Targeting BMAL1 reverses drug resistance of acute myeloid leukemia cells and promotes ferroptosis through HMGB1-GPX4 signaling pathway.

PURPOSE: Acute myeloid leukemia (AML) is a refractory hematologic malignancy that poses a serious threat to human health. Exploring alternative therapeutic strategies capable of inducing alternative modes of cell death, such as ferroptosis, holds great promise as a viable and effective intervention. METHODS: We analyzed online database data and collected clinical samples to verify the expression and function of BMAL1 in AML. We conducted experiments on AML cell proliferation, cell cycle, ferroptosis, and chemotherapy resistance by overexpressing/knocking down BMAL1 and using assays such as MDA detection and BODIPY 581/591 C11 staining. We validated the transcriptional regulation of HMGB1 by BMAL1 through ChIP assay, luciferase assay, RNA level detection, and western blotting. Finally, we confirmed the results of our cell experiments at the animal level. RESULTS: BMAL1 up-regulation is an observed phenomenon in AML patients. Furthermore, there existed a strong correlation between elevated levels of BMAL1 expression and inferior prognosis in individuals with AML. We found that knocking down BMAL1 inhibited AML cell growth by blocking the cell cycle. Conversely, overexpressing BMAL1 promoted AML cell proliferation. Moreover, our research results revealed that BMAL1 inhibited ferroptosis in AML cells through BMAL1-HMGB1-GPX4 pathway. Finally, knocking down BMAL1 can enhance the efficacy of certain first-line cancer therapeutic drugs, including venetoclax, dasatinib, and sorafenib. CONCLUSION: Our research results suggest that BMAL1 plays a crucial regulatory role in AML cell proliferation, drug resistance, and ferroptosis. BMAL1 could be a potential important therapeutic target for AML.

A novel function of STAT3ß in suppressing interferon response improves outcome in acute myeloid leukemia.

Signal transducer and activator of transcription 3 (STAT3) is frequently overexpressed in patients with acute myeloid leukemia (AML). STAT3 exists in two distinct alternatively spliced isoforms, the full-length isoform STAT3α and the C-terminally truncated isoform STAT3ß. While STAT3α is predominantly described as an oncogenic driver, STAT3ß has been suggested to act as a tumor suppressor. To elucidate the role of STAT3ß in AML, we established a mouse model of STAT3ß-deficient, MLL-AF9-driven AML. STAT3ß deficiency significantly shortened survival of leukemic mice confirming its role as a tumor suppressor. Furthermore, RNA sequencing revealed enhanced STAT1 expression and interferon (IFN) signaling upon loss of STAT3ß. Accordingly, STAT3ß-deficient leukemia cells displayed enhanced sensitivity to blockade of IFN signaling through both an IFNAR1 blocking antibody and the JAK1/2 inhibitor Ruxolitinib. Analysis of human AML patient samples confirmed that elevated expression of IFN-inducible genes correlated with poor overall survival and low STAT3ß expression. Together, our data corroborate the tumor suppressive role of STAT3ß in a mouse model in vivo. Moreover, they provide evidence that its tumor suppressive function is linked to repression of the STAT1-mediated IFN response. These findings suggest that the STAT3ß/α mRNA ratio is a significant prognostic marker in AML and holds crucial information for targeted treatment approaches. Patients displaying a low STAT3ß/α mRNA ratio and unfavorable prognosis could benefit from therapeutic interventions directed at STAT1/IFN signaling.

FLT3-ITD regulation of the endoplasmic reticulum functions in acute myeloid leukemia.

The FLT3-ITD mutation represents the most frequent genetic alteration in newly diagnosed acute myeloid leukemia (AML) patient and is associated with poor prognosis. Mutation result in the retention of a constitutively active form of this receptor in the endoplasmic reticulum (ER) and the subsequent modification of its downstream effectors. Here, we assessed the impact of such retention on ER homeostasis and found that mutant cells present lower levels of ER stress due to the overexpression of ERO1α, one of the main proteins of the protein folding machinery at the ER. Overexpression of ERO1α resulted essential for ITD mutant cells survival and chemoresistance and also played a crucial role in shaping the type of glucose metabolism in AML cells, being the mitochondrial pathway the predominant one in those with a higher ER stress (non-mutated cells) and the glycolytic pathway the predominant one in those with lower ER stress (mutated cells). Our data indicate that FLT3 mutational status dictates the route for glucose metabolism in an ERO1α depending on manner and this provides a survival advantage to tumors carrying these ITD mutations.

The effect of bone marrow mesenchymal stromal cell exosomes on acute myeloid leukemia's biological functions: a focus on the potential role of LncRNAs.

Acute myeloid leukemia represents a group of malignant blood disorders that originate from clonal over-proliferation and the differentiation failure of hematopoietic precursors, resulting in the accumulation of blasts in the bone marrow. Mesenchymal stromal cells (MSCs) have been shown to exert diverse effects on tumor cells through direct and indirect interaction. Exosomes, as one of the means of indirect intercellular communication, are released from different types of cells, including MSCs, and their various contents, such as lncRNAs, enable them to exert significant impacts on target cells. Our study aims to investigate the effects of BM-MSC exosomes on the cellular and molecular characterization of HL-60 AML cells, particularly detecting the alterations in the expression of lncRNAs involved in AML leukemogenesis, cell growth, drug resistance, and poor prognosis. BM-MSCs were cultured with serum-free culture media to isolate exosomes from their supernatants. The validation of exosomes was performed in three stages: morphological analysis using TEM, size evaluation using DLS, and CD marker identification using flow cytometry. Subsequently, the HL-60 AML cells were treated with isolated BM-MSC exosomes to determine the impact of their contents on leukemic cells. Cell metabolic activity was evaluated by the MTT assay, while cell cycle progression, apoptosis, ROS levels, and proliferation were assessed by flow cytometry. Furthermore, RT-qPCR was conducted to determine the expression levels of lncRNAs and apoptosis-, ROS-, and cell cycle-related genes. MTT assay and flow cytometry analysis revealed that BM-MSC exosomes considerably suppressed cell metabolic activity, proliferation, and cell cycle progression. Also, these exosomes could effectively increase apoptosis and ROS levels in HL-60 cells. The expression levels of p53, p21, BAX, and FOXO4 were increased, while the BCL2 and c-Myc levels decreased. MALAT1, HOTAIR, and H19 expression levels were also significantly decreased in treated HL-60 cells compared to their untreated counterparts. BM-MSC exosomes suppress cell cycle progression, proliferation, and metabolic activity while simultaneously elevating the ROS index and apoptosis ratio in HL-60 cells, likely by reducing the expression levels of MALAT1, HOTAIR, and H19. These findings suggest that BM-MSC exosomes might serve as potential supportive therapies for leukemia.

Identification of triciribine as a novel myeloid cell differentiation inducer.

Differentiation therapy using all-trans retinoic acid (ATRA) for acute promyelocytic leukemia (APL) is well established. However, because the narrow application and tolerance development of ATRA need to be improved, we searched for another efficient myeloid differentiation inducer. Kinase activation is involved in leukemia biology and differentiation block. To identify novel myeloid differentiation inducers, we used a Kinase Inhibitor Screening Library. Using a nitroblue tetrazolium dye reduction assay and real-time quantitative PCR using NB4 APL cells, we revealed that, PD169316, SB203580, SB202190 (p38 MAPK inhibitor), and triciribine (TCN) (Akt inhibitor) potently increased the expression of CD11b. We focused on TCN because it was reported to be well tolerated by patients with advanced hematological malignancies. Nuclear/cytoplasmic (N/C) ratio was significantly decreased, and myelomonocytic markers (CD11b and CD11c) were potently induced by TCN in both NB4 and acute myeloid leukemia (AML) M2 derived HL-60 cells. Western blot analysis using NB4 cells demonstrated that TCN promoted ERK1/2 phosphorylation, whereas p38 MAPK phosphorylation was not affected, suggesting that activation of the ERK pathway is involved in TCN-induced differentiation. We further examined that whether ATRA may affect phosphorylation of ERK and p38, and found that there was no obvious effect, suggesting that ATRA induced differentiation is different from TCN effect. To reveal the molecular mechanisms involved in TCN-induced differentiation, we performed microarray analysis. Pathway analysis using DAVID software indicated that "hematopoietic cell lineage" and "cytokine-cytokine receptor interaction" pathways were enriched with high significance. Real-time PCR analysis demonstrated that components of these pathways including IL1ß, CD3D, IL5RA, ITGA6, CD44, ITGA2B, CD37, CD9, CSF2RA, and IL3RA, were upregulated by TCN-induced differentiation. Collectively, we identified TCN as a novel myeloid cell differentiation inducer, and trials of TCN for APL and non-APL leukemia are worthy of exploration in the future.

Oncogene EVI1 drives acute myeloid leukemia via a targetable interaction with CTBP2.

Acute myeloid leukemia (AML) driven by the activation of EVI1 due to chromosome 3q26/MECOM rearrangements is incurable. Because transcription factors such as EVI1 are notoriously hard to target, insight into the mechanism by which EVI1 drives myeloid transformation could provide alternative avenues for therapy. Applying protein folding predictions combined with proteomics technologies, we demonstrate that interaction of EVI1 with CTBP1 and CTBP2 via a single PLDLS motif is indispensable for leukemic transformation. A 4× PLDLS repeat construct outcompetes binding of EVI1 to CTBP1 and CTBP2 and inhibits proliferation of 3q26/MECOM rearranged AML in vitro and in xenotransplant models. This proof-of-concept study opens the possibility to target one of the most incurable forms of AML with specific EVI1-CTBP inhibitors. This has important implications for other tumor types with aberrant expression of EVI1 and for cancers transformed by different CTBP-dependent oncogenic transcription factors.

Valrubicin-loaded immunoliposomes for specific vesicle-mediated cell death in the treatment of hematological cancers.

We created valrubicin-loaded immunoliposomes (Val-ILs) using the antitumor prodrug valrubicin, a hydrophobic analog of daunorubicin. Being lipophilic, valrubicin readily incorporated Val-lLs that were loaded with specific antibodies. Val-ILs injected intravenously rapidly reached the bone marrow and spleen, indicating their potential to effectively target cancer cells in these areas. Following the transplantation of human pediatric B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), or acute myeloid leukemia (AML) in immunodeficient NSG mice, we generated patient-derived xenograft (PDX) models, which were treated with Val-ILs loaded with antibodies to target CD19, CD7 or CD33. Only a small amount of valrubicin incorporated into Val-ILs was needed to induce leukemia cell death in vivo, suggesting that this approach could be used to efficiently treat acute leukemia cells. We also demonstrated that Val-ILs could reduce the risk of contamination of CD34+ hematopoietic stem cells by acute leukemia cells during autologous peripheral blood stem cell transplantation, which is a significant advantage for clinical applications. Using EL4 lymphoma cells on immunocompetent C57BL/6 mice, we also highlighted the potential of Val-ILs to target immunosuppressive cell populations in the spleen, which could be valuable in impairing cancer cell expansion, particularly in lymphoma cases. The most efficient Val-ILs were found to be those loaded with CD11b or CD223 antibodies, which, respectively, target the myeloid-derived suppressor cells (MDSC) or the lymphocyte-activation gene 3 (LAG-3 or CD223) on T4 lymphocytes. This study provides a promising preclinical demonstration of the effectiveness and ease of preparation of Val-ILs as a novel nanoparticle technology. In the context of hematological cancers, Val-ILs have the potential to be used as a precise and effective therapy based on targeted vesicle-mediated cell death.

miR-133a and miR-135a Regulate All-Trans Retinoic Acid-Mediated Differentiation in Pediatric Acute Myeloid Leukemia by Inhibiting CDX2 Translation and Serve as Prognostic Biomarkers.

Background: Acute myeloid leukemia (AML) is a type of blood cancer characterized by excessive growth of immature myeloid cells. Unfortunately, the prognosis of pediatric AML remains unfavorable. It is imperative to further our understanding of the mechanisms underlying leukemogenesis and explore innovative therapeutic approaches to enhance overall disease outcomes for patients with this condition. Methods: Quantitative reverse-transcription PCR was used to quantify the expression levels of microRNA (miR)-133a and miR-135a in 68 samples from 59 pediatric patients with AML. Dual-luciferase reporter transfection assay, Cell Counting Kit-8 assay, and western blot analysis were used to investigate the functions of miR-133a and miR-135a. Results: Our study found that all-trans-retinoic acid (ATRA) promoted the expression of miR-133a and miR-135a in AML cells, inhibited caudal type homeobox 2 (CDX2) expression, and subsequently inhibited the proliferation of AML cells. Additionally, miR-133a and miR-135a were highly expressed in patients with complete remission and those with better survival. Conclusions: miR-133a and miR-135a may play an antioncogenic role in pediatric AML through the ATRA-miRNA133a/135a-CDX2 pathway. They hold promise as potentially favorable prognostic indicators and novel therapeutic targets for pediatric AML.

Systems Biology for Drug Target Discovery in Acute Myeloid Leukemia.

Combining new therapeutics with all-trans-retinoic acid (ATRA) could improve the efficiency of acute myeloid leukemia (AML) treatment. Modeling the process of ATRA-induced differentiation based on the transcriptomic profile of leukemic cells resulted in the identification of key targets that can be used to increase the therapeutic effect of ATRA. The genome-scale transcriptome analysis revealed the early molecular response to the ATRA treatment of HL-60 cells. In this study, we performed the transcriptomic profiling of HL-60, NB4, and K562 cells exposed to ATRA for 3-72 h. After treatment with ATRA for 3, 12, 24, and 72 h, we found 222, 391, 359, and 1032 differentially expressed genes (DEGs) in HL-60 cells, as well as 641, 1037, 1011, and 1499 DEGs in NB4 cells. We also found 538 and 119 DEGs in K562 cells treated with ATRA for 24 h and 72 h, respectively. Based on experimental transcriptomic data, we performed hierarchical modeling and determined cyclin-dependent kinase 6 (CDK6), tumor necrosis factor alpha (TNF-alpha), and transcriptional repressor CUX1 as the key regulators of the molecular response to the ATRA treatment in HL-60, NB4, and K562 cell lines, respectively. Mapping the data of TMT-based mass-spectrometric profiling on the modeling schemes, we determined CDK6 expression at the proteome level and its down-regulation at the transcriptome and proteome levels in cells treated with ATRA for 72 h. The combination of therapy with a CDK6 inhibitor (palbociclib) and ATRA (tretinoin) could be an alternative approach for the treatment of acute myeloid leukemia (AML).

Influence of Human Bone Marrow Mesenchymal Stem Cells Secretome from Acute Myeloid Leukemia Patients on the Proliferation and Death of K562 and K562-Lucena Leukemia Cell Lineages.

Leukemias are among the most prevalent types of cancer worldwide. Bone marrow mesenchymal stem cells (MSCs) participate in the development of a suitable niche for hematopoietic stem cells, and are involved in the development of diseases such as leukemias, to a yet unknown extent. Here we described the effect of secretome of bone marrow MSCs obtained from healthy donors and from patients with acute myeloid leukemia (AML) on leukemic cell lineages, sensitive (K562) or resistant (K562-Lucena) to chemotherapy drugs. Cell proliferation, viability and death were evaluated, together with cell cycle, cytokine production and gene expression of ABC transporters and cyclins. The secretome of healthy MSCs decreased proliferation and viability of both K562 and K562-Lucena cells; moreover, an increase in apoptosis and necrosis rates was observed, together with the activation of caspase 3/7, cell cycle arrest in G0/G1 phase and changes in expression of several ABC proteins and cyclins D1 and D2. These effects were not observed using the secretome of MSCs derived from AML patients. In conclusion, the secretome of healthy MSCs have the capacity to inhibit the development of leukemia cells, at least in the studied conditions. However, MSCs from AML patients seem to have lost this capacity, and could therefore contribute to the development of leukemia.

Oxidative stress is two-sided in the treatment of acute myeloid leukemia.

INTRODUCTION: Oxidative stress caused by elevated ROS, as a novel therapeutic mechanism, has been implicated in various tumors including AML. AML cells are chronically under oxidative stress, yet overreliance on ROS production makes tumor cells increasingly vulnerable to further damage. Reducing the cytotoxic effect of ROS on normal cells while killing leukemia stem cell (LSC) with high levels of reactive oxygen species is a new challenge for oxidative stress therapy in leukemia. METHODS: By searching literature databases, we summarized recent relevant studies. The relationship of ROS on AML genes, signaling pathways, and transcription factors, and the correlation of ROS with AML bone marrow microenvironment and autophagy were summarized. In addition, we summarize the current status of research on ROS and AML therapeutics. Finally, we discuss the research progress on redox resistance in AML. RESULTS: This review discusses the evidence showing the link between redox reactions and the progression of AML and compiles the latest research findings that will facilitate future biological studies of redox effects associated with AML treatment. CONCLUSION: We believe that exploiting this unique oxidative stress property of AML cells may provide a new way to prevent relapse and drug resistance.

Age-specific induction of mutant p53 drives clonal hematopoiesis and acute myeloid leukemia in adult mice.

The investigation of the mechanisms behind p53 mutations in acute myeloid leukemia (AML) has been limited by the lack of suitable mouse models, which historically have resulted in lymphoma rather than leukemia. This study introduces two new AML mouse models. One model induces mutant p53 and Mdm2 haploinsufficiency in early development, showing the role of Mdm2 in myeloid-biased hematopoiesis and AML predisposition, independent of p53. The second model mimics clonal hematopoiesis by inducing mutant p53 in adult hematopoietic stem cells, demonstrating that the timing of p53 mutation determines AML vs. lymphoma development. In this context, age-related changes in hematopoietic stem cells (HSCs) collaborate with mutant p53 to predispose toward myeloid transformation rather than lymphoma development. Our study unveils new insights into the cooperative impact of HSC age, Trp53 mutations, and Mdm2 haploinsufficiency on clonal hematopoiesis and the development of myeloid malignancies.

Measuring response to therapy in AML: Difference from normal flow cytometry vs RQ-PCR.

Multiple technologies have been used to monitor response to therapy in acute myeloid leukemia (AML) to improve detection of leukemia over the standard of practice, morphologic counting of blasts. The two techniques most frequently used in a routine clinical setting, flow cytometry and RQ-PCR, differ in their targets, sensitivity, and ability to detect residual disease. Both flow cytometry and RQ-PCR detect the expression of abnormal gene products, at the protein level or RNA level, respectively. Flow cytometry can be applied to a broad range of AML cases while RQ-PCR is limited to specific genetic abnormalities identified in subsets of AML. This article compares the results when both techniques were used in a reference laboratory to monitor AML over the course of treatment, comparing quantitative and qualitative results.

Leukemia Cutis in Relapsed Acute Myeloid Leukemia: A Call for Distinct Classification.

BACKGROUND Acute myeloid leukemia is characterized by dysregulated proliferation and maturation arrest of myeloid precursors, precipitating a spectrum of complications. Among these, leukemia cutis refers specifically to ectopic deposition and proliferation of malignant myeloid cells within the skin. This infiltration pathogenesis remains unclear. Although there are numerous reports of leukemia cutis in the setting of acute myeloid leukemia or primary acute myeloid leukemia, there are no specific reports of leukemia cutis in the setting of relapsed acute myeloid leukemia. CASE REPORT A 59-year-old woman, with a history of remission from poor-risk acute myeloid leukemia, previously treated with chemotherapy and allogenic bone marrow transplant, presented with shortness of breath, lethargy, anemia, thrombocytopenia, and subcutaneous nodules on lower extremities. Leukemia cutis was diagnosed, in the setting of relapsed acute myeloid leukemia. After unsuccessful salvage chemotherapy and being deemed unsuitable for further treatment, she pursued palliative care and died a month later. CONCLUSIONS Our case highlights a lack of reporting or making a distinction of those patients with relapsed acute myeloid leukemia and leukemia cutis. Consequently, it can be deduced that patients who simultaneously have relapsed acute myeloid leukemia and leukemia cutis are expected to fare worse in terms of clinical outcomes than those with primary acute myeloid leukemia and leukemia cutis. Relapsed acute myeloid leukemia patients with leukemia cutis should be classified as a distinct group, warranting further research into aggressive therapeutic targets and survival rates, while emphasizing the need for more vigilant follow-up and lower biopsy thresholds for cutaneous lesions in patients with treated hematologic malignancies.

Metformin as an Enhancer for the Treatment of Chemoresistant CD34+ Acute Myeloid Leukemia Cells.

Acute myeloid leukemia is the second most frequent type of leukemia in adults. Due to a high risk of development of chemoresistance to first-line chemotherapy, the survival rate of patients in a 5-year period is below 30%. One of the reasons is that the AML population is heterogeneous, with cell populations partly composed of very primitive CD34+CD38- hematopoietic stem/progenitor cells, which are often resistant to chemotherapy. First-line treatment with cytarabine and idarubicin fails to inhibit the proliferation of CD34+CD38- cells. In this study, we investigated Metformin's effect with or without first-line conventional chemotherapy, or with other drugs like venetoclax and S63845, on primitive and undifferentiated CD34+ AML cells in order to explore the potential of Metformin or S63845 to serve as adjuvant therapy for AML. We found that first-line conventional chemotherapy treatment inhibited the growth of cells and arrested the cells in the S phase of the cell cycle; however, metformin affected the accumulation of cells in the G2/M phase. We observed that CD34+ KG1a cells respond better to lower doses of cytarabine or idarubicin in combination with metformin. Also, we determined that treatment with cytarabine, venetoclax, and S63845 downregulated the strong tendency of CD34+ KG1a cells to form cell aggregates in culture due to the downregulation of leukemic stem cell markers like CD34 and CD44, as well as adhesion markers. Also, we found that idarubicin slightly upregulated myeloid differentiation markers, CD11b and CD14. Treatment with cytarabine, idarubicin, venetoclax, metformin, and S63845 upregulated some cell surface markers like HLA-DR expression, and metformin upregulated CD9, CD31, and CD105 cell surface marker expression. In conclusion, we believe that metformin has the potential to be used as an adjuvant in the treatment of resistant-to-first-line-chemotherapy AML cells. Also, we believe that the results of our study will stimulate further research and the potential use of changes in the expression of cell surface markers in the development of new therapeutic strategies.

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.

Preferential HDAC6 inhibitors derived from HPOB exhibit synergistic antileukemia activity in combination with decitabine.

Histone deacetylase 6 (HDAC6) is an emerging drug target to treat oncological and non-oncological conditions. Since highly selective HDAC6 inhibitors display limited anticancer activity when used as single agent, they usually require combination therapies with other chemotherapeutics. In this work, we synthesized a mini library of analogues of the preferential HDAC6 inhibitor HPOB in only two steps via an Ugi four-component reaction as the key step. Biochemical HDAC inhibition and cell viability assays led to the identification of 1g (highest antileukemic activity) and 2b (highest HDAC6 inhibition) as hit compounds. In subsequent combination screens, both 1g and especially 2b showed synergy with DNA methyltransferase inhibitor decitabine in acute myeloid leukemia (AML). Our findings highlight the potential of combining HDAC6 inhibitors with DNA methyltransferase inhibitors as a strategy to improve AML treatment outcomes.

FLT3-selective PROTAC: Enhanced safety and increased synergy with Venetoclax in FLT3-ITD mutated acute myeloid leukemia.

Acute myeloid leukemia (AML) patients carrying Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations often face a poor prognosis. While some FLT3 inhibitors have been used clinically, challenges such as short efficacy and poor specificity persist. Proteolytic targeting chimera (PROTAC), with its lower ligand affinity requirement for target proteins, offers higher and rapid targeting capability. Gilteritinib, used as the ligand for the target protein, was connected with different E3 ligase ligands to synthesize several series of PROTAC targeting FLT3-ITD. Through screening and structural optimization, the optimal lead compound PROTAC Z29 showed better specificity than Gilteritinib. Z29 induced FLT3 degradation through the proteasome pathway and inhibited tumor growth in subcutaneous xenograft mice. We verified Z29's minimal impact on platelets in a patient-derived xenografts (PDX) model compared to Gilteritinib. The combination of Z29 and Venetoclax showed better anti-tumor effects, lower platelet toxicity, and lower hepatic toxicity in FLT3-ITD+ models. The FLT3-selective PROTAC can mitigate the platelet toxicity of small molecule inhibitors, ensuring safety and efficacy in monotherapy and combination therapy with Venetoclax. It is a promising strategy for FLT3-ITD+ patients, especially those with platelet deficiency or liver damage.