Treatment Of Acute Myeloid Leukemia 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.

Pityriasiform drug eruption associated with venetoclax for acute myeloid leukaemia.

Venetoclax is a targeted antileukaemic therapy that has emerged as the primary treatment of acute myeloid leukaemia in patients of advanced age or who would otherwise be ineligible for standard chemotherapy. Despite the documented evidence of cutaneous side effects of venetoclax, few reports have clarified presenting cutaneous features beyond the descriptors 'rash' and 'pruritus'. In this report, we describe the development of a pityriasiform drug eruption following venetoclax-based induction therapy for acute myeloid leukaemia. This study provides further evidence to characterise the range of cutaneous adverse events that are associated with venetoclax-based therapy. Further studies are needed to elucidate the epidemiology and pathophysiology of venetoclax-induced cutaneous toxicities.

Abstract 642: Flavopiridol inhibits cell proliferation, migration, and invasion via downregulation of FOXM1 in triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) represent 15-20% of all breast cancers and is the subtype of breast cancer that is not responsive to targeted breast cancer treatments such as anti-estrogens (tamoxifen) or HER2 targeted antibodies due to the lack of ER, PR, and HER2 receptors. TNBC is associated with poor prognosis, early relapse, and distant metastasis and drug resistance. With the use of standard chemotherapeutics in the neoadjuvant setting, unfortunately the majority of patients (~60%) with TNBC do not achieve complete remission and 40% of the patients with regional (Stage II) and 90% of the patients with distant metastasis (Stage IV) die within 5-years. Currently, there is no effective targeted therapy for TNBC patients for targeting oncogenic pathways. Although recently the FDA approved sacituzumab (govitecan), a Trop-2-receptor directed antibody conjugated with chemotherapeutic agent topoisomerase inhibitor in metastatic TNBC, and response rate was 30% and the median duration of response is 7.7 months, and the majority of patients did not maintain response longer than 12 months. Therefore, identifying novel molecular targets and developing alternative therapeutic strategies are urgently needed. FOXM1 is a protooncogenic transcription factor suppressed by p53 and drives expression of genes, which play a crucial role in promoting cancer cell proliferation, metastasis, progression, tumorigenesis of TNBC. We previously demonstrated for the first time that in vivo targeting of FOXM1 suppresses TNBC tumor growth in mice. Here, analyzing TCGA patient database we found that FOXM1 expression correlates with shorter patient survival and prognosis in TNBC patients. Overall data suggest that FOXM1 is a potential molecular target. However, currently, there is no FDA approved inhibitor of FOXM1. To identify potential inhibitors, utilizing silico docking and molecular dynamics studies we screened the FDA-approved compounds found that flavopiridol interacts with FOXM1. Flavopiridol is a small molecule inhibitor for CDKs and is approved by the FDA for the treatment of Acute Myeloid Leukemia. We demonstrated that flavodilol inhibits FOXM1 expression at nanomolar concentration and cell proliferation, migration, invasion, and induces apoptosis in TNBC cells. We are currently testing favopridol in MDA-MB-231 (human) and 4T1 mouse mammary) TNBC tumor models. In conclusion, our studies suggest that flavopiridol is a highly potent FOXM1 inhibitor and is a promising agent for repurposing to target FOXM1 and for the treatment of TNBC. Citation Format: Sayra Dilmac, Nermin Kahraman, Ferah Comert Onder, Zuhal Hamurcu, Bulent Ozpolat. Flavopiridol inhibits cell proliferation, migration, and invasion via downregulation of FOXM1 in triple negative breast cancer [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 642.

Abstract 6206: Molecular stratification of AML: Integrative genomic and immune profiling points to high risk subgroup and targetable molecular drivers

Abstract Background: Acute Myeloid Leukemia (AML) is a complex hematological malignancy characterized by clinical diversity. The ability to accurately subtype patients can inform prognosis and guide therapeutic approaches. With the integration of multi-omics data, it is possible to elucidate the molecular mechanisms underpinning unique AML subtypes, with intent to identify potential drug targeting pathways in a precision medicine strategy. Method: RNAseq datasets from Beat-AML (discovery, N=462), TCGA-AML (validation, N=173), and Leucegene (validation, N=452) were analyzed through non-negative matrix factorization to delineate distinct transcriptomic-driven molecular subtypes within AML. A curated set of the top 1,000 most variable genes was utilized to minimize analytical noise. GSVA was used for pathway analyses across Hallmark, KEGG, Reactome, and the immune microenvironment was characterized via xCell. Recurring DNA variants were examined in each subtype. Clinical outcomes of each subtype were analyzed using the Kaplan-Meier method and assessed by a log-rank test. These insights were subsequently validated in two independent cohorts - TCGA AML and Leucegene. The most influential components of immunological effector mechanisms, which characterize poor prognostic subpopulation, were assessed using bootstrap forest partitioning as a feature selection technique. Results: Our Beat-AML analysis identified 5 distinct clusters. Cluster 3 consisted of 20% of AML cases (n=86), and displayed a worse overall survival with a HR (95% CI) of 1.41 (1.03, 1.92, P=0.03). This cluster was characterized by three co-mutations: FLT3, NPM1, and DNMT3A. Pathway analysis highlighted biological processes key to antibody therapy intervention, specifically complement pathways, NK cell cytotoxicity, and FCGR-mediated phagocytosis as enriched within this subpopulation. Concurrently, oncogenic (PI3K/AKT/MTOR, TGFB, NOTCH) and metabolic pathways (fatty acid and oxidative phosphorylation) were activated. Immune profiling indicated increased levels of M2 macrophages. Using bootstrap forest partitioning, C2 and ITGAM were identified as key genes defining cluster 3. These observations were confirmed in TCGA AML and Leucegene cohorts. Conclusion: Our comprehensive analysis identified a distinct high-risk AML subgroup characterized by unique genomic, transcriptomic, and immunological patterns. The combined presence of NPM1/FLT3-ITD and DNMT3A mutations in cluster 3—each previously attributed with varied prognostic outcomes—warrants in-depth biological investigation. With these consistent AML subgroup results identified across three independent cohorts (total of 1,087 patients), we highlight the complexities within the molecular landscape of AML and emphasize the need for tailored therapeutic approaches - a transformative shift in AML treatment. Citation Format: Kubra Karagoz, Lauren Brady, Brandon Higgs, Han Si. Molecular stratification of AML: Integrative genomic and immune profiling points to high risk subgroup and targetable molecular drivers [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 6206.

Abstract 2548: Retrospective prediction of clinical response to standard-of-care therapies in acute myeloid leukemia by an ex vivo drug sensitivity assay using cryopreserved primary samples

Abstract Background: Assessing drug efficacy in primary acute myeloid leukemia (AML) patient cells is crucial for pinpointing drug targets and rationally selecting therapies for patients. We have previously shown that our flow cytometry-based predictive precision medicine platform (PPMP) predicts patient outcomes for standard-of-care (SOC) AML treatments using fresh blood or bone marrow samples with high precision. Here, we wanted to test the feasibility and efficacy of the PPMP on cryopreserved patient-derived leukemic samples. However, cryopreserved leukemic samples are known for exhibiting low viability and cell recovery, rendering their practical utilization often challenging. In this study, we adapt an existing methodology for cryopreservation for patient-derived leukemic cells to investigate the potential of cryopreserved samples as an alternative to fresh samples for ex vivo prediction of treatment outcomes on our PPMP. Methods: We screened freshly isolated white blood cells from AML patients on the PPMP to assess their response to multiple SOC single agents and combinations. The assay was also run without drug treatments immediately after receiving the fresh sample (≤ 2 days from draw) to establish a phenotype baseline. The cryopreserved samples were processed using a similar protocol, with post-thaw culture times of either 24 or 48 hours in two types of cytokine-containing media. A comparative analysis between untreated fresh and cryopreserved samples determined the optimal post-thaw culture time for cryopreserved samples. Once culture time was optimized, we screened cryopreserved samples on the PPMP with identical SOC drug conditions as the fresh samples and compared the response of leukemic blasts to drug treatment as well as impacts on other phenotypic markers. Results: Based on our preliminary data, we found that culturing cryopreserved cells for 24 hours post-thaw was sufficient to maintain a minimum cell viability of 80% while closely maintaining a phenotype that recapitulated that of the corresponding fresh samples. In response to drug treatments within the ex vivo assays, preliminary data support a correlation (Decitabine single agent, R2=0.81) between the response profiles of cryopreserved and fresh patient samples as gauged from AUC calculations. Conclusion: While the preferred method for screening is to use fresh patient samples, many practical constraints make it challenging or unfeasible. The ability of Notable Lab’s high-throughput platform to use cryopreserved samples opens the doors to enhanced retrospective studies leveraging clinically annotated data from biobanks or clinical trials. These retrospective studies provide a substantial reduction in the time needed to conclude such investigations. Citation Format: Lokesh Patil, Christine J. Gu, Wade Anderson, Nicola Long, Kara Johnson, Cristina Tognon, Markus D. Lacher. Retrospective prediction of clinical response to standard-of-care therapies in acute myeloid leukemia by an ex vivo drug sensitivity assay using cryopreserved primary samples [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 2548.

Abstract 2532: Deciphering multiple protein complexes in BCL2 family to measure apoptotic dependencies in acute myeloid leukemia

Abstract The BCL2 protein family controls apoptosis by shifting the dynamic equilibrium state of complex formation. BH3 mimetics disrupt this balance by disturbing the interaction of a specific anti-apoptotic protein, leading to the initiation of apoptosis. However, relying solely on molecular data for the specific anti-apoptotic protein can be challenging due to the functional redundancy within the BCL2 protein family and their complex interaction network. Therefore, comprehensive profiling information considering protein complexes is essential to determine apoptotic dependencies, particularly in a clinical context. In this study, we adapted a Single-molecule Protein Interaction Detection (SPID) platform that can assess levels of protein complexes in a quantitative manner. Out of the 47 metrics included in our Apoptosis Signaling 47 panel, we utilized 22 assays to measure apoptosis dependencies in acute myeloid leukemia (AML) cells obtained from 32 patients. By combining these metrics, we developed a predictive model for the ex vivo response to ABT-199 treatment, focusing on three key biomarkers: BCL2-BAX complex, BCL2-BIM binding potential, and BCLxl-BAK level. From a receiver operating curve (ROC) analysis, the model exhibited high accuracy with a value of 0.94 area under the curve (AUC). We further validated our model by comparing the predicted response to the therapeutic outcomes in 10 AML patients according to the 2017 ELN guidelines, and achieved 100% sensitivity (4 out of 4 identified correctly) and 83.3% specificity (5 out of 6 identified correctly). Overall, our platform provides an effective method to explore apoptosis dependencies in clinical samples based on the information regarding protein complexes within the BCL2 protein family. Three key molecular biomarkers can be translated into a gauge for predicting therapeutic outcomes of BH3 mimetics, suggesting an improvement of precision medicine in AML treatment. Citation Format: Hongwon Lee, Changju Chun, Byungsan Choi, Saem Hong, Yunseo Lee, Youngil Koh, Tae-Young Yoon. Deciphering multiple protein complexes in BCL2 family to measure apoptotic dependencies in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2532.

Abstract 6712: LBL-043, a novel LILRB4xCD3 T cell engager, for the treatment of LILRB4 positive leukemia

Abstract Background: Acute myeloid leukemia (AML) is a malignant clonal disorder linked to a broad spectrum of molecular alterations. It is still an incurable cancer, the prognosis is even worse in elderly patients, where overall survival (OS) at 1 year is approximately 10-15%. Now, several CD3 bispecific immunotherapies that harness T cells against AML are at various stages of preclinical and clinical development, but most of them are hampered by the lack of specific targets to distinguish tumor cells from normal hematopoietic stem cells (HSC). Leukocyte immunoglobulin-like receptor B4 (LILRB4) is an immune checkpoint inhibitory receptor, which is highly expressed on FAB M4 and M5 AML cells with a lack of expression on normal HSC and progenitor cells. LILRB4 expression level inversely correlates with OS of patients with M4 and M5 AML. All of these indicate that LILRB4 represents a promising target for developing a T cell redirecting bispecific antibody to treat patients with AML, especially for more aggressive M4 and M5 subtype. Herein a novel bispecific antibody targeting LILRB4 and CD3, LBL-043, is developed to specifically kill LILRB4 positive tumor cells by engaging T cells. Methods: LBL-043 was constructed using our proprietary LeadsBodyTM T cell engager platform, which is a 2:1 format bispecific antibody with two VHH arms targeting LILRB4 with high affinity and one scFv arm targeting CD3 with fine-tuned low affinity. The binding affinity of LBL-043 to LILRB4 and CD3 was determined with Fortebio, while the activity was measured using several cell based assays including reporter gene and TDCC assays. The anti-tumor activity of LBL-043 was investigated in B-NDG/hu-PBMC reconstructed mice implanted with MOLM-13 (human AML-M5a) tumor model. Results: The binding affinity of LBL-043 to LILRB4 and CD3 protein was 0.724 nM and 35.7 nM, respectively. In CD3 reporter gene assays, LBL-043 could activate the NFAT reporter signaling through LILRB4 binding dependent CD3 cross-linking with an EC50 value of 2.459 nM. LBL-043 was shown a potent TDCC activity in different LILRB4 expression level MOLM-13 cells and HL-60 cells with T cell activation and cytokine release. In B-NDG/hu-PBMC reconstructed mice implanted with MOLM-13 tumor cells, LBL-043 was shown significant anti-tumor activity. Simultaneously, LBL-043 can effectively improve the survival rate and maintain relatively stable body weight in the study compared to the vehicle group. Conclusion: LBL-043, a novel bispecific antibody targeting CD3 and LILRB4 with affinity differentiation, induces T cell killing of AML cells by simultaneously binding LILRB4 expressed on tumor cells and CD3 on T cells, redirecting T cells to efficiently killing of AML cells. It is also shown a great anti-tumor efficacy in CDX mouse models. These data support LBL-043 as a novel therapeutic bispecific antibody for the treatment of LILRB4 positive AML and CMML leukemia patients. Citation Format: Yujia Dang, Xiao Huang, Yurong Qin, Xiaoya Liu, Min Chen, Duqing Jiang, Guojin Wu, Mi Ye, Jianming Sun, Baohui Wang, Jing Guan, Tingting Li, Jordan Zhu, Shoupeng Lai, Xiaoqiang Kang, Hong Ling. LBL-043, a novel LILRB4xCD3 T cell engager, for the treatment of LILRB4 positive leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6712.

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

Abstract Dysregulated RNA splicing is a molecular feature that characterizes almost all tumor types. Splicing is altered in cancer owing to both recurrent mutations and deregulated expression of trans-acting factors that catalyze and regulate splicing. Therefore, splicing can be targeted for the development of new drugs to treat cancers, particularly those with mutations in splicing factors. In myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), genes that encode splicing factors are among those with the highest mutation rates. These mutations are heterozygous and largely mutually exclusive, suggesting the possibility of synthetic lethality due to multiple splicing factor mutations. Such synthetic lethality can be applied as an innovative strategy to develop drugs for the treatment of MDS and AML. We have reported the development of a CLK kinase inhibitor, CTX-712, and evaluated its anti-leukemic activities both in vitro and in vivo. The mechanism of action of CTX-712 involves the suppression of the phosphorylation of multiple SR proteins, including SRSF2/3/4/6, alteration of nuclear speckle morphology, and switching of binding partners of SRSF2. RNA-seq analysis revealed that CTX-712 induced global alterations in splicing, typically resulting in exon exclusion (exon skipping) of cassette exons. Notably, the anti-leukemic effects of CTX-712 positively correlated with the degree of altered splicing, suggesting that CTX-712 inhibits RNA splicing. [Cancer Res (2022) 82 (12_Supplement): 5494., Blood (2022) 140 (Supplement 1): 489-490]. However, patient stratification markers for CTX-712 based on the above mechanism are still undefined. To identify biomarkers for CTX-712, we performed a comprehensive analysis by integrating both wet and dry DNA/RNA sequencing on more than 60 ex vivo primary samples from MDS/AML patients and 23 MDS/AML-derived xenografts (PDX). Unexpectedly, regardless of their splicing factor-mutation status, various types of primary AML cells and PDX models were highly sensitive to CTX-712. Both the primary cells and PDX models showed significant responses to CTX-712 and greater changes in splicing. Additionally, we performed biomarker analysis by comparing gene mutations and RNA splicing patterns between the sensitive and insensitive models and associating them with the anti-tumor effect. We identified several potential biomarkers that can indicate sensitivity to CTX-712. Mechanistic studies that aim to determine why these biomarkers could make cancer more sensitive to splicing perturbation by CTX-712 are ongoing. In conclusion, our results demonstrated a significant effect of CTX-712 on MDS/AML-derived models regardless of their splicing factor-mutation status. Additionally, we present novel biomarkers that could predict sensitivity to CTX-712. Citation Format: Akinori Yoda, Daisuke Morishita, Yotaro Ochi, Akio Mizutani, Hirokazu Tozaki, Yoshihiko Satoh, Takuto Mori, June Takeda, Hideki Makishima, Masahiro Nakagawa, Yasuhito Nannya, Seishi Ogawa. Biomarkers for CLK inhibitor CTX-712 treatment response in myeloid neoplasms: Paving the way toward clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2081.

Differentiation syndrome associated with treatment with IDH2 inhibitor enasidenib: pooled analysis from clinical trials

AbstractTreatment with enasidenib, a selective mutant isocitrate dehydrogenase isoform 2 (IDH2) inhibitor, has been associated with the development of differentiation syndrome (DS) in patients with acute myeloid leukemia (AML). Studies on the incidence and clinical features of DS are limited in this setting, and diagnosis is challenging because of nonspecific symptoms. This study assessed the incidence, diagnostic criteria, risk factors, and correlation with clinical response of DS based on the pooled analysis of 4 clinical trials in patients with IDH2-mutated AML treated with enasidenib as monotherapy, or in combination with azacitidine or with chemotherapy. Across the total AML population, 67 of 643 (10.4%) had ≥1 any-grade DS event, with highest incidence in patients who received enasidenib plus azacitidine and lowest incidence in patients who received enasidenib plus chemotherapy (13/74 [17.6%] and 2/93 [2.2%]). The most common symptoms of DS were dyspnea/hypoxia (80.6%) and pulmonary infiltrate (73.1%). Median time to onset of first DS event across all studies was 32 days (range, 4-129). Most patients (88.1%) received systemic steroids for treatment of DS. Evaluation of baseline risk factors for DS identified higher levels of bone marrow blasts and lactate dehydrogenase as independent factors associated with increased grade 3 to 5 DS risk. Overall, these results suggest that DS associated with IDH inhibition is manageable, given the benefits of enasidenib treatment in IDH2-mutated AML. We further characterized enasidenib-related DS in these patients and identified risk factors, which could be used for DS management in clinical practice. These trials were registered at www.ClinicalTrials.gov as # NCT01915498, NCT02577406, NCT02677922, and NCT02632708.

Abstract 5852: Tumor gene expression patterns affecting response to BCl-2 inhibitor venetoclax in acute myeloid leukemia

Abstract Background: Venetoclax (VEN), a B-cell lymphoma-2 (BCL-2) inhibitor, is an important drug in acute myeloid leukemia (AML) treatment regimens. However, resistance to VEN-based therapy is a common challenge in AML management. Some factors contributing to resistance include dysregulation of BCL-2 family proteins, mutations in the TP53 gene, activating kinase mutations, and oxidative phosphorylation activity. Despite these insights, other mechanisms of resistance remain elusive. We aim to analyze tumor gene expression patterns associated with treatment response in patients who received VEN-based therapy. Methods: Molecular and clinical data of the patients were obtained as part of the Oncology Research Information Exchange Network (ORIEN) Avatar dataset. RNA-Seq data from diagnostic Bone Marrow were processed to remove batch effects, and differential expression analysis was performed using DESeq2, while correcting for age at sample collection and sex of patients. Results: We included 16 individuals treated with VEN-based therapy. Median age at the time of specimen collection was 65 years (interquartile range, 58-69), 9 (56%) male, 11 (69%) had active disease despite treatment with VEN. VEN-based therapy was used as 1st line, 2nd line, 3rd line, 4th line or more in 3, 4, 4, 2, and 3 patients, respectively. We found a total of 332 genes differentially expressed (fdr<0.05 and abs(log2FoldChange)>2.0) among patients having no evidence of active disease and those with no active disease at the time of last contact. Among those genes, we found HOXA7 and PRAME of particular interest. HOXA7 has been previously implicated in Bromodomain and Extraterminal Domain Inhibitors (BETi) inhibitor resistance in AML. This makes HOXA7 a potentially universal mechanism of chemotherapy resistance in AML. PRAME has been associated with leukemia cell survival, and its knockdown has been shown to induce apoptosis in K562 cells, which is only expressed in AML cells but not in normal hematopoietic cells. Pathway analysis found that the top two enriched pathways for the differentially expressed genes were MET activates PTK2 signaling (R-HSA-8874081) and Metabolism of Angiotensinogen to Angiotensins(R-HSA-2022377). Conclusions: This study highlights some molecular mechanisms that give rise to AML resistance to VEN. Our results indicate that HOXA7 may potentially be a universal mechanism of chemotherapy resistance not limited to VEN-based therapy. Similarly, pathways enriched in resistant cells could be targeted to prevent or treat resistance to VEN-based therapy. These and other findings may help us identify resistance mechanisms in all AML cases vs those specific to VEN-based therapy, allowing the development of treatment strategies based on treatment mechanism. Also, analysis of these mechanisms can aid therapeutic decision-making and the development of effective treatment strategies to improve outcomes in AML. Citation Format: Juan Antonio Raygoza Garay, Seongseok Yun, Afaf Osman, Reshma Ramlal, David Feith, Michelle Churchman, Prajwal Dhakal. Tumor gene expression patterns affecting response to BCl-2 inhibitor venetoclax in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5852.

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

Abstract Despite significant advancements in acute myeloid leukemia (AML) treatment, patients with relapsed/refractory (r/r) AML have limited therapeutic options. Novel approaches are urgently needed. CAR T-cell therapy has shown remarkable success in hematological malignancies. However, its application in AML has been hampered by the absence of highly AML-specific antigens, making current CAR T-cell AML therapies under development poorly tolerated and effective. This pre-clinical study introduces a novel CAR T-cell therapy, CCTx-001, specifically designed to target IL-1RAP, a 78-kDa transmembrane glycoprotein. IL-1RAP, is highly expressed on AML cells but not on healthy hematopoietic stem and progenitor cells (CD34+ HSPC). Additionally, transcriptomic analyses reveal a correlation between high IL-1RAP expression and poor prognosis in AML patients. IL-1RAP's predominant presence on myeloid leukemia cells, coupled with their dependence to IL-1RAP-related pathways for their survival, presents a promising avenue for CAR T-cell therapy without the risk of treatment-induced antigen loss. CCTx-001, an autologous CAR T-cell therapy, was developed to target IL-1RAP on AML cells. It comprises CD8+ and CD4+ T-cells transduced with a highly specific anti-IL-1RAP Single-Chain Variable Fragment (scFv), an IgG1 hinge region, CD28, 4-1BB co-stimulatory domains, and a CD3ζ activation domain. In vitro experiments confirmed CCTx-001's efficacy in killing AML cell lines and primary AML cells of varying IL-1RAP expression levels, also demonstrating cytotoxicity and cytokine release that was dependent on the (E:T) ratio and target expression. Furthermore, in vivo studies using immunocompromised NCG mice confirmed CCTx-001's ability to target IL-1RAP and inhibit the growth of IL-1RAP-positive human AML tumor xenografts, leading to increased survival time. Biodistribution analyses confirmed the successful adoptive transfer, expansion and persistence of CCTx-001 CAR T-cells in vivo, only when injected into IL-1RAP-expressing tumor-bearing mice, which correlated with reduced levels of circulating AML cells. The promising preclinical data presented herein warrant the evaluation of CCTx-001 CAR T-cell therapy in clinical trials for r/r AML patients. CCTx-001 CAR T-cell therapy offers a potential breakthrough in addressing the unmet medical needs of AML patients with resistant or refractory disease. Citation Format: Edouard De Dreuzy, Soha Reda El Sayed, Delphine Genin, Clementine Primus, Lucie Bouquet, Alexis Collette, Olivier Favre-Bulle, Erik Rutjens, Jens Hasskarl, Ksenija Pavletic, Marina Deschamps, Christophe Ferrand, Richard C. A. Sainson. CCTx-001 CAR T-cells targeting the AML specific antigen IL-1RAP show robust pre-clinical anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6322.

Abstract 3231: Pharmacological inhibition of the m6A RNA reader, YTHDC1, as a novel approach to targeting biomolecular condensates in cancer

Abstract Epigenetic dysregulation is a hallmark of cancer, and tumors have evolved complex mechanisms to fine-tune oncogenic gene expression. In acute myeloid leukemia (AML), one such regulatory mechanism involves post transcriptional modification of RNAs. The most abundant post transcriptional modification is methylation of adenosine at the N6 position (m6A). This modification is catalyzed by the m6A “writer”, METTL3 and METTL14 complex, and is removed by “erasers” such as FTO and ALKBH5. m6A RNAs can regulate a variety of cellular processes which can impact tumor phenotypes, including RNA stability, splicing, and nuclear export; and METTL3 catalytic inhibitors are currently in clinical development for AML and other cancers (NCT05584111).The m6A “reader”, YTHDC1, has been recognized as a critical contributor to the proliferation and survival of AML cells. As the primary nuclear m6A reader, YTHDC1 localizes with m6A marked RNAs in phase-separated assemblies, termed biomolecular condensates, helping to maintain the stability of oncogenic transcripts such as MYC. Here, we have employed our proprietary microfluidics based screening platform (termed PhaseScanTM) to identify modulators of YTHDC1 biomolecular condensates. Evaluation of early screening hits helped identify that disruption of the interaction between YTHDC1 and m6A RNA is a functionally relevant mechanism of action (MoA) that results in dissolution of YTHDC1 condensates. Using computational and knowledge based approaches, we have identified other structurally distinct inhibitors that occupy the RNA binding pocket of YTHDC1 and disrupt its interaction with m6A RNA at nanomolar potencies. These compounds disrupt cellular YTHDC1 condensates, suppress oncogenic gene expression, and result in anti proliferative and pro-differentiation phenotypes in AML cell lines. Compounds are selective for YTHDC1 over other YTH family members and are also selective in dissolving YTHDC1 condensates over other nuclear and cytosolic condensate systems. Our optimized chemical matter has suitable ADME properties to support in vivo studies. Thus, targeting YTHDC1 biomolecular condensates represents a novel approach to the treatment of AML. Citation Format: Richard C. Centore, Mark Charles, Mansi Arora, Yiwen Chen, Matthew Watson, Charlotte Kelley, Magdalena Czekalska, Marius Rebmann, Mahmoud Ghandi, Jerome Cattin, Nagakumar Bharatham, Prathima Radhakrishnan, Alex Howarth, Gale Rudlaff, William E. Arter, Seema Qamar, Kadi Saar, Douglas Williamson, Andrew Seeber, Tuomas Knowles, Shilpi Arora. Pharmacological inhibition of the m6A RNA reader, YTHDC1, as a novel approach to targeting biomolecular condensates in cancer [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 3231.

Abstract 5718: TGN-1062, a dual CDK7 and FLT3 Inhibitor, shows potent anti-AML activity and synergizes with Venetoclax

Abstract Purpose: Acute Myeloid leukemia (AML) is an aggressive malignancy of the hematopoietic system with poor survival rates. The best responses to treatment are achieved for young and fit patients who can tolerate chemotherapy followed by stem cell transplantation compared to older patients unfit to take intense chemotherapy. Since its approval venetoclax, a BCL2 inhibitor, has been the standard treatment for older patients in combination with a hypomethylating agent (HMA), such as azacitidine or decitabine, with a 76% complete response (CR). Despite this success as a frontline treatment, there is limited response for patients with relapse and refractory (r/r) and development of novel agents is required to improve overall therapeutic response. MYC has been shown to be frequently activated in AML and plays an important role in the induction of leukemogenesis and leukemic progression. While BCL2 inhibition via venetoclax has, in part, been shown to control c-MYC activation, the addition of another inhibitor which controls MYC may prove beneficial. CDK7 plays a dual role in tumor progression by regulating the cell cycle and transcription. CDK7 has also been shown to be associated with the overexpression of oncogenic driver genes such as BCL2 and MYC. Previously, we have presented the development of a potent and reversible CDK7 inhibitor, TGN-1062. Here, we have generated data supporting the potential benefit of CDK7 inhibition in combination with standard of care therapy for AML. Methods: AML lines were co-treated with TGN-1062, venetoclax, and/or an HMA, assessed for viabilities with CellTiter-Glo, and analyzed with the Bliss synergy model. AML CDX or PDX cells were implanted subcutaneously or transplanted via tail vein, respectively, and treated with TGN-1062 and/or venetoclax. Subcutaneous tumors were measured to determine tumor growth inhibition (TGI) and correlating drug accumulation levels were measured in tumors and plasma using mass spectrometry. Leukemic burden and survival were evaluated at the end of treatment for transplanted models. Immunohistochemistry (IHC), Western blotting, and RT-PCR were performed to determine changes in relevant biomarkers in tumors and/or AML cell lines. Results: Previously we have reported that TGN-1062 was effective in suppressing the growth of MV4-11 AML model. We extend these studies and show that TGN-1062 has activity at lower doses in both in vitro and in vivo AML models. In addition, we show synergy between TGN-1062 and other agents for AML treatment, including venetoclax or an HMA. Western blotting demonstrated significant changes in apoptotic markers. Survival of mice bearing AML was significantly increased with TGN-1062 treatment and combination treatment with venetoclax produced the longest survival. Conclusion: Targeting CDK7 with TGN-1062 in combination with venetoclax or an HMA is a promising therapeutic approach for treatment of AML and needs further investigation. Citation Format: Trason Thode, Le Xuan Truong Nguyen, Brian Durbin, Alexis Weston, Serina Ng, Tithi Ghosh Halder, Taylor Bargenquast, Raffaella Soldi, Srinivas Kasibhatla, Vincent Chung, Victoria Villaflor, Mohan Kaadige, Guido Marcucci, Sunil Sharma. TGN-1062, a dual CDK7 and FLT3 Inhibitor, shows potent anti-AML activity and synergizes with Venetoclax [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 5718.

CAR’TCR-T cells co-expressing CD33-CAR and dNPM1-TCR as superior dual-targeting approach for AML treatment

Acute myeloid leukemia (AML), a fast-progressing hematological malignancy affecting myeloid cells, is typically treated with chemotherapy or hematopoietic stem cell transplantation. However, approximately half of the patients face relapses and five-year survival rates are poor. With the goal to facilitate dual-specificity, boosting anti-tumor activity, and minimizing the risk for antigen escape, this study focused on combining chimeric antigen receptor (CAR) and T cell receptor (TCR) technologies. CAR’TCR-T cells, co-expressing a CD33-CAR and a transgenic dNPM1-TCR, revealed increased and prolonged anti-tumor activity in vitro, particularly in case of low target antigen expression. The distinct transcriptomic profile suggested enhanced formation of immunological synapses, activation, and signaling. Complete elimination of AML xenografts in vivo was only achieved with a cell product containing CAR’TCR-T, CAR-T and TCR-T cells, representing the outcome of co-transduction with two lentiviral vectors encoding either CAR or TCR. A mixture of CAR-T and TCR-T cells, without CAR’TCR-T cells, did not prevent progressive tumor outgrowth and was comparable to treatment with CAR-T and TCR-T cells individually. Overall, our data underscore the efficacy of co-expressing CAR and transgenic TCR in one T cell, and might open a novel therapeutic avenue not only for AML but also other malignancies.

Abstract 3218: Combination drug screen identifies novel epigenetic therapies to enhance menin inhibitor efficacy in KMT2A rearranged and NPM1 mutant AML

Abstract Background: Acute myeloid leukemia (AML) with KMT2A-rearrangements have a poor prognosis and so there is an urgent need for novel therapies. Menin inhibitors have shown promising anti-leukemic effects in KMT2A-rearranged and NPM1-mutated AML. However, the efficacy of menin inhibitors as single agents may be limited by the development of resistance mutations. Combining menin inhibitors with other epigenetic drugs may present a strategy to enhance their therapeutic potential. In this study, we performed a combination drug screen using an epigenetic drug library in KMT2A-rearranged (MV4;11) and NPM1-mutant (OCI-AML3) AML cell lines to identify synergistic therapies that can be used in conjunction with menin inhibitors. Methods: During assay optimization, MV4;11 and OCI-AML3 cells were treated with the menin inhibitor DSP-5336, and their viability was assessed. Quality control metrics were applied with Z’ values consistently greater than 0.5 for all assay plates and then the cells were subjected to a small molecule compound library screen containing a set of 932 epigenetic drugs. The screening process included a systematic evaluation of each drug's effect on cell viability in combination with the DSP-5336. Results: The combination drug screen demonstrated robust performance in both MV4;11 and OCI-AML3 cell lines, with reliable quality control metrics. Among the epigenetic drugs tested, five compounds were identified as potential hits that synergistically enhanced the efficacy of DSP-5336 across both cell lines.The five identified compounds exhibited enhanced anti-leukemic effects when combined with the menin inhibitor in either one of the cell lines. The targets of these drugs varied, including histone deacetylases, histone acetyltransferases and BRD4 inhibition, suggesting many alternate mechanisms of action involving epigenetic modulation. Remarkably, one of the 5 hits C301-3161, a KAT2A inhibitor, showed limited activity in either cell line alone but increased cell killing with DSP-5336 in both cell lines, highlighting its potential as a specific therapeutic for combining with DSP-5336 in AML patients. Conclusions: Our findings suggest combining menin inhibitors with other epigenetic drugs may improve AML treatment outcomes. The combination drug screen successfully identified four novel compounds that synergistically enhance menin inhibitor efficacy in both KMT2A-rearranged and NPM1-mutant AML cell lines. These compounds hold significant promise as potential therapeutic candidates for further investigation. Future studies should focus on elucidating the underlying mechanisms of action for each hit and evaluating their effects in preclinical animal models. Ultimately, this research may pave the way for the development of innovative combination therapeutic strategies for AML patients with KMT2A-rearragned and NPM1-mutant AML. Citation Format: Tulasigeri M. Totiger, Sana Chaudhry, Skye Montoya, Maurizio Affer, Anya K. Sondhi, Alexandra Chirino, Claude-Henry Volmar, Shaun Brothers, Justin Watts, Justin Taylor. Combination drug screen identifies novel epigenetic therapies to enhance menin inhibitor efficacy in KMT2A rearranged and NPM1 mutant 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 3218.

Abstract 1363: Blocking inhibitory receptor ILT3 by an antibody with enhanced Fc function promoted adaptive immunity against hematopoietic malignancy independent of ADCC

Abstract Immunoglobulin-like transcript 3 (ILT3) is an inhibitory receptor that is expressed by mature monocytes, dendritic cells (DC), plasma blasts, and the malignant cells arising thereof including acute myeloid leukemia (AML), B cell lymphoma and multiple myeloma (MM). We developed an anti-ILT3 blocking monoclonal antibody (mAb) 17G8 to test the hypothesis that blockade of ILT3 on hematopoietic malignant cells might drive anti-cancer responses through several mechanisms such as cell migration, ADCC, and T cell driven immune responses. Blockade of ILT3 using 17G8 without Fc effector function impaired AML cell line THP1 dissemination into distant sites in vivo and using 17G8 with enhanced Fc function through afucosylation further reduced the tumor burden. This effect of afucosylated 17G8 appears to be mediated by a mechanism other than ADCC, since afucosylated 17G8 showed modest tumor inhibition in ICR mice carrying ILT3 positive AML and MM xenografts. In immune competent mice carrying murine syngeneic AML cell C1498 expressing human ILT3, 17G8 also showed modest anti-tumor effects as a single agent, however, when combined with anti-PD1, afucosylated 17G8 increased CD8+ effector memory T cells, reduced proportion of cells with an exhausted phenotype and significantly improved survival when compared to different controls. Since T cells don’t express ILT3, the effects of 17G8 on T cells are hypothesized to be mediated by blocking ILT3 expressed on myeloid cells. The expression of ILT3 on immature human monocyte-derived DC (moDC) is higher than on mature moDC. Blocking ILT3 with afucosylated 17G8 on immature moDC potentiated maturation of moDC when compared to the controls in vitro. Surprisingly, afucosylated 17G8 stimulated IFNγ production from moDC during maturation. The enhanced DC function induced by afucosylated 17G8 was subsequently found to be translated into enhanced T cell function in an allogeneic mixed lymphocyte reaction (allo-MLR) assay, in which afucosylated 17G8 significantly increased IFNγ production when combined with anti-PD1. The activation of effector T cells was also monitored through a cytotoxicity assay by mixing THP-1 cells with human PBMC in culture. Afucosylated 17G8 induced strong cytotoxicity on THP-1 cells with minimal effects on normal ILT3 positive cells such as monocytes or moDC, suggesting that blockade of ILT3 with afucosylated 17G8 induced more potent T cell mediated allogeneic cytotoxicity than innate cell mediated ADCC killing. In summary, ILT3 is a highly selective M4/M5 AML target. Developing a blocking antibody with enhanced Fc function could add an additional therapeutic strategy of inducing host anti-tumor immunity to current AML treatments which induce direct tumor killing, such as chemotherapy, targeted therapy, and antibody drug conjugate. Citation Format: Shiming Ye, Dong Zhang, Min-Zu Wu, Harini Raghu, Diane Cohen, Mally Romero, Siu-Sze Tan, Donghee Choi, Jonathan Hickson, John Engelhardt, Alex Shoemaker. Blocking inhibitory receptor ILT3 by an antibody with enhanced Fc function promoted adaptive immunity against hematopoietic malignancy independent of ADCC [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 1363.

Abstract 479: Enhanced payload delivery for acute myeloid leukemia treatment through CD37-targeting DNA nanobots

Abstract Acute Myeloid Leukemia (AML) treatment faces considerable challenges, in particular the delivery of therapeutics with sufficient potency and specificity. Traditional Antibody-Drug Conjugates (ADCs) offer a strategic approach to cancer therapy by combining the targeting capabilities of monoclonal antibodies with the cell-killing effect of cytotoxic drugs. However, the limitations in drug-to-antibody ratio (DAR) and the challenges in controlling the release kinetics have often hindered their therapeutic window suggesting novel approaches are necessary. Our approach integrates DNA nanobot technology to facilitate targeted delivery of anthracycline agents directly to CD37-expressing leukemic cells. CD37, a tetraspanin superfamily antigen, has shown promise as a target for AML due to its selective expression on immune cells and favorable internalization properties. By harnessing DNA origami assembly techniques, we developed a customizable platform capable of accommodating a significantly higher payload of chemotherapeutic agents than traditional ADCs. This increased payload potential, paired with the precision targeting of CD37, presents a potent therapeutic strategy with the possibility of reduced systemic toxicity. Here we present preclinical evidence demonstrating targeted delivery of our DNA nanobot drug delivery device and anthracycline payload to CD37+ AML target cells in vitro. In addition, we show targeted efficacy in vitro and in vivo of our anthracycline-loaded DNA nanobot-enabled delivery system, which significantly outperformed free anthracycline in AML target cells. Collectively, our findings suggest an improvement in the therapeutic index of anthracycline and antimitotic agents as well as the ability to combine multiple drug pathways in a single delivery device. The implications of this work extend beyond AML, offering a versatile platform that could revolutionize ADC chemistry and targeted drug delivery. We propose that our DNA nanobot delivery system signifies a pivotal advancement in the field of targeted cancer therapy, with the potential to overcome longstanding barriers in the treatment of AML and potentially other malignancies. Citation Format: Nicholas Vantangoli, Patrick D. Halley, Jeffrey R. Spitzner, John C. Byrd, Karilyn T. Larkin, Carlos E. Castro, Christopher R. Lucas. Enhanced payload delivery for acute myeloid leukemia treatment through CD37-targeting DNA nanobots [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 479.

Red blood cell membrane-coated FLT3 inhibitor nanoparticles to enhance FLT3-ITD acute myeloid leukemia treatment

FMS-like tyrosine kinase 3 (FLT3) is a viable and important therapeutic target for acute myeloid leukemia (AML). FLT3 internal tandem duplication (FLT3-ITD) mutations have been identified in approximately 30% of AML patients, and are associated with unfavorable prognosis, higher risk of relapse, drug resistance, and poor clinical outcome. Even FLT3 inhibitors have demonstrated promising efficacy, they cannot cure AML or even significantly extend the lives of patients with FLT3-ITD mutations. This is partly because of poor water solubility, insufficient membrane penetration and short half-life of small molecule inhibitors. Besides, the presence of enzymes like CYP3A4 in bone marrow accelerate the elimination and metabolism of FLT3 inhibitors, resulting in low plasma concentrations and side effects. Here we report the erythrocyte membrane-camouflaged FLT3 inhibitor nanoparticles to enhance FLT3-ITD AML treatment. Briefly, we physically coextruded red blood cell (RBC) membrane vesicles with nanoparticles derived from FLT3 inhibitor F30 to obtain F30@RBC-M, which exhibited comparable potent FLT3-ITD inhibitory effects compared to free F30in vitro, while displaying a higher potent antitumor efficacy in xenograft models due to the prolonged circulation properties. Furthermore, administration of F30@RBC-M significantly extended the survival of mice in a transplanted mouse model than F30 free drug. These findings suggest that RBC membrane-coated nanoparticles derived from FLT3 inhibitors hold promise as a tool to enhance the therapeutic efficacy to treat FLT3-ITD AML.

Differential Gene Regulatory Network Analysis between Azacitidine-Sensitive and -Resistant Cell Lines.

Azacitidine, a DNA methylation inhibitor, is employed for the treatment of acute myeloid leukemia (AML). However, drug resistance remains a major challenge for effective azacitidine chemotherapy, though several studies have attempted to uncover the mechanisms of azacitidine resistance. With the aim to identify the mechanisms underlying acquired azacitidine resistance in cancer cell lines, we developed a computational strategy that can identify differentially regulated gene networks between drug-sensitive and -resistant cell lines by extending the existing method, differentially coexpressed gene sets (DiffCoEx). The technique specifically focuses on cell line-specific gene network analysis. We applied our method to gene networks specific to azacitidine sensitivity and identified differentially regulated gene networks between azacitidine-sensitive and -resistant cell lines. The molecular interplay between the metallothionein gene family, C19orf33, ELF3, GRB7, IL18, NRN1, and RBM47 were identified as differentially regulated gene network in drug resistant cell lines. The biological mechanisms associated with azacitidine and AML for the markers in the identified networks were verified through the literature. Our results suggest that controlling the identified genes (e.g., the metallothionein gene family) and "cellular response"-related pathways ("cellular response to zinc ion", "cellular response to copper ion", and "cellular response to cadmium ion", where the enriched functional-related genes are MT2A, MT1F, MT1G, and MT1E) may provide crucial clues to address azacitidine resistance in patients with AML. We expect that our strategy will be a useful tool to uncover patient-specific molecular interplay that provides crucial clues for precision medicine in not only gastric cancer but also complex diseases.

CLPs-miR-103a-2-5p inhibits proliferation and promotes cell apoptosis in AML cells by targeting LILRB3 and Nrf2/HO-1 axis, regulating CD8 + T cell response

BackgroundLILRB3, a member of the leukocyte immunoglobulin-like receptor B (LILRB) family, has immunosuppressive functions and directly regulates cancer development, which indicates that LILRB3 is an attractive target for cancer diagnosis and therapy. Novel therapeutic treatments for acute myeloid leukemia (AML) are urgent and important, and RNA therapeutics including microRNAs (miRNAs) could be an effective option. Here, we investigate the role of dysregulated miRNA targeting LILRB3 in the AML microenvironment.MethodsPotential miRNAs binding to the 3ʹ-untranslated region (3ʹ-UTR) of the LILRB3 mRNA were predicted by bioinformatics websites. Then, we screened miRNAs targeting LILRB3 by quantitative real-time PCR, and the dual luciferase reporter assay. The expression of LILRB3 and microRNA (miR)-103a-2-5p in AML were determined and then their interactions were also analyzed. In vitro, the effects of miR-103a-2-5p were determined by CCK8, colony formation assay, and transwell assay, while cell apoptosis and cell cycle were analyzed by flow cytometry. Cationic liposomes (CLPs) were used for the delivery of miR-103a-2-5p in the AML mouse model, which was to validate the potential roles of miR-103a-2-5p in vivo.ResultsLILRB3 was upregulated in AML cells while miR-103a-2-5p was dramatically downregulated. Thus, a negative correlation was found between them. MiR-103a-2-5p directly targeted LILRB3 in AML cells. Overexpressed miR-103a-2-5p significantly suppressed the mRNA and protein levels of LILRB3, thereby inhibiting AML cell growth and reducing CD8 + T cell apoptosis. In addition, overexpressed miR-103a-2-5p reduced both the relative expression of Nrf2/HO-1 pathway-related proteins and the ratio of GSH/ROS, leading to the excessive intracellular ROS that may promote AML cell apoptosis. In the mouse model, the delivery of miR-103a-2-5p through CLPs could inhibit tumor growth.ConclusionsMiR-103a-2-5p serves as a tumor suppressor that could inhibit AML cell proliferation and promote their apoptosis by downregulating LILRB3 expression, suppressing the Nrf2/HO-1 axis, and reducing the ratio of GSH/ROS. Besides, our findings indicate that miR-103a-2-5p may enhance the CD8 + T cell response by inhibiting LILRB3 expression. Therefore, the delivery of miR-103a-2-5p through CLPs could be useful for the treatment of AML.