusion: High MERTK expression in monocytic AML suggested that MERTK is a promising therapeutic target in this subtype. RGX-019-MMAE significantly induced AML cell death and produced a synergistic anti-leukemic effect in combination with chemotherapeutic agents and targeted therapy in vitro, suggesting its potential for targeting patients with high-risk AML. Citation Format: Anudishi Tyagi, Maryam Siddiqui, Isabel Kurth, Shugaku Takeda, Amanda Eckstrom, Jenny Borgman, Abhishek Maiti, Venkata Lokesh Battula. Targeting myeloid epithelial tyrosine kinase (MERTK) receptor in acute myeloid leukemia using a novel antibody-drug conjugate, RGX-019-MMAE [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6341.

Comprehensive characterization of IFNγ signaling in acute myeloid leukemia reveals prognostic and therapeutic strategies

Interferon gamma (IFNγ) is a critical cytokine known for its diverse roles in immune regulation, inflammation, and tumor surveillance. However, while IFNγ levels were elevated in sera of most newly diagnosed acute myeloid leukemia (AML) patients, its complex interplay in AML remains insufficiently understood. We aim to characterize these complex interactions through comprehensive bulk and single-cell approaches in bone marrow of newly diagnosed AML patients. We identify monocytic AML as having a unique microenvironment characterized by IFNγ producing T and NK cells, high IFNγ signaling, and immunosuppressive features. IFNγ signaling score strongly correlates with venetoclax resistance in primary AML patient cells. Additionally, IFNγ treatment of primary AML patient cells increased venetoclax resistance. Lastly, a parsimonious 47-gene IFNγ score demonstrates robust prognostic value. In summary, our findings suggest that inhibiting IFNγ is a potential treatment strategy to overcoming venetoclax resistance and immune evasion in AML patients.

Myelomonocytic and monocytic acute myeloid leukemia demonstrate comparable poor outcomes with venetoclax-based treatment: a monocentric real-world study.

Venetoclax (VEN), a BCL-2 inhibitor, has transformed treatment strategies for elderly and unfit acute myeloid leukemia (AML) patients by significantly improving response rates and survival. However, the predictive factors for VEN efficacy differ from traditional chemotherapy. The clinical relevance of the FAB (French-American-British) monocytic subtype, including M4 and M5, has been debated as a marker for VEN resistance. This real-world study examined 162 newly diagnosed (ND) and 85 relapsed/refractory (R/R) AML patients who received VEN-based therapy at West China Hospital, Sichuan University, from January 2019 to January 2023. We retrospectively collected clinical and treatment data from electronic medical records. The median age of the cohort was 55.5years (range: 16.5-83.5). The composite complete remission (cCR) rate in the entire cohort was 60.7%. Specifically, among newly diagnosed (ND) patients, FAB monocytic subtypes exhibited lower cCR compared to non-monocytic subtypes (55.1% vs. 76.3%, P = 0.007). Additionally, there were no significant differences observed between M4 and M5 subtypes, both in the ND group (61.7% vs. 40.9%, p = 0.17) and the R/R group (38.2% vs. 40%, p > 0.9). Furthermore, the median follow-up was 238 (range: 7-1120) days. ND patients with monocytic subtypes had shorter overall survival compared to non-monocytic subtypes (295days vs. not reached, p = 0.0017). Conversely, R/R patients showed no such difference (204 vs. 266days, p = 0.72). In summary, our study suggests that the FAB monocytic subtype can predict VEN resistance and shorter survival in ND AML patients. Moreover, there is no significant distinction between M4 and M5 subtypes.

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.

N/KRAS-Mutant AML LSCs Originate from Committed Myelomonocytic Progenitors and Drive Clinical Resistance to Venetoclax

Driver mutations in acute myeloid leukemia (AML) often exhibit distinct temporal acquisition patterns, but the biological basis for this, if any, remains unknown. RAS mutations occur invariably late in AML, upon progression or relapsed/refractory disease, in contrast to their function as early drivers in solid tumors. We developed synthetic leukemogenesis models in human induced pluripotent stem cell (iPSC)- and primary cord blood (CB)-derived human hematopoietic stem/progenitor cells (HSPCs) by introducing the prototypical NRAS G12D mutation alone or with other mutational combinations, using CRISPR, and engraftment of a transplantable leukemia into NSG mice as the readout. RAS mutations alone were not sufficient for leukemogenesis, but required specific cooperating mutations. Subsequently, by creating temporally controlled engineered models, we found that RAS mutations are obligatory late events that can only drive leukemic transformation when acquired after, but not before, cooperating mutations. We provide the mechanistic explanation for this in a requirement for mutant RAS to specifically transform committed granulocyte-monocyte progenitors (GMPs) harboring previously acquired driver mutations, through sorting and transplantation and multi-omics experiments. In contrast, acquisition of RAS mutations in earlier progenitors (common myeloid progenitors, CMP), with or without cooperating mutations, blocked the emergence of GMPs and abolished engraftment or gave rise to a myeloproliferative neoplasm. These results indicate that advanced RAS-mutant (MT) leukemic clones have a different cell type of origin from the ancestral clone. Next, using single-cell transcriptomics in cells isolated from xenografts of two AML-iPSC lines generated from an AML patient with a subclonal KRAS G12D mutation, one capturing the RAS WT major clone and one the KRAS G12D subclone, we show that KRAS G12D leukemia stem cells (LSCs) give rise to leukemia with a more monocytic immunophenotype, while the ancestral clone within the same patient generates more leukemic cells with primitive and neutrophil markers. These results were corroborated by Genotyping of Transcriptomes (GoT) in another AML patient with subclonal NRAS G12D mutation. Recent studies have linked monocytic AML and, independently, RAS pathway mutations to poor responses to Venetoclax (VEN)-containing regiments. In view of the causal link we found between RAS mutations and monocytic disease, we evaluated the associations between monocytic AML, RAS mutations and response to VEN. We reanalyzed data from a cohort of 118 older/unfit newly diagnosed AML patients treated on a prospective clinical trial with VEN and decitabine (DEC) (NCT03404193). All outcomes were comparable between monocytic and non-monocytic groups, including overall response rate, duration of response (DOR) and overall survival. On the other hand, patients with N/KRAS mutations had significantly shorter DOR. These results support the presence of N/KRAS mutations and not the monocytic stage as predictors of inferior responses to VEN regimens. To definitely test this, we treated defined cell types generated from RAS WT and RAS MT iPSC lines - CD34+ LSC-like and CD11b+/CD68+/CD14+ monocytic cells - with VEN. In agreement with previous studies, monocytes were resistant and RAS WT LSCs were sensitive to VEN. However, strikingly RAS MT LSCs were VEN-resistant. Consistent with these, single-cell transcriptomics in patient-derived AML-iPSC cells in vivo, GoT in primary AML cells and bulk transcriptomics in synthetic iPSC-AML cells showed downregulation of BCL2 and upregulation of MCL1 in the RAS MT LSCs. This study provides the first example of a mechanistic explanation of why the timing of mutational acquisition in AML is so strongly biased. It shows for the first time that interaction between an oncogenic driver and the non-genetic developmental hematopoietic hierarchy imposes a specific LSC cell-of-origin restriction and in turn shapes the hierarchical structure of the resulting AML and critically determines therapeutic outcomes in patients. Importantly, our results have direct implications for clinical practice, as they support that RAS MT LSCs drive clinical resistance to VEN and that treatment with VEN in patients with RAS mutations can accelerate disease progression by selection of RAS MT LSCs (Figure).

Single-Cell Proteogenomics Analysis of AML Samples Treated with Decitabine and Venetoclax Reveals Divergent Treatment Escape Mechanisms

Background: The combination therapy of hypomethylating agents and venetoclax (HMA+VEN) has emerged as a standard of care for acute myeloid leukemia (AML) patients unfit for intensive chemotherapy. Nonetheless, significant clinical challenges persist, including relapse and treatment resistance. Previous studies have revealed genetic ( RAS/RTK pathway mutations and TP53 mutations) and phenotypic (monocytic differentiation) resistance mechanisms in the context of HMA+VEN therapy. Despite these advancements, our comprehensive understanding of the HMA+VEN resistance mechanisms remains incomplete. To gain further insights into the resistance mechanism in HMA+VEN therapy, we implemented single-cell proteogenomics analysis on longitudinally collected samples from AML patients treated with decitabine+venetoclax (DAC+VEN) trial. Methods: We performed a high-throughput, single-cell targeted DNA-antibody sequencing (DAb-seq) using Tapestri platform from Mission Bio Inc. Sixty-three AML samples sequentially collected from 33 unique patients who participated in DAC+VEN clinical trial (NCT03404193) were analyzed. Our custom DNA panel covered 37 recurrently mutated genes in AML, whereas the custom antibody panel included either 15 cell surface proteins (N = 20 patients) or 48 proteins (N = 13 patients). Cellular phenotypes were inferred based on the patterns of cell surface protein expression. Median 2,767 cells were sequenced per sample, with total 196,719 cells sequenced for this study. Results: The median age of the cohort was 70 years (range 21-84). Twenty-four (73%) patients had primary AML and 9 (27%) had secondary or therapy-related AML. Based on the FAB classification, 13 patients had M0/M1/M2, 6 had M4/M5, one had M7, and the rest had unknown subtypes. The most frequent gene mutation at baseline was DNMT3A (45%), followed by NPM1 (39%), TET2 (30%), and TP53 (24%). Among the 33 patients, 32 had evaluable treatment response; 27 (82%) were defined as responders by achieving CR or CRi, whereas 5 (15%) were non-responders. Six patients experienced relapse after responding to the therapy, and their baseline-relapse pairs were analyzed by the single-cell platform. Using the composition of cellular phenotype data, AML samples were classified into either stem-like (N = 5), progenitor-like (N = 1), predominantly monocytic (N = 4), predominantly erythroid (N = 2) and mixed population AML (mixed population of stem, progenitor, monocyte or erythroid-like population, N = 21). Genotype-phenotype analysis showed a significant correlation between the KRAS mutation and the mixed phenotype (p= 0.0403), as well as between the U2AF1 mutation and the mixed phenotype (p= 0.0471). There was no significant association between treatment response and AML phenotype. Analysis of six baseline-relapse pairs showed that relapse was associated with phenotypic shift of AML cells. Stem-like cells were largely eradicated at relapse and replaced by more differentiated cells. Among the six baseline-relapse pairs, three showed monocytic shift, one showed erythroid shift and two showed mixed monocytic/erythroid shift. Phenotypic shifts at relapse were not always associated with expansion of new genetic mutations, except for in two cases: one had monocytic shift accompanied with expansion of KRAS mutation, and another had erythroid shift accompanied with expansion of NRAS mutation. Conclusions: Phenotypic transition from stem-like cells to more differentiated cells (either monocytic or erythroid) was frequently observed in AML relapse following DAC+VEN therapy. These findings are consistent with the known dependency of these cells on non-BCL-2 anti-apoptotic proteins such as MCL-1 and BCL-xL. Close monitoring of these phenotypic alterations throughout the course of HMA+VEN treatment may potentially facilitate the precise identification of cellular populations predisposed to relapse.

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).

Defining a Targetable Leukemia Intrinsic Dependency on Noncanonical PI3Kgamma Signaling

There are overlapping clinical and pathologic characteristics between poor prognosis subsets of acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN), but common dependencies are ill-defined. We hypothesized that identifying novel shared targets and biomarkers could elucidate biology and nominate new therapies for both malignancies. We performed genome-wide CRISPR interference in BPDCN cells and identified 327 dependency genes, which were then analyzed informatically and functionally in AML, ALL, and BPDCN cells and patient-derived xenografts (PDXs). We defined a leukemia subset characterized by PIK3R5 activation and sensitivity to phosphoinositide 3-kinase gamma (PI3Kγ) inhibition via a noncanonical downstream pathway. The PI3Kγ complex includes an enzymatic subunit p110γ encoded by PIK3CG and a regulatory subunit p101 encoded by PIK3R5. Intriguingly, despite elevation of both p110γ and p101 proteins in this leukemia subset, only the PIK3R5 mRNA is overexpressed. To investigate this disconnection, we measured protein half-life and found that p110γ stability is positively regulated by PIK3R5/p101, associated with reduced p110γ ubiquitination. This suggested that increased expression of PIK3R5/p101 leads to posttranslational p110γ elevation by protecting it from ubiquitin proteasome-mediated degradation. To identify the upstream activator of PIK3R5, we analyzed RNA-seq data in >200 leukemia PDXs and found that AMLs with activated PIK3R5 are enriched for an innate immune response signature (IIRS). Supporting this model and indicating potential clinical relevance, analysis of TCGA AML patients revealed that the IIRS, which includes PIK3R5, was associated with M4/M5 monocytic AML and poor prognosis, independent of age, cytogenetics, or somatic mutations. Furthermore, in cells with low baseline expression, PIK3R5 was induced by the Toll-like receptor agonist resiquimod, showing that PIK3R5 is activated by inflammatory signaling and consistent with a monocytic/dendritic lineage association. By integrated analysis of ATAC-seq and ChIP-seq in PIK3R5 high vs low leukemias, as well as gene dependency scores, we identified the transcription factor PU.1 as responsible for PIK3R5 activation. Treatment with resiquimod enhanced the interaction between PU.1 and the PIK3R5 promoter, while depletion of PU.1 nullified the ability of resiquimod to activate PIK3R5. To elucidate molecular events downstream of p101/PI3Kγ, we studied consequences of pathway inactivation using PIK3R5 interference or the PI3Kγ-selective inhibitor eganelisib. Surprisingly, there was no effect of PI3Kγ inactivation on AKT phosphorylation or canonical PI3K-AKT-mTOR mRNA targets. Therefore, we performed phosphoproteomics and found that PAK1 S144, a phosphorylation site not previously associated with PI3K signaling, was suppressed by PIK3R5/PI3Kγ inhibition ( panel a). Selective inhibitors of PAK1, but not AKT, replicated the effect of eganelisib. Furthermore, introduction of constitutively active PAK1, but not inactive PAK1, eliminated sensitivity of leukemias to eganelisib, supporting the functional relationship between PAK1 phosphorylation and PI3Kγ dependency. PI3Kγ inhibitors are being tested in solid tumor trials to repolarize macrophages and potentiate immune checkpoint blockade, but their cancer cell-intrinsic role has not been explored. To assess PI3Kγ inhibition in vivo, we first performed intradermal xenografting assays using BPDCN derived from a patient's skin tumor. Eganelisib achieved comparable efficacy to cytarabine chemotherapy and the combination of eganelisib and cytarabine exerted significant synergy (combination index=0.63, p<0.0001). Next, we measured the effect of PI3Kγ inhibition on survival in disseminated PDX models. As predicted, single agent eganelisib significantly prolonged survival in AML, ALL, and BPDCN with high PIK3R5, while having no benefit in cases with low PIK3R5. To our surprise, the combination of eganelisib/cytarabine synergistically increased survival in all cases, regardless of baseline PIK3R5 ( panel b). We conclude that inflammatory signaling-activated PIK3R5 is a biomarker for sensitivity to PI3Kγ inhibition via an unappreciated PI3Kγ-PAK1 axis. Synergy with cytarabine unmasked eganelisib sensitivity in additional leukemias, expanding the therapeutic potential of targeting PI3Kγ.

IFNγ Signaling in Acute Myeloid Leukemia Mediates Immunomodulation and Reveals Therapeutic Strategies

Background: Interferon gamma (IFNγ) is a critical cytokine known for its diverse roles in immune regulation, inflammation, and tumor surveillance. Studies have reported the dichotomous nature of IFNγ signaling in both the pathogenesis of cancer and immunotherapy response. However, its complex interplay with acute myeloid leukemia (AML) remains insufficiently understood. Methods: Three independent RNA-seq datasets were integrated to disentangle the IFNγ signaling in AML. We also performed single cell RNA sequencing (scRNA) on 20 newly diagnosed AML patients to discern relative contribution of cells and cellular communications for IFNγ signaling. Results: Single-sample gene set enrichment analysis was used to examine the transcriptional programs linked to IFNγ signaling in 672 newly diagnosed adult AML patients. We observed higher IFNγ signaling score in subgroups of patients including diploid with monocytic differentiation, inv16 core-binding-factor AML, and del7/7q. Notably, sorted CD34 + cells from 17 healthy donors had markedly lower levels of IFNγ signaling scores than did those from AML patients, making it a predominant feature in AML. Additionally, we found significant positive correlations between IFNγ signaling score and expression of its downstream targets HLA class 1 and 2 as well as T cell dysfunction score, T cell exhaustion score, and T cell senescence score, consistent with the fact that chronic IFNγ can drive T cell dysfunction. In scRNA, we observed that AML cells in patients with diploid monocytic AML had the highest expression of IFNγ signaling followed by del7/7q, while the non-monocytic, diploid AML cells had the lowest (Figure 1A). We then explored whether IFNγ signaling is differentially activated across the AML hierarchies and found the spectrum along the hierarchy with the primitive cells displaying significant higher IFNγ signaling than GMP while reaching the highest in Mono-like state. Transcription factor analysis revealed high regulon activity of interferon regulator factors (IRFs) in AML cells, with elevated levels of IRF1 and IRF5 regulons in del7/7q and del5/5q, respectively and elevated IRF8 regulon in diploid AML cells with monocytic differentiation, consistent with its role as a lineage-determinant factor promoting monocytic differentiation. Cell-cell interactions revealed IFNγ from CD8 T cells and NK cells as a top interaction to AML cells. These observations indicate that the activation levels of IFNγ signaling in AML cells are associated with distinct cellular states and hierarchies, and that disparate regulons of IFNγ signaling characterize distinct patient subgroups. The high level of IFNγ signaling in monocytic patient prompted us to assess its correlation with drug response as monocytic subclones are suggested to have inherent resistance to venetoclax. In BEAT-AML ex vivo drug test data, we found a strong positive correlation between IFNγ signaling score and venetoclax resistance, indicating that IFNγ signaling confers venetoclax resistance. This correlation was validated in an independent cohort. However, the IFNγ signaling score did not predict survival outcomes in our bulk cohort. Therefore, we defined a light weighted IFNγ signature to improve the prognostic sensitivity using the least absolute shrinkage and selection operator model. After regression, 47 genes related to survival were retained, forming a parsimonious IFNγ signature. The new parsimonious IFNγ score revealed a tight positive correlation with HLA class 1 and 2 scores. Importantly, this parsimonious IFNγ score was able to predict patient outcomes in our bulk cohort, whereby a higher score predicted worse survival (Figure 1B). Ultimately, these results suggest that the IFNγ pathway activation is associated with resistance to venetoclax-based therapy and can predict patient outcomes independently of known risk factors, making it a promising target for therapeutic intervention. Conclusions: Characterization of inflammation in AML using independent bulk and scRNA profiling led to the identification of novel drug targets and mechanisms of resistance to targeted therapy. We identified monocytic AML as having a unique microenvironment characterized by high IFNγsignaling in AML cells and immunosuppressive features. IFNγ signaling scores correlated strongly with venetoclax resistance. A parsimonious IFNγ gene signature demonstrated robust prognostic value.

ATP1A1/BCL2L1 predicts the response of myelomonocytic and monocytic acute myeloid leukemia to cardiac glycosides

Myelomonocytic and monocytic acute myeloid leukemia (AML) subtypes are intrinsically resistant to venetoclax-based regimens. Identifying targetable vulnerabilities would limit resistance and relapse. We previously documented the synergism of venetoclax and cardiac glycoside (CG) combination in AML. Despite preclinical evidence, the repurposing of cardiac glycosides (CGs) in cancer therapy remained unsuccessful due to a lack of predictive biomarkers. We report that the ex vivo response of AML patient blasts and the in vitro sensitivity of established cell lines to the hemi-synthetic CG UNBS1450 correlates with the ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1)/BCL2 like 1 (BCL2L1) expression ratio. Publicly available AML datasets identify myelomonocytic/monocytic differentiation as the most robust prognostic feature, along with core-binding factor subunit beta (CBFB), lysine methyltransferase 2A (KMT2A) rearrangements, and missense Fms-related receptor tyrosine kinase 3 (FLT3) mutations. Mechanistically, BCL2L1 protects from cell death commitment induced by the CG-mediated stepwise triggering of ionic perturbation, protein synthesis inhibition, and MCL1 downregulation. In vivo, CGs showed an overall tolerable profile while impacting tumor growth with an effect ranging from tumor growth inhibition to regression. These findings suggest a predictive marker for CG repurposing in specific AML subtypes.

Abstract A13: Development of LILRB4 biparatopic synthetic T-cell receptor and antigen receptor (STAR)-T cells for the treatment of acute myeloid leukemia (AML)

Abstract Background: Acute myeloid leukemia (AML) is one of the most common hematopoietic malignancies. Adoptive cellular immunotherapy like chimeric antigen receptor (CAR) T therapy has demonstrated remarkable antitumor activity against B cell malignancies. However, CAR-T therapy exhibited very limited efficacy in treating AML due to the heterogeneity of AML cells and their similarity to normal hematopoietic cells and lack of leukemic cell-specific targets. LILRB4 (also known as ILT3, CD85k) is an immunosuppressive receptor that is highly expressed on monocyte AML blasts (FAB M4 and M5 AML subtypes), which makes it an ideal therapeutic target for monocytic AML. Here we generated LILRB4-specific nanobody-based STAR-T (Synthetic T-Cell Receptor and Antigen Receptor-T) cells, aiming to develop a new treatment strategy to improve outcomes for monocytic AML. Methods: LILRB4-specific nanobodies were obtained from alpacas immunized with the resombinant LILRB4 extracellular domain. These nanobodies were then fused to IgG1 Fc, expressed and purified from CHO cells and used for measuring the affinity, epitope binning, target binding specificity by SPR, BLI and flow cytometry. The LILRB4 STAR was designed as a double-chain TCR-based receptor with nanobodies fused to the N-terminus of the modified mouse TCR-Cα or TCR-Cβ, the two chains were linked with Furin-P2A sequence. Based on STAR structure, LILRB4 single epitopic STAR was generated by fusing an nanobody to the TCR-Cβ, whereas LILRB4 biparatopic STAR contained two nanobodies fused to the TCR-Cα and TCR-Cβ separately. In vitro assays and xenograft models were used to assess the potential of LILRB4 STAR-T cells for elimination of leukemic disease. Results and Conclusions: Multiple STAR-T cells were generated using a lentiviral-based vector and tested for their tumor lysis activity. Among multiple nanobodies tested, two nanobodies that displayed high antigen binding affinity and specificity were identified for further studies. They recognized two distinctive epitopes of LILRB4, and neither of them had competitive binding with APOE or FN1, known ligands of LILRB4. We generated both single paratopic and bi-paratopic STAR constructs and engineered the corresponding STAR-T cells. In vitro and in vivo cytotoxicity assays demonstrated that all LILRB4 STAR-T cells had high cytotoxic activity against LILRB4+ AML cell lines. In addition, LILRB4 biparatopic STAR-T cells exhibited the highest anti-leukemic activity and the best expansion in vivo. Therefore, the LILRB4 STAR-T cells described here may be further developed for the treatment of AML and represent a new immune cell therapy strategy for LILRB4+ AML patients. Citation Format: Wei Rui, Lei Lei, Zezhong Zhang, Chunyan Wu, Yu Xia, Yingying Liu, Xiaojing Pang, Ruifang Du, Lan Wang, Dengyue Sheng, Jing Guo, Yanyan Zhang, Xueqiang Zhao, James Pan. Development of LILRB4 biparatopic synthetic T-cell receptor and antigen receptor (STAR)-T cells for the treatment of acute myeloid leukemia (AML) [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 A13.

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.

Abstract 3185: Development of LILRB4 biparatopic synthetic T-cell receptor and antigen receptor (STAR)-T cells for the treatment of acute myeloid leukemia (AML)

Abstract Background: Acute myeloid leukemia (AML) is one of the most common hematopoietic malignancies. Adoptive cellular immunotherapy like chimeric antigen receptor (CAR) T therapy has demonstrated remarkable antitumor activity against B cell malignancies. However, CAR-T therapy exhibited very limited efficacy in treating AML due to the heterogeneity of AML cells and their similarity to normal hematopoietic cells and lack of leukemic cell-specific targets.LILRB4 (also known as ILT3, CD85k) is an immunosuppressive receptor that is highly expressed on monocyte AML blasts (FAB M4 and M5 AML subtypes), which makes it an ideal therapeutic target for monocytic AML. Here we generated LILRB4-specific nanobody-based STAR-T (Synthetic T-Cell Receptor and Antigen Receptor-T) cells, aiming to develop a new treatment strategy to improve outcomes for monocytic AML. Methods: LILRB4-specific nanobodies were obtained from alpacas immunized with the recombinant LILRB4 extracellular domain. These nanobodies were then fused to IgG1 Fc, expressed and purified from CHO cells and used for measuring the affinity, epitope binning, target binding specificity by SPR, BLI and flow cytometry. The LILRB4 STAR was designed as a double-chain TCR-based receptor with nanobodies fused to the N-terminus of the modified mouse TCR-Cα or TCR-Cβ, the two chains were linked with Furin-P2A sequence. Based on STAR structure, LILRB4 single epitopic STAR was generated by fusing an nanobody to the TCR-Cβ,whereas LILRB4 biparatopic STAR contained two nanobodies fused to the TCR-Cα and TCR-Cβ separately. In vitro assays and xenograft models were used to assess the potential of LILRB4 STAR-T cells for elimination of leukemic disease. Results and Conclusions: Multiple STAR-T cells were generated using a lentiviral-based vector and tested for their tumor lysis activity. Among multiple nanobodies tested, two nanobodies that displayed high antigen binding affinity and specificity were identified for further studies. They recognized two distinctive epitopes of LILRB4, and neither of them had competitive binding with APOE or FN1, known ligands of LILRB4. We generated both single paratopic and bi-paratopic STAR constructs and engineered the corresponding STAR-T cells. In vitro and in vivo cytotoxicity assays demonstrated that all LILRB4 STAR-T cells had high cytotoxic activity against LILRB4+ AML cell lines. In addition, LILRB4 biparatopic STAR-T cells exhibited the highest anti-leukemic activity and the best expansion in vivo. Therefore, the LILRB4 STAR-T cells described here may be further developed for the treatment of AML and represent a new immune cell therapy strategy for LILRB4+ AML patients. Citation Format: Wei Rui, Lei Lei, Zezhong Zhang, Chunyan Wu, Yu Xia, Yingying Liu, Xiaojing Pang, Ruifang Du, Lan Wang, Dengyue Sheng, Jing Guo, Yanyan Zhang, Xueqiang Zhao, James Pan. Development of LILRB4 biparatopic synthetic T-cell receptor and antigen receptor (STAR)-T cells for the treatment of acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3185.

High-expression of the innate-immune related gene UNC93B1 predicts inferior outcomes in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy with dismal prognosis. Identification of better biomarkers remained a priority to improve established stratification and guide therapeutic decisions. Therefore, we extracted the RNA sequence data and clinical characteristics of AML from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression database (GTEx) to identify the key factors for prognosis. We found UNC93B1 was highly expressed in AML patients and significantly linked to poor clinical features (p < 0.05). We further validated the high expression of UNC93B1 in another independent AML cohort from GEO datasets (p < 0.001) and performed quantitative PCR of patient samples to confirm the overexpression of UNC93B1 in AML (p < 0.005). Moreover, we discovered high level of UNC93B1 was an independent prognostic factor for poorer outcome both in univariate analysis and multivariate regression (p < 0.001). Then we built a nomogram model based on UNC93B1 expression, age, FAB subtype and cytogenetic risk, the concordance index of which for predicting overall survival was 0.729 (p < 0.001). Time-dependent ROC analysis for predicting survival outcome at different time points by UNC93B1 showed the cumulative 2-year survival rate was 43.7%, and 5-year survival rate was 21.9%. The differentially expressed genes (DEGs) between two groups divided by UNC93B1 expression level were enriched in innate immune signaling and metabolic process pathway. Protein-protein interaction (PPI) network indicated four hub genes (S100A9, CCR1, MRC1 and CD1C) interacted with UNC93B1, three of which were also significantly linked to inferior outcome. Furthermore, we discovered high UNC93B1 tended to be infiltrated by innate immune cells, including Macrophages, Dendritic cells, Neutrophils, Eosinophils, and NK CD56dim cells. We also found UNC93B1 had a significantly positive correlation with CD14, CD68 and almost all Toll-like receptors. Finally, we revealed negatively correlated expression of UNC93B1 and BCL2 in AML and conjectured that high-UNC93B1 monocytic AML is more resistant to venetoclax. And we found high MCL-1 expression compensated for BCL-2 loss, thus, we proposed MCL-1 inhibitor might overcome the resistance of venetoclax in AML. Altogether, our findings demonstrated the utility of UNC93B1 as a powerful poor prognostic predictor and alternative therapeutic target.

Phase 1b Trial of Anti-Immunoglobulin-like Transcript 3 Monoclonal Antibody MK-0482 Monotherapy in Relapsed/Refractory Acute Myeloid Leukemia and Chronic Myelomonocytic Leukemia

Background: Immunoglobulin-like transcript 3 (ILT3) is a cell surface inhibitory receptor expressed on various immune cells including dendritic cells, monocytic myeloid cells, and macrophages. The expression of ILT3 on dendritic cells and myeloid cells is thought to be involved in immune suppression and antigen-specific immune tolerance, contributing to an immunosuppressive tumor microenvironment. Acute myeloid leukemia (AML) is a type of cancer characterized by bone marrow that makes a large number of abnormal blood cells. In monocytic AML, ILT3 is highly expressed, and its signaling can mediate T-cell suppression and promote tumor infiltration (Deng M et al. Nature. 2018;562:605-609). Chronic myelomonocytic leukemia (CMML) is a myeloid neoplasm characterized by dysplasia, abnormal production and accumulation of monocytic cells, and with an elevated risk of transforming into AML. Based on the M2GEN database, ILT3 is also highly expressed in CMML. Thus, targeting ILT3 may exhibit antitumor activity in patients with AML and CMML. Currently, limited effective treatments are available for AML and CMML, especially in the relapse and refractory setting. MK-0482, a humanized IgG4 anti-ILT3 monoclonal antibody, has been evaluated as monotherapy and in combination with pembrolizumab at doses ≤2250 mg every 3 weeks (Q3W) in patients with advanced solid tumors. Patients treated with MK-0482 had a manageable safety profile, and modest antitumor activity was observed with MK-0482 plus pembrolizumab (Gutierrez M et al. J Clin Oncol. 2022;40. Abstract 2505). In this phase 1b study (NCT05038800), we are evaluating the safety and the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of MK-0482 in relapsed or refractory (r/r) AML and CMML. Study Design and Methods: Adult patients with AML with myelomonocytic or monoblastic/monocytic differentiation or CMML and with confirmed refractory or relapsed disease after treatment with available therapies will be eligible for enrollment. All patients must have an ECOG performance status of 0-2, adequate organ function, and a white blood cell count ≤20 × 109/L. Patients with active central nervous system leukemia, isolated extramedullary disease (ie no leukemic involvement), acute promyelocytic leukemia, or who have previously received allogeneic stem cell or organ transplant <60 days of treatment will be excluded. In part 1 (dose escalation), MK-0482 will be evaluated at dose levels ranging from 7.5 mg to 750 mg Q3W for ≤35 cycles with an accelerated titration design followed by a modified toxicity probability interval design in ~20 patients. After the recommended phase 2 dose (RP2D) is determined, 10 to 15 additional patients with AML with myelomonocytic or monoblastic/monocytic differentiation will be enrolled in part 2 (dose expansion). The primary objective of the study is to determine safety and tolerability and RP2D. Secondary objectives include characterizing the PK profile of MK-0482 monotherapy and antileukemia activities in AML as assessed by complete remission (CR), composite CR, and objective response per the 2017 European Leukemia Net AML response criteria. Exploratory objectives include assessment of receptor occupancy in peripheral blood and tumor biopsies, molecular biomarkers, and antileukemic activities in patients with CMML. Dose-limiting toxicities will be observed for 21 days after the first dose in cycle 1 and adverse events (AEs) will be assessed through 30 days after treatment discontinuation (90 days for serious AEs; 30 days if new anticancer therapy is initiated) per National Cancer Institute Common Terminology Criteria for Adverse Events, version 5.0. Disease assessment will be performed by investigator at baseline and prior to day 1 of cycles 3, 5, and 7, followed by every 3 cycles until disease progression or confirmed CR. Samples for PK analyses will be collected before and after treatment on day 1 of cycles 1-4, 6, 8, and every 4 cycles thereafter. The study is currently ongoing.

Preclinical Characterization of NGM936, a Novel Bispecific T Cell Engager Targeting ILT3 for the Treatment of Acute Myeloid Leukemia with Monocytic Differentiation

Acute myeloid leukemia (AML) with monocytic differentiation [M4 and M5 AML per the French-American-British (FAB) classification] accounts for 25-30% of all AML cases and has a particularly poor prognosis, with a <15% five-year overall survival rate and an increased risk of bone marrow and extramedullary relapse after stem cell transplant compared with other AML subtypes. The use of T cell engagers in AML has been limited by the lack of suitable target antigens with high specificity for AML cells and minimal expression on healthy bone marrow progenitors. Immunoglobulin-like transcript 3 (ILT3, also known as LILRB4) is a highly specific marker of AML with monocytic differentiation and is not expressed on non-monocytic blood cells or hematopoietic progenitor cells. This highly restricted expression profile makes ILT3 a promising target for monocytic AML, with the potential to enable effective elimination of AML cells with less toxicity than current therapies. Here, we report the pre-clinical characterization of NGM936, an ILT3-based T cell engager for the treatment of monocytic AML and other ILT3+ hematologic malignancies. NGM936 is a monovalent scFv-Fab bispecific on an effectorless human IgG1 backbone (LALA) with a knob-in-hole Fc design for heterodimeric assembly. NGM936 demonstrates sub-nM affinity towards ILT3 of human origin (KD = 0.2 nM at 25°C by surface plasmon resonance) and is not cross-reactive towards other LILRA/B family members. A panel of more than 30 ILT3 x CD3 engagers in various formats was generated and screened in assays to measure both T cell-dependent cytotoxicity and cytokine release. NGM936 was identified for its ability to potently induce T cell-dependent cytotoxicity (TDCC) against ILT3+ AML cells while inducing minimal cytokine release. In both whole blood cytokine release assays and in TDCC assays in which cytokine secretion was measured after target engagement, induction of TNF-α, IL-6, IFN-γ, and IL-2 by NGM936 was considerably lower than that induced by a vibecotamab biosimilar (CD123 x CD3). NGM936 induced potent cytotoxicity when both expanded and naïve T cells were used as effectors. Importantly, NGM936 efficiently ablated tumor cells with a range of ILT3 expression, from ~1500 copies/cell to ~40,000 copies/cell. In addition, NGM936 failed to induce T cell-dependent cytotoxicity against CD34+ hematopoietic stem cells or non-monocytic immune cells, consistent with the lack of ILT3 expression on these cell types. In ex vivo cultures of primary M5 AML bone marrow, NGM936 induced a dose-dependent depletion of AML cells and a concordant increase in T cell proliferation and activation. Finally, NGM936 induced a dose-dependent depletion of circulating tumor cells in a xenograft model in which irradiated, immunodeficient NSG mice were engrafted with human PBMCs and human ILT3+ AML cells. NGM936 thus represents a promising new treatment strategy for monocytic AML, with the potential to eliminate monocytic leukemia cells while minimizing the myelotoxicity associated with ablation of healthy bone marrow.