MV4-11 Acute Myeloid Leukemia Cells Research Articles

CXCR4-Mediated Codelivery of FLT3 and BCL-2 Inhibitors for Enhanced Targeted Combination Therapy of FLT3-ITD Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is often associated with poor prognosis and survival. Small molecule inhibitors, though widening the treatment landscape, have limited monotherapy efficacy. The combination therapy, however, shows suboptimal clinical outcomes due to low bioavailability, overlapping systemic toxicity and drug resistance. Here, we report that CXCR4-mediated codelivery of the BCL-2 inhibitor venetoclax (VEN) and the FLT3 inhibitor sorafenib (SOR) via T22 peptide-tagged disulfide cross-linked polymeric micelles (TM) achieves synergistic treatment of FLT3-ITD AML. TM-VS with a VEN/SOR weight ratio of 1/4 and T22 peptide density of 20% exhibited an extraordinary inhibitory effect on CXCR4-overexpressing MV4-11 AML cells. TM-VS at a VEN/SOR dosage of 2.5/10 mg/kg remarkably reduced leukemia burden, prolonged mouse survival, and impeded bone loss in orthotopic MV4-11-bearing mice, outperforming the nontargeted M-VS and oral administration of free VEN/SOR. CXCR4-mediated codelivery of BCL-2 and FLT3 inhibitors has emerged as a prospective clinical treatment for FLT3-ITD AML.

Abstract LB026: Exploring MCT4 inhibitor Ful-23 and cytarabine synergy in acute myeloid leukemia therapeutics

Abstract In the ongoing pursuit of addressing the formidable challenge presented by Acute Myeloid Leukemia (AML) treatment, this comprehensive study delves into the intricate antileukemic effects of Ful-23, our newly developed Monocarboxylate Transporter 4 (MCT4) inhibitor. Positioned as a promising candidate, Ful-23's unique mechanisms and potential synergies with established chemotherapeutic agents, such as Cytarabine, are elucidated in this research. Intracellular alkalinization stands as a crucial factor contributing to the evasion of chemotherapy by cancer cells. It serves as the primary determinant for the heightened resistance exhibited by cancer cells against chemotherapy interventions. The investigation reveals the multifaceted impact of Ful-23 on AML cells, showcasing its capacity to induce intracellular lactate accumulation and acidification. These metabolic alterations correlate with a substantial inhibition of proliferation, as evidenced by an IC50 of approximately 0.55 μM, and a marked induction of apoptosis, particularly notable in the MV411 AML cell line. These findings underscore Ful-23's potential as a potent antileukemic agent with distinct cellular effects. A collaborative approach involves the combination of low concentrations of Ful-23 (0.312 μM) with Cytarabine (0.312 μM), revealing a significant enhancement in cytarabine-induced proliferation inhibition and apoptosis when Ful-23 is introduced concurrently. Importantly, the translational impact of these findings extends to a zebrafish model, utilizing both MV411 cells and transplanted primary AML patient-derived bone marrow cells. In the zebrafish AML model, the combination treatment of Ful-23 and Cytarabine demonstrates a significant enhancement in their respective antitumor effects compared to monotherapy, indicating a pronounced synergistic effect. This additional validation strengthens the clinical relevance of the observed synergistic antileukemic interactions between Ful-23 and Cytarabine. Essentially, this investigation advances our understanding of AML treatment dynamics, emphasizing the crucial role of MCT4 inhibition and elucidating the mechanism by which it leads to intracellular acidification in synergistic therapeutic approaches for this intricate and formidable disease. Citation Format: Yong Wu, Ke Wu, Jay Vadgama. Exploring MCT4 inhibitor Ful-23 and cytarabine synergy in acute myeloid leukemia therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB026.

Autophagy Induction Constitutes a Targetable Vulnerability in FLT3-Mutant Acute Myeloid Leukemia Cells Treated with FLT3-Inhibitors

Recently, we and others identified autophagy induction as a protective mechanism in FLT3-ITD mutant acute myeloid leukemia (AML) cells upon treatment with FLT3-inhibitors (FLT3i), constituting an increasing issue in clinical practice. Here, we investigated the molecular consequences of autophagy induction upon FLT3i treatment, exploring resistance mechanisms and interrogated whether autophagy inhibition can improve FLT3i antileukemic therapy. Drug response of primary FLT3-ITD AML samples ex vivo treated with FLT3i, quizartinib (AC220, n= 59) and midostaurin (PKC412, n=64), was associated with their transcriptional profile to unravel which molecular programs were enriched in poor responders. Both gene set enrichment analysis (GSEA) and single sample GSEA (ssGSEA) revealed enriched molecular signatures compatible with autophagy activation in poor responders to FLT3i, while sensitive samples enriched molecular signatures consonant with mitochondrial function. Enrichment scores for programs associated with “regulation of autophagy” and “autophagosome organization” were positively correlated to ex vivo response area under the curve values for AC220 and PKC412. To functionally address the molecular pathways associated with autophagy activation upon FLT3i treatment, MOLM-13 and MV4-11 AML cells ( FLT3-ITD) and primary AML samples, were treated with AC220 and PKC412 alone or in combination with the autophagy inhibitor chloroquine (CQ, 5-10μM). In FLT3-ITD AML models, treatment with FLT3i significantly induced the formation of acidic vesicular organelles, suggesting autophagy induction. We also observed a reduction of autophagy regulators in a dose- and time-dependent manner, evidenced by the conversion of LC3B-I to LC3B-II, with degradation of LC3B-II and p62. Furthermore, FLT3i treatment resulted in phosphorylation inhibition of AKT Ser473, mTOR Ser2448 and its downstream protein p70S6K Thr421/Ser424, and phosphorylation reduction of ULK1 Ser757. Combination of FLT3i with CQ (10 μM) significantly reduced cell viability and survival for both PKC412 and AC220 inhibitors. To rule out the off target effects associated with CQ treatment, we performed a knockdown (KD) on the key autophagy mediator ATG5 using shRNA-mediated inhibition in FLT3-ITD cells. In MOLM13 cells, FLT3i treatment in ATG5-KD cells significantly reduced the IC50 (control: 31nM vs. ATG5-KD: 17nM) and increased apoptosis induction (control: 41% vs. ATG5-KD: 58%). We validated these observations in ex vivo treated primary AML samples, where we observed that in FLT3-ITD but not in FLT3 wild-type samples, the combination of FLT3i (PKC412 and AC220) plus autophagy inhibitors (CQ) reduced cell proliferation and survival. In summary, we demonstrated that autophagy induction is a biological process intrinsically associated with drug resistance in FLT3-ITD mutant AMLs when treated with FLT3i. Hence, autophagy inhibition can be leveraged to increase the antileukemic effect of FLT3i and highlights the autophagy as a major acquired biological process associated with FLT3i therapy.

A Novel Natural Derivative GL-V9 Inducing Mitotic Catastrophe and Displaying a Synergistic Anti-Tumor Effect with EZH2 Inhibitor in Acute Myeloid Leukemia

Background GL-V9 is a flavonoid derivative from the natural product wogonin with elegant anti-tumor activity, while the effect of GL-V9 in acute leukemia has no report. Enhancer of zeste homolog 2 (EZH2) catalyzes trimethylation on the K27 residue of histone H3 (H3K27Me3), which facilitates non-homologous DNA end joining (NHEJ). EZH2 inhibitor could induce chromatin decondensation and promote the sensitivity of tumor cells towards DNA damage drugs. In the present study, we evaluated the anti-leukemia activity of GL-V9 and its synergy with EZH2 inhibitor DZNeP in acute myeloid leukemia (AML). Methods Cell proliferation and cytotoxicity were measured by Cell Counting Kit-8(CCK-8) in U937, THP-1, and MV4-11 AML cells. RNA-seq was performed in U937 cells treated with 2μM DZNeP, MV4-11 cells treated with 4μM GL-V9, and vehicle for 48 hours. The clone formation rate was measured by a plate clone formation assay. Cell cycle and apoptosis were measured by flow cytometry analysis. Morphology examination was conducted with Fluorescence (IF) microscopy . Expression of target genes was detected by western blot and RT-qPCR. Results GL-V9 had a dose-/time-dependent effect on cell viability with IC50 at 3-4μM in U937 and THP-1 cells (Fig.1A); it also significantly induced the G2/M arrest and apoptosis of the cells (Fig.1B, C). IF staining showed GL-V9 triggers micro-, multi-nucleation, and cytoskeleton abnormalities, the feature of mitotic catastrophe in U937 cells 4hrs after the treatment (Fig1.D). These data indicated that GL-V9 exerts the anti-leukemia effect by inducing mitotic catastrophe. We further analyzed the whole genome transcriptome upon GL-V9 treatment in U937 cells, and GSEA analysis of the differentially expressed genes (DEGs) showed that GL-V9 treatment negatively correlated with genes involved in sister chromatid separation, microtubule cytoskeleton organization, DNA double-strand break repair, mismatch repair, and cell cycle checkpoint signaling (Fig1.E). These data revealed that GL-V9 may induce DNA damage-mediated mitotic catastrophe in the AML cells. H3K27me3 and EZH2 play an important role in the early DNA damage response. We thus carried out the combination therapy of EZH2 inhibitor DZNeP with GL-V9. Results showed that co-treatment of GL-V9 with DZNeP exerted synergistic effects on reduced cell viability and clone formation versus single-drug in U937 and MV4-11 cells (Fig2.AB). DZNeP induced G0/G1 arrest; and GL-V9 induced G2/M arrest; whereas the combination significantly decreased the number of cells in the S-phase (Fig2.C). Also, the percentage of the apoptotic cells significantly increased upon the GL-V9+DZNeP treatment versus the controls in the cells (Fig2.D) as well as in ASXL1 mutant AML-M5 and AML-M2 primary cells (Fig2.E). Consistently, the combination significantly induced the expression changes of cell cycle regulator CCNE2 and p21, and dramatically up-regulated apoptotic effectors PARP, Caspase-3, and DNA damage marker γH2AX versus single-drug (Fig2.F). Furthermore, the overlapped DEGs were identified with 242 genes regulated in homo-direction but 25 regulated in the opposite treated with GL-V9 or DZNeP, these genes mainly influenced DNA replication, chromatin structures, and DNA damage response (Fig2.G). GL-V9 and/or DZNeP down-regulated the EZH2 expression, induced the decrease of p-AKT and c-Myc, and the increase of p53 in U937 cells (Fig. 2H). These data indicate that GL-V9+DZNeP may exert the anti-leukemia effect by inducing DNA damage via targeting EZH2/ PI3K/AKT signaling and the p53-c-Myc axis in the cells. EZH2 inhibition modifies the chromatin structure and decondensed the H3K27me3-marked chromatin, making AML cells more accessible to the nuclear-attacked agent GL-V9. The mechanism model is depicted in Fig. 2I. Conclusions GL-V9 is an effective novel nature compound against AML and exhibited strong synergistic effects with the epigenomic drug EZH2 inhibitor DZNeP potentially by inducing DNA damage-mediated mitotic catastrophe through targeting EZH2/ PI3K/AKT signaling and p53-cMyc axis. Our results provide pre-clinical evidence for the new combination as a promising clinical option for AML patients.

Abstract A35: Overcoming Venetoclax Resistance in Acute Myeloid Leukemia (AML) via the Novel Imipridone ONC213

Abstract Older adults make up most of the AML patient population and have an especially dismal prognosis due to limited therapeutic options. The Bcl-2 inhibitor, venetoclax (VEN), was approved for use in combination with low-dose cytarabine or a hypomethylating agent [azacitidine (AZA) or decitabine] in AML patients who are ≥75 years old or ineligible for intensive chemotherapy (IC). Due to superior survival data and tolerability, the combination of VEN and AZA has become a new standard of care for older patients and those unfit for IC. Though this was a promising therapeutic option for these patients, resistance to this therapy results in a 1-year overall survival rate of only 30-40% and the median overall survival following treatment failure is less than three months, highlighting the urgent need of new therapies to address this. Previous studies have found VEN resistance can be overcome by targeting mitochondria and Mcl-1. Our studies on ONC213, a novel imipridone, have found that it has potent antileukemic activity against both AML cell lines and primary patient samples. It reduces Mcl-1 and targets mitochondria of AML cells – and was thus a promising agent to overcome VEN resistance. When used in combination, ONC213 and VEN are highly synergistic in both AML cell lines and primary patient samples, including those intrinsically resistant to VEN. The combination also targets AML progenitor cells determined by colony formation assays and shows in vivo efficacy in a cell line derived xenograft mouse model (median survival of 57 days vs 37 days in control mice, p=0.002). To determine if ONC213 could overcome acquired resistance to VEN and AZA, we developed VEN and AZA double resistant ML-2 and MV4-11 AML cells (designated ML-2/VEN+AZA-R and MV4-11/VEN+AZA-R) by stepwise increasing concentrations of VEN and AZA at 1:3 fixed ratio over an extended period. ML-2/VEN+AZA-R and MV4-11/VEN+AZA-R AML cells demonstrate 437- and 1156-fold, respectively, increase in VEN+AZA (1:3 ratio) IC50 compared to parental cells (5,285.3 nM vs 12.1 nM and 6,704.3 nM vs 5.8 nM, respectively). ONC213 can resensitize resistant cells to VEN and the combination of ONC213 and VEN is highly synergistic in both ML-2/VEN+AZA-R and MV4-11/VEN+AZA-R cells as evident by a combination index (CI) value of < 0.05. ONC213 alone and in combination with VEN suppress oxidative phosphorylation (OXPHOS) and Mcl-1 in AML cells, including those with inherent resistance to VEN or with acquired resistance to VEN+AZA. Overexpression of Mcl-1 can partially rescue cells from combination therapy. These results suggest that ONC213 suppresses OXPHOS and Mcl-1 to overcome VEN resistance. Here we demonstrate that VEN resistance can be addressed through dual targeting of mitochondria and Mcl-1 using the novel imipridone, ONC213. ONC213 resensitizes VEN + AZA resistant cells to VEN and offers a new approach to combat VEN+AZA resistance in AML. Citation Format: Jenn L. Carter, Yongwei Su, Holly Edwards, Lisa Polin, Joshua E. Allen, Varun V. Prabhu, Jay Yang, Maik Huettermann, Yubin Ge. Overcoming Venetoclax Resistance in Acute Myeloid Leukemia (AML) via the Novel Imipridone ONC213 [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 A35.

Codelivery of BCL2 and MCL1 Inhibitors Enabled by Phenylboronic Acid-Functionalized Polypeptide Nanovehicles for Synergetic and Potent Therapy of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the most refractory hematologic malignancy characterized by acute onset, rapid progression, and high recurrence rate. Here, codelivery of BCL2 (ABT199) and MCL1 (TW37) inhibitors using phenylboronic acid-functionalized polypeptide nanovehicles to achieve synergetic and potent treatment of AML is adopted. Leveraging the dynamic boronic ester bonds, BN coordination, and π-π stacking, the nanovehicles reveal remarkably efficient and robust drug coencapsulation. ABT199 can induce a series of pro-apoptotic reactions by promoting the dissociation of the pro-apoptotic protein Bim from BCL2, while the released Bim is often captured by MCL1 protein overexpressed in AML. TW37 has a strong inhibitory ability to MCL1, thereby can restrain the depletion of Bim protein. Dual inhibitor-loaded nanoparticles (NPAT) reveal excellent stability, acid/enzyme/H2 O2 -triggered drug release, and significant cytotoxicity toward MOLM-13-Luc and MV-411 AML cells with low half maximal inhibitory concentrations of 1.15 and 7.45ng mL-1 , respectively. In mice bearing MOLM-13-Luc or MV-411 AML cancer, NPAT reveal significant inhibition of tumor cell infiltration in bone marrow and main organs, potent suppression of tumor growth, and remarkably elevated mouse survival. With facile construction, varying drug combination, superior safety, synergetic efficacy, the phenylboronic acid-functionalized smart nanodrugs hold remarkable potential for AML treatment.

The Multi-CDK Inhibitor Dinaciclib Shows Synergistic Activity with the BET Inhibitor PLX51107 and Reverses BET Resistance through Inhibition of Canonical Wnt Signaling in Acute Myeloid Leukemia (AML)

PLX51107 is a novel BET inhibitor (BETi) with antineoplastic activity in pre-clinical models of both aggressive B-cell malignancies and AML. Differentiating it from other BET inhibitors currently under investigation, PLX51107 has a unique binding site in the acetylated lysine binding pocket of BRD4. Through interaction with pTEFb, which is comprised of CDK9 and several cyclin T1 heterodimers, BRD4 promotes phosphorylation of RNA polymerase II, ultimately influencing gene transcription. Dinaciclib is a potent inhibitor of multiple cyclin dependent kinases including CDK9 and has been shown to have pre-clinical activity in AML. Therefore, we hypothesized that PLX51107 would have synergistic activity when combined with dinaciclib. We found synergistic effects on proliferation in primary AML cells when PLX51107 was combined with dinaciclib in stromal co-cultures across a range of dose combinations below the IC50 of either compound alone. Synergistic activity was also present in cytokine support culture conditions, colony forming assays, and synergy assays utilizing COMbenefit software. Interestingly, the highly conserved canonical β-catenin dependent Wnt signaling pathway is a known BETi resistance mechanism in AML. Therefore, destruction of β-catenin and inhibition of Wnt signaling could represent a novel therapeutic target to circumvent development of BET-mediated resistance. We found dinaciclib treatment reduced protein levels of total β-catenin in primary AML cells and AML cell lines (Figure 1A). Phosphorylated β-catenin and non-phosphorylated "active" β-catenin protein levels were also reduced following dinaciclib treatment (Figure 1A). In addition, mRNA expression of multiple components of the Wnt pathway, including the Wnt co-receptor LRP6, AXIN2, FZD1, and many Wnt target genes were significantly decreased following dinaciclib and combination treatment. Moreover, in a luciferase based Wnt reporter cell line, we found dinaciclib significantly reduced Wnt3a induced luciferase activity, suggesting a direct effect on β-catenin mediated transcription of Wnt target genes. Collectively, this data suggests dinaciclib has multiple inhibitory effects on canonical β-catenin dependent Wnt signaling. We found combination treatment of PLX51107 and dinaciclib produced synergistic reduction in expression of both Wnt signaling components and Wnt target genes. Conversely, β-catenin and Wnt target gene expression levels were either not significantly changed or even increased when treated with PLX51107 alone. In order to examine the effects of dinaciclib in the setting of BET resistance, we developed PLX51107 resistant MOLM-13 and MV4-11 AML cell lines via serial drugging. These resistant cell lines remained sensitive to dinaciclib treatment and to cytotoxic chemotherapy drugs including cytarabine and etoposide while demonstrating resistance to PLX51107 via Annexin PI and MTS assays. We found sensitivity to dinaciclib treatment in these resistant cell lines remained unaffected by BETi resistance and continued to demonstrate inhibition of Wnt signaling, similar to the effects seen in parental cells. Therefore, dinaciclib represents a novel strategy to overcome potential therapy emergent BETi resistance. We next examined the efficacy of PLX51107 in combination with dinaciclib in vivo, utilizing luciferase labeled MOLM-13 AML cells xenotransplanted into NOD / SCID / IL2rgnull (NSG) immunodeficient mice. Daily oral dosing with 20 mg/kg PLX51107 resulted in prolonged survival (median 42 days) compared to vehicle treated control animals (median 28 days, p< 0.001). Weekly intraperitoneal 10 mg/kg dinaciclib did not significantly improve survival (median 30 days, p=0.09) in MOLM-13 xenograft mice. However, mice receiving combination therapy survived a median of 56.5 days, significantly longer than with either drug alone (p<0.05) (Figure 1B). We also found decreased disease burden by bioluminescence and by histopathology in combination treated animals, compared to both vehicle and single agent controls. In conclusion, we have demonstrated in vitro and in vivo synergistic activity of the BET inhibitor PLX51107 in combination with dinaciclib in pre-clinical models of AML and have identified novel inhibition of Wnt / β-catenin signaling by dinaciclib, thereby providing a strategy to circumvent resistance to BET inhibitors in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Thymoquinone Inhibits JAK/STAT and PI3K/Akt/ mTOR Signaling Pathways in MV4-11 and K562 Myeloid Leukemia Cells.

Constitutive activation of Janus tyrosine kinase-signal transducer and activator of transcription (JAK/STAT) and Phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathways plays a crucial role in the development of acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). Thymoquinone (TQ), one of the main constituents of Nigella sativa, has shown anti-cancer activities in several cancers. However, the inhibitory effect mechanism of TQ on leukemia has not been fully understood. Therefore, this study aimed to investigate the effect of TQ on JAK/STAT and PI3K/Akt/mTOR pathways in MV4-11 AML cells and K562 CML cells. FLT3-ITD positive MV4-11 cells and BCR-ABL positive K562 cells were treated with TQ. Cytotoxicity assay was assessed using WSTs-8 kit. The expression of the target genes was evaluated using RT-qPCR. The phosphorylation status and the levels of proteins involved in JAK/STAT and PI3K/Akt/mTOR pathways were investigated using Jess western analysis. TQ induced a dose and time dependent inhibition of K562 cells proliferation. TQ significantly downregulated PI3K, Akt, and mTOR and upregulated PTEN expression with a significant inhibition of JAK/STAT and PI3K/Akt/mTOR signaling. In conclusion, TQ reduces the expression of PI3K, Akt, and mTOR genes and enhances the expression of PTEN gene at the mRNA and protein levels. TQ also inhibits JAK/STAT and PI3K/Akt/mTOR pathways, and consequently inhibits proliferation of myeloid leukemia cells, suggesting that TQ has potential anti-leukemic effects on both AML and CML cells.

A Novel BRD Family PROTAC Inhibitor dBET1 Exerts Great Anti-Cancer Effects by Targeting c-MYC in Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) represents an aggressive hematopoietic malignancy with a prognosis inferior to that of other leukemias. Recent targeted therapies offer new opportunities to achieve better treatment outcomes. However, due to the complex heterogeneity of AML, its prognosis remains dismal. In this study, we first identified the correlation between high expression of BRD4 and overall survival of patients with AML. Targeted degradation of BRD2, BRD3, and BRD4 proteins by dBET1, a proteolysis-targeting chimera (PROTAC) against the bromodomain and extra-terminal domain (BET) family members, showed cytotoxic effects on Kasumi (AML1-ETO), NB4 (PML-RARa), THP-1 (MLL-AF9), and MV4-11 (MLL-AF4) AML cell lines representing different molecular subtypes of AML. Furthermore, we determined that dBET1 treatment arrested cell cycling and enhanced apoptosis and c-MYC was identified as the downstream target. Collectively, our results indicated that dBET1 had broad anti-cancer effects on AML cell lines with different molecular lesions and provided more benefits to patients with AML.

Abstract 3973: Transcriptomic profiling of in-vitro modelled drug resistant AML cell lines unravel metabolic and stemness gene signatures

Abstract Disease recurrence is the major cause of treatment failure and mortality in acute myeloid leukemia (AML). A small fraction of leukemic cells that withstand chemotherapeutic insult can rewire cellular processes to re-emerge with lethal disease. Approaches to decipher drug resistance mechanisms in these drug persister cells can pave the way for newer therapies and improve survival in patients. To uncover drug resistant mechanisms that are clinically relevant, we generated in-house Cytarabine (AraC) and Arsenic trioxide (ATO) resistant cells from naïve MV4-11 AML cell line by incremental dose exposure method. Single cell clones were generated from AraC (MV411-AraC-R)/ATO (MV411-ATO-R) drug tolerant cells. Both the AraC-R and ATO-R clones were resistant to AraC (600-fold) and ATO (2-fold) respectively compared to the naïve cells. AraC-R cells were marginally cross resistant to daunorubicin (DNR) and ATO. Interestingly, the ATO-R cells were highly cross resistant to both AraC (600-fold) and DNR (4-fold) demonstrating altered drug resistance mechanism. We next subjected the AraC-R, ATO-R and the naïve cells to RNAseq analysis to identify transcriptomic changes post resistance development. 1218 transcripts in AraC-R cells and 7654 transcripts in ATO-R cells were differentially expressed compared to the naïve cells, suggesting ATO exposure alters the transcriptome drastically compared to AraC. We performed gene set enrichment analysis (GSEA) with these differentially expressed gene signatures. AraC-R gene signatures were highly enriched (NES-2.8 &amp; 2.0 FDR-0.00) for oxidative phosphorylation and mitochondrial related signatures, consistent with previous report suggesting AraC exposed residual cells are highly enriched for oxidative phosphorylation and are not enriched for stemness gene signatures (Farge et al). ATO-R gene signatures were positively enriched (NES- 2.0 &amp; 1.4 FDR 0.005 &amp; 0.03) for Leukemic stem cell gene signatures (Eppert_LSC and Gentles_LSC) and relapse (NES- 2.0 &amp; 1.5 FDR 0.0 &amp; 0.01) gene signatures (CALCRL_RIC and Hackles_relapse) and negatively enriched for mitochondrial gene signatures showing reduced reliance on mitochondrial metabolism. Since AraC-R cells showed high dependency on mitochondrial metabolism, we treated these cells with venetoclax (VEN) a known oxphos inhibitor for 48 hours and assessed the viability. Ven markedly sensitized the AraC-R cells compared to naïve cells (IC50 13nM and 323nM), while ATO-R cells were markedly resistant to Ven (IC50 8049nM) corroborating our transcriptomic findings. These finding suggest transcriptome profiling of in-vitro modelled drug resistant cell lines can mimic clinically relevant drug resistant mechanism and could aide in identifying alternate therapies to circumvent disease relapse. Further metabolomic characterization and mechanistic studies are ongoing to substantiate these findings. Citation Format: Raveen Stephen Illangeswaran, Daniel Zechariah Jebanesan, Kezia Kanimozhi Sivakumar, Rakhi Thalayattu Vidhyadharan, Vikram Mathews, Shaji Ramachandran Velayudhan, Poonkuzhali Balasubramanian. Transcriptomic profiling of in-vitro modelled drug resistant AML cell lines unravel metabolic and stemness gene signatures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3973.

Thymoquinone Inhibits Growth of Acute Myeloid Leukemia Cells through Reversal SHP-1 and SOCS-3 Hypermethylation: In Vitro and In Silico Evaluation.

Epigenetic silencing of tumor suppressor genes (TSGs) plays an essential role in cancer pathogenesis, including acute myeloid leukemia (AML). All of SHP-1, SOCS-1, and SOCS-3 are TSGs that negatively regulate JAK/STAT signaling. Enhanced re-expression of TSGs through de-methylation represents a therapeutic target in several cancers. Thymoquinone (TQ) is a major component of Nigella sativa seeds with anticancer effects against several cancers. However, the effects of TQ on DNA methylation are not entirely understood. This study aimed to evaluate the ability of TQ to re-express SHP-1, SOCS-1, and SOCS-3 in MV4-11 AML cells through de-methylation. Cytotoxicity, apoptosis, and cell cycle assays were performed using WSTs-8 kit, Annexin V-FITC/PI apoptosis detection kit, and fluorometric-red cell cycle assay kit, respectively. The methylation of SHP-1, SOCS-1, and SOCS-3 was evaluated by pyrosequencing analysis. The expression of SHP-1, SOCS-1, SOCS-3, JAK2, STAT3, STAT5A, STAT5B, FLT3-ITD, DNMT1, DNMT3A, DNMT3B, TET2, and WT1 was assessed by RT-qPCR. The molecular docking of TQ to JAK2, STAT3, and STAT5 was evaluated. The results revealed that TQ significantly inhibited the growth of MV4-11 cells and induced apoptosis in a dose- and time-dependent manner. Interestingly, the results showed that TQ binds the active pocket of JAK2, STAT3, and STAT5 to inhibit their enzymatic activity and significantly enhances the re-expression of SHP-1 and SOCS-3 through de-methylation. In conclusion, TQ curbs MV4-11 cells by inhibiting the enzymatic activity of JAK/STAT signaling through hypomethylation and re-expression of JAK/STAT negative regulators and could be a promising therapeutic candidate for AML patients.

Clinical Translation of SC-DARIC33: A Pharmacologically Controlled CD33-Targeted Anti-AML CAR T Cell Product Regulated By Low Nanomolar Concentrations of Rapamycin

Background: Acute myeloid leukemia (AML) chimeric antigen receptor (CAR) T cell therapies are at early stages of testing in human clinical trials. We previously described the design of a CD33-specific dimerizing agent regulated immunoreceptor complex (DARIC33) that, in the presence of rapamycin (RAPA), switches from an “OFF” to “ON” state that activates T cells in response to tumor antigen. Here, we describe RAPA controlled activation and anti-AML activity of DARIC33 using human AML xenograft NSG mouse models and GMP-compliant T cell manufacturing methodologies. We find that low nanomolar whole blood concentrations of RAPA, below levels used for immunosuppression, are needed for DARIC33 to become active in vivo and exhibit potent CD33-specific anti-AML activity.Methods: Clinical scale, DARIC33 and control cell products were manufactured by Seattle Children's Therapeutics (SC-DARIC33) by stimulating equal numbers of CD4+ and CD8+ T cells with anti-CD3/CD28 microbeads in closed gas-permeable culture vessels followed by lentiviral vector transduction and expansion in serum-free media supplemented with IL7, IL15 and IL21. The activation and anti-AML activity of SC-DARIC33 assayed in vitro by measuring cytokine production or lysis of chromium labeled target cells in the presence or absence of RAPA. NSG mice inoculated with either 1x10 6 luciferase expressing MV4-11 (CD33+) or 0.5x10 6 Raji cells expressing a huCD33 transgene (Raji.CD33, CD19+/CD33+) were treated with SC-DARIC33 and RAPA. Concentrations of RAPA in mouse blood were quantified by LC-MS/MS (Charles River).Results: Donor matched SC-DARIC33 and control CD19 CAR T cell products exhibited similar surface markers of engraftment fitness (CD62L+CD45RA+) and capacity for anti-tumor (CD27+CCR7+) effector function. Following coculture of SC-DARIC33 and Raji.CD33 cells without RAPA, concentrations of IL-2, TNF-α or IFN-γ were not increased in comparison to Raji.CD33 cells cocultured with mock T cell products. However, when exposed to 1 nM RAPA and Raji.CD33 targets, SC-DARIC33 produced cytokines in quantities similar to (Fig A). To determine the concentration of RAPA required to activate SC-DARIC33 in patients, DARIC33 T cells, CD33+ AML targets, and graded concentrations of RAPA were added to allogeneic human whole blood samples and plasma was recovered after 24 hours of incubation. RAPA addition increased IFN-g release with an apparent EC50 = 2.6nM (Fig B).Anti-AML SC-DARIC33 activity and whole blood [RAPA WB] in tumor bearing mice were determined and compared to pediatric RAPA pharmacokinetic models to select appropriate clinical RAPA dose schedules. NSG mice inoculated with Raji.CD33 tumors treated with either 1x10 7 CD19 CAR T cells or 3x10 7 SC-DARIC33 T cells, followed by RAPA 0.1 mg/kg IP QOD, exhibited stringent control of tumor growth, demonstrating a 3:1 cell dose equivalency (Fig C). AML progression was also inhibited when NSG mice were inoculated with MV411 AML cells and subsequently treated with 10 7 SC-DARIC33 followed by 0.01 mg/kg RAPA QD (Fig D). Blood samples from tumor bearing mice obtained on day 15, 2 hours post RAPA administration, showed [RAPA WB] = 2.3 ± 1.3 ng/mL, overlapping with the in vitroand indicating very low concentrations of RAPA effectively modulate the “OFF-to-ON” state transition. Population PK models simulating various RAPA doses and schedules in pediatric patients found oral daily dosing of 0.5 mg/m 2 RAPA will achieve [RAPA WB] = 1-3 ng/mL in most patients (Fig E).Conclusion: Evaluation of GMP cell products and RAPA PK demonstrate that very low doses of RAPA are sufficient to regulate SC-DARIC33. To establish safety of SC-DARIC33 in humans, an upcoming phase 1 trial clinical trial evaluating SC-DARIC33 in pediatric AML patients will test escalating cell doses followed by low-dose RAPA administration from post T cell infusion days 2-21 using a Bayesian optimal interval (BOIN) design. Peripheral blood samples will be monitored for CD33+ myeloid cell recovery after cessation of RAPA dosing. These data will establish safety and support the feasibility of SC-DARIC33 CAR T cells to be reversibly modulated in an “OFF-ON-OFF” fashion by intermittent low-dose RAPA administration. [Display omitted] DisclosuresPrice: bluebird, bio: Current Employment. Zhang: bluebird, bio: Current Employment. Sundaram: bluebird, bio: Current Employment. Lewis: bluebird, bio: Current Employment. Bilic: C4 Therapeutics: Current Employment. Xia: bluebird, bio: Current Employment. Krostag: bluebird, bio: Ended employment in the past 24 months. So: bluebird, bio: Current Employment. Martin: bluebird, bio: Current Employment. Leung: bluebird, bio: Ended employment in the past 24 months. Astrakhan: bluebird, bio: Current Employment. Pogson: bluebird, bio: Current Employment. Jarjour: bluebird, bio: Current Employment. Jensen: bluebird, bio: Research Funding.

Inhibition of microRNA 10a, an Autophagy Modulator, Increases Chemosensitivity to Ara-C in Acute Myeloid Leukemia

Aims: Leukemic relapse due to chemoresistance is a major obstacle in curing acute myeloid leukemia (AML), and the activation of pro-survival autophagy pathways has been implicated in this process. The microRNA-10a (miR-10a), a post-transcriptional regulator of several cell cycle and autophagy-related genes, is highly expressed in normal karyotype (NK) AML, especially those with nucleophosmin1 mutations (NPM1c+ ). This study aimed to investigate whether miR-10a modulation could affect autophagy and chemosensitivity in these cells.Results: Lentiviral transduction of a miR-10a inhibitor into the miR-10a-high NPM1c+ OCI-AML3 cell line significantly inhibited autophagy as signified by a marked decrease in autophagy marker LC3-II accumulation, compared to cells transduced with a scrambled control inhibitor (SCR). This was accompanied by significantly increased sensitivity to Ara-C (IC50 = 8.2µM vs 20.0µM; p <0.0001) characterised by a 2-fold increase in apoptosis (p = 0.035; quantified by Annexin V binding). Conversely, overexpression of miR-10a in the miR-10a-low, NPM1-wildtype MV4-11 AML cell line resulted in increased autophagy as demonstrated by a 3-fold increase in LC3-II (p = 0.001) and conferred pro-survival effects, including reduced apoptosis (p = 0.04) and markedly decreased Ara-C sensitivity (IC50 = 8.7 µM vs 2.7µM; p = 0.03).We previously demonstrated that miR-10a directly targets TFAP2C, a key inhibitor of CDKN1A-encoding cell cycle inhibitor p21. p21 is an important promoter of cell cycle arrest that precedes autophagy in response to stress signalling from the p53 signalling network. miR-10a inhibition was associated with a marked reduction in p21 expression, particularly in those cells in which p53 was activated either by treatment with MDM2 antagonist Nutlin-3a (p = 0.02) or by serum deprivation (p =0.005), and was associated with aberrant cell cycling in response to Ara-C treatment. Inhibition of CDKN1A specifically by siRNA showed similar effects to miR-10a inhibition, with increased cell death in response to Ara-C treatment (p <0.05).The observed increase in chemosensitivity in vitro was replicated in vivo in NSG mice engrafted with miR-10a inhibitor-transduced OCI-AML3 cells and treated with a course of Ara-C (50mg/kg for 3 weeks; n = 6 per treatment group). Treated mice had significantly improved survival (83% of miR-10a inhibited mice surviving at 30 days post-engraftment vs 50% of controls; hazard ratio = 6.6; p = 0.02) and had significantly fewer leukemic cells detectable in the bone marrow 24h after the end of treatment (26.7% of cells compared to 54.5% in animals engrafted with control inhibitor-transduced OCI-AML3 cells; p = 0.04).Conclusion: To our knowledge, this study provides for the first time evidence that miR-10a confers chemoresistance to Ara-C in NK-AML, likely via its effects on the p21/p53-mediated activation of autophagy and may in part explain treatment failure observed in the clinic. DisclosuresStoelzel:medac: Other: Travel support.

Preclinical Characterization of TP‐0903, a Novel Multikinase Inhibitor, in TP53 Mutant Acute Myeloid Leukemia

Objective Acute myeloid leukemia (AML) with mutations in the tumor suppressor gene TP53 confers a dismal prognosis with 1-year overall survival of <5%. Effective treatment options are limited and current standard-of-care includes the hypomethylating agents (HMA) decitabine and azacytidine. While inhibition of kinases involved in cell cycle regulation has been shown to induce synthetic lethality in a variety of TP53 mutant cancers, this strategy has not been evaluated in mutant TP53 AML. Previously, we demonstrated that TP-0903 is a novel multikinase inhibitor with low nM activity against AURKA/B, CHEK1/2, and other cell cycle regulators (Jeon JY et al. JCI Insight 2020), thus providing scientific rationale to evaluate TP-0903 activity in TP53 mutant AML. Methods To generate an in vitro model of TP53 mutant AML, we isolated single-cell clones containing mutant (R248W) or wild-type (WT) TP53 from the established MV4-11 AML cell line; regulation of p53 targets (MDM2, p21) following gamma irradiation and inhibition of p-AURKA and p-CHEK1 by TP-0903 were assessed by immunoblot. Using these and additional TP53 mutant AML cell lines (Kasumi-1, HL-60), in vitro efficacy of TP-0903 alone and in combination with HMA was assessed in viability (MTT) and apoptosis (Annexin V) assays. In vivo efficacy studies were conducted in NSG mice following intravenous injection of HL-60 or luciferase-tagged MV4-11/TP53-R248W cells. Mice (5-10 per treatment cohort) were treated with vehicle, TP-0903 (50 mg/kg orally; 5 days on/2 days off), decitabine (0.2-0.4 mg/kg i.p.; 4 days on/10 days off) or the combination. Whole body bioluminescence imaging was performed weekly and median survival was determined. Results Compared to the clone with WT TP53, we observed a lack of MDM2 and p21 induction in MV4-11/TP53-R248W cells following gamma irradiation. In vitro, TP-0903 inhibited pAURKA and pCHEK1 at 50 nM, inhibited cell viability (IC50 values, 12-40nM), and induced apoptosis at 20-50nM. The combination of TP-0903 with HMA was additive to synergistic in all AML cell lines evaluated. In the HL-60 xenograft model, the TP-0903/decitabine combination prolonged median survival (75 days) compared to cohorts of mice treated with TP-0903 (63 days), decitabine (55 days), or vehicle (46 days) (P<0.0001). In the MV4-11/TP53-R248W xenograft model, bioluminescence imaging showed that TP-0903 alone or in combination with decitabine was more effective in suppressing the outgrowth of leukemia cells compared to mice treated with vehicle or decitabine alone (P<0.05); survival analysis is ongoing. Conclusions TP-0903 was effective in all evaluated preclinical models of TP53 mutant AML. Together, these results provide scientific premise for the initiation of a Phase 1b/2 trial of TP-0903 in combination with decitabine in TP53 mutant/complex karyotype AML under the umbrella Beat AML Master Trial.

Biological Evaluation of Selected Flavonoids as Inhibitors of MNKs Targeting Acute Myeloid Leukemia.

Excessive eIF4E phosphorylation by mitogen-activated protein kinase (MAPK)-interacting kinases 1 and 2 (MNK1 and MNK2; collectively, MNKs) has been associated with oncogenesis. The overexpression of eIF4E in acute myeloid leukemia (AML) is related to cancer cell growth and survival. Thus, the inhibition of MNKs and eIF4E phosphorylation are potential therapeutic strategies for AML. Herein, a structure-based virtual screening approach was performed to identify potential MNK inhibitors from natural products. Three flavonoids, apigenin, hispidulin, and luteolin, showed MNK2 inhibitory activity with IC50 values of 308, 252, and 579 nM, respectively. A structure-activity relationship analysis was performed to disclose the molecular interactions. Furthermore, luteolin exhibited substantial inhibitory efficacy against MNK1 (IC50 = 179 nM). Experimental results from cellular assays showed that hispidulin and luteolin inhibited the growth of MOLM-13 and MV4-11 AML cells by downregulating eIF4E phosphorylation and arresting the cell cycle at the G0/G1 phase. Therefore, hispidulin and luteolin showed promising results as lead compounds for the potential treatment for AML.

BCL-2 Inhibitor Venetoclax Enhances Temozolomide Sensitivity in AML

Introduction: Many older patients with acute myeloid leukemia (AML) are ineligible for intensive chemotherapy due to frailty and co-morbidities; for such patients, existing treatments are often ineffective and new treatments are needed. Temozolomide (TMZ) is an alkylating agent that causes DNA methylation at O6 guanine, generating single strand break leading to apoptosis. However, the efficacy of TMZ depends on the DNA repair protein O6-methylguanine methyltransferase (MGMT), that maintains the genomic integrity by removing the O6-methyl group and restoring guanine nucleobase, thereby enhancing resistance to TMZ. Previous clinical trials in AML found that responses to TMZ correlated with low MGMT expression; however, even in those with low MGMT expression complete response rates were only in the 25% range. BCL-2, an anti-apoptotic protein, is overexpressed in AML cells. Direct inhibition by the selective BCL-2 inhibitor venetoclax (Venet) promotes apoptosis. This study evaluated the ability of Venet to enhance TMZ sensitivity in AML cells, including those with MGMT overexpression. Methods: KG1, MV4-11 and MOLM13 AML cell lines were studied, as well as bone marrow blast cells collected from AML patients. Western blot was used to measure MGMT and BCL-2 expression. The cells were incubated with TMZ at varying concentrations in combination with a fixed concentration of Venet. After 48 hours, cell viability and apoptosis assays were performed using spectrophotometry and flow cytometry, respectively. Synergy was evaluated by the Chou-Talalay method. Cleaved-PARP was measured by Western blot in selected combination doses after 3 hours in MV4-11 and MOLM13 and after 6 hours in KG1. Results: KG1 cells expressing high MGMT demonstrated strong resistance to TMZ; however, co-incubation with 1 uM Venet resulted in a marked enhancement of sensitivity to TMZ. Similarly, in MV4-11 and MOLM13 cell lines, which demonstrated very low or absent MGMT expression. Venet 2.5 nM in combination with TMZ markedly increased the cytotoxicity to TMZ. A synergistic effect was demonstrated in all cell lines with combination index (CI) &lt; 1. Cells overexpressed annexin V and propidium iodide (PI) apoptotic marker after drug combination in all cell lines. Apoptotic effect with the drug combination was verified by cleaved-PARP expression. Most (6/8) AML patient samples which were resistant to TMZ in vitro became sensitized to TMZ in combination with 1 uM Venet, including those with moderate to high MGMT expression. Conclusion: Venetoclax synergizes with TMZ and induces cytotoxicity in all AML cell lines and in most AML patient samples, including those in whom MGMT was highly expressed, by activating apoptotic pathways to trigger cell death. This combination represents a potentially promising new treatment. Further studies evaluating this combination in animal models are in progress. Disclosures Brandwein: Roche: Research Funding; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Otsuka: Honoraria; Jazz Pharma: Consultancy, Honoraria. OffLabel Disclosure: Temozolomide for AML

Role of HDAC8 Upregulation in Resistance of FLT3-ITD AML Stem Cells to FLT3 Inhibitors

The FLT3-ITD mutation occurs in 25-30% of acute myeloid leukemia (AML) incidence, and is associated with higher relapse rate and inferior survival. FLT3 inhibitors have shown potent antileukemic effect, but the responses are not sustained in most FLT3-ITD AML patients. Histone deacetylases (HDACs) are crucial for cancer stem cell maintenance through epigenetically regulating gene expression, modulating the function of non-histone proteins via post-translational modification, and maintaining genome stability. Here we investigated the potential role of HDACs in FLT3 inhibitors resistance and FLT3-ITD AML stem cells maintenance. We firstly evaluated mRNA expression alterations of 11 HDACs in FLT3-ITD+ MV4-11 and MOLM-13 AML cells upon FLT3 inhibitor AC220 (Quizartinib) treatment, and found that HDAC8 was the most significantly upregulated gene. Inhibition of FLT3 signaling via siRNA-mediated knockdown of FLT3 also resulted in increased expression of HDAC8. Treatment of primary FLT3-ITD+ CD34+ AML cells with AC220 confirmed HDAC8 upregulation. Targeting HDAC8 using specific HDAC8 inhibitor 22d or knockdown of HDAC8 readily resulted in significant apoptosis of cell lines and primary AML samples, and inhibited colony growth and replating activity in primary FLT3-ITD+ CD34+ AML cells. Moreover, these antileukemic effects were even more pronounced upon 22d and AC220 combination. To explore the mechanism of HDAC8 mediated FLT3 inhibitor resistance, we investigated transcriptomic change of MV4-11 cells upon 22d treatment by RNA-seq. KEGG pathway analysis revealed that the p53 signaling pathway was significantly upregulated. Upstream regulator prediction by Ingenuity pathway analysis also showed p53 as the key activated upstream transcription factor. Western blot analysis on acetylated p53 (Lys382) demonstrated that targeting FLT3 signaling had little effect on the p53 acetylation, while suppression of HDAC8 markedly increased p53 acetylation, and this effect was even more significant when both FLT3 and HDAC8 were inhibited simultaneously. Knockdown of p53 could partially rescued FLT3-ITD AML cells from 22d and/or AC220 induced apoptosis, and offset the inhibitory effect on growth and colony formation. We next studied the mechanism of HDAC8 upregulation upon FLT3 signally inhibition. We found FOXO1 and FOXO3 (FOXOs) binding motifs in HDAC8 promoter. FLT3 signaling inhibition promoted the binding of FOXOs to HDAC8 promoter. Knockdown both FOXOs abrogated HDAC8 upregulation upon AC220 treatment, and caused growth inhibition and apoptosis of FLT3-ITD AML cells. Finally, to translate our experimental results into clinical usage, we tested the in vivo effect of 22d, AC220, and the combination of both on patient-derived xenograft (PDX) model engrafted with four primary FLT3-ITD AML samples. 22d or AC220 treatment reduced the percentage and total number of human CD45+ leukemic cells in murine bone marrow, and prolonged the survival of leukemic mice. The combination of 22d and AC220 significantly reduced AML cell engraftment compared with either 22d or AC220 alone, and further elongated the survival period. Human CD34+ cells in BM were markedly reduced in mice treated with 22d or AC220 alone, with further reduction seen in combination. Secondary transplantation of BM cells from mice receiving combination treatment resulted in significantly reduced engraftment compared with AC220 treatment alone, indicating reduced LSC capacity of residual cells. These results show that combination of 22d and AC220 enhances targeting of primitive AML stem cells in vivo. In conclusion, upon FLT3 inhibitor treatment, FLT3-ITD AML cells may upregulate the expression of HDAC8 through FOXOs activation, which in turn inhibit the function of tumor suppressor gene p53, and promote the maintenance of FLT3-ITD AML stem cells and drug resistance. Targeting FLT3 and HDAC8 simultaneously may be promising approach to eliminating FLT3-ITD AML stem cells, and to cure this subset of leukemia. Disclosures No relevant conflicts of interest to declare.

Effect of Mangiferin on Proliferation of FLT3-ITD Mutation Positive Acute Myeloid Leukemia Cell MV4-11 and Its Mechanism

To investigate the effects of mangiferin on proliferation, apoptosis and cycle of FLT3-ITD mutation-positive acute myeloid leukemia cells and its mechanism. The effects of different concentration of mangiferin on proliferation of MV4-11 cells were detected by CCK8 method. Apoptosis, cell cycle and FLT3 transmembrane protein expression were detected by flow cytometry. FLT3 mRNA expression was detected by real-time fluorescent quantitative polymerase chain reaction (PCR) . Mangiferin obviously inhibited MV4-11 proliferation in a concentration and time dependent manner (48 h，r=0.922；72 h，r=0.959；96 h，r=0.973). The ratio of G0/G1 phase in cell cycle increased with the enhancement of concentration of mangiferin in MV4-11 cells for 48 h, and the ratio of S phase decreased with enhasment of concentration. The increase of apoptosis was more obvious. The expression of FLT3 transmembrane protein significantly decreased after the actior of IC50 concentration of mangiferin in MV4-11 cells for 48 h. The results of qRT-PCR showed that the expression of FLT3 mRNA significantly decreased after treatment of MN4-11 cells with mangiferin (P＜0.05). Mangiferin inhibits MV4-11 cell proliferation, arrests cell cycle progression and promotes apoptosis, which may be related with the inhibition of FLT3 activity by mangiferin and the subsequent signaling pathways involved in apoptosis and proliferation of cells.

Inhibition of USP9X induces apoptosis in FLT3-ITD-positive AML cells cooperatively by inhibiting the mutant kinase through aggresomal translocation and inducing oxidative stress.

FLT3-ITD and FLT3-TKD are the most frequent mutations in acute myeloid leukemia (AML) with the former associated with a poor prognosis. Here we show that inhibition of the deubiquitinase USP9X by its inhibitor WP1130 or EOAI3402143 (G9) induces apoptosis preferentially in cells transformed by these mutant kinases, including FLT3-ITD-positive AML cell line MV4-11 and primary AML cells. Mechanistically, WP1130 induced aggresomal translocation of the mutant kinases, particularly FLT3-ITD in its activated and autophosphorylated conformation, to block the downstream signaling events, which was aggravated by knock down of USP9X. Moreover, USP9X physically associated with FLT3-ITD to inhibit its K63-linked polyubiquitination, while FLT3-ITD induced tyrosine phosphorylation and degradation of USP9X through the ubiquitin/proteasome pathway. WP1130 or G9 also induced oxidative stress to stimulate stress-related MAP kinase pathways and DNA damage responses to activate in cooperation with inhibition of FLT3-ITD signaling the intrinsic mitochondria-mediated apoptotic pathway, which was synergistically enhanced by BH3 mimetics and prevented by overexpression of Bcl-xL or Mcl-1. Thus, USP9X represents a promising target for novel therapies against therapy-resistant FLT3-ITD-positive AML.

8-chloro-adenosine activity in FLT3-ITD acute myeloid leukemia.

Nucleoside analogs represent the backbone of several distinct chemotherapy regimens for acute myeloid leukemia (AML) and combination with tyrosine kinase inhibitors has improved survival of AML patients, including those harboring the poor-risk FLT3-ITD mutation. Although these compounds are effective in killing proliferating blasts, they lack activity against quiescent leukemia stem cells (LSCs), which contributes to initial treatment refractoriness or subsequent disease relapse. The reagent 8-chloro-adenosine (8-Cl-Ado) is a ribose-containing, RNA-directed nucleoside analog that is incorporated into newly transcribed RNA rather than in DNA, causing inhibition of RNA transcription. In this report, we demonstrate antileukemic activities of 8-Cl-Ado in vitro and in vivo and provide mechanistic insight into the mode of action of 8-Cl-Ado in AML. 8-Cl-Ado markedly induced apoptosis in LSC, with negligible effects on normal stem cells. 8-Cl-Ado was particularly effective against AML cell lines and primary AML blast cells harboring the FLT3-ITD mutation. FLT3-ITD is associated with high expression of miR-155. Furthermore, we demonstrate that 8-Cl-Ado inhibits miR-155 expression levels accompanied by induction of DNA-damage and suppression of cell proliferation, through regulation of miR-155/ErbB3 binding protein 1(Ebp1)/p53/PCNA signaling. Finally, we determined that combined treatment of NSG mice engrafted with FLT3-ITD + MV4-11 AML cells with 8-Cl-Ado and the FLT3 inhibitor AC220 (quizartinib) synergistically enhanced survival, compared with that of mice treated with the individual drugs, suggesting a potentially effective approach for FLT3-ITD AML patients.