Acute Myeloid Leukemia Cell Lines MV4-11 Research Articles

Correlation of miR-155 Expression with Drug Sensitivity of FLT3-ITD+ Acute Myeloid Leukemia Cell Line and Its Mechanism

To investigate the correlation of miR-155 expression with drug sensitivity of FLT3-ITD+ acute myeloid leukemia (AML) cell line and its potential regulatory mechanism. By knocking out miR-155 gene in FLT3-ITD+ AML cell line MV411 through CRISPR/Cas9 gene-editing technology, monoclonal cells were screened. The genotype of these monoclonal cells was validated by PCR and Sanger sequencing. The expression of mature miRNA was measured by RT-qPCR. The treatment response of doxorubicin, quizartinib and midostaurin were measured by MTT assay and IC50 of these drugs were calculated to identify the sensitivity. Transcriptome sequencing was used to analyze change of mRNA level in MV411 cells after miR-155 knockout, gene set enrichment analysis to analyze change of signaling pathway, and Western blot to verify expressions of key molecules in signaling pathway. Four heterozygotes with gene knockout and one heterozygote with gene insertion were obtained through PCR screening and Sanger sequencing. RT-qPCR results showed that the expression of mature miR-155 in the monoclonal cells was significantly lower than wild-type clones. MTT results showed that the sensitivity of MV411 cells to various anti FLT3-ITD+ AML drugs increased significantly after miR-155 knockout compared with wild-type clones. RNA sequencing showed that the mTOR signaling pathway and Wnt signaling pathway were inhibited after miR-155 knockout. Western blot showed that the expressions of key molecules p-mTOR, Wnt5α and β-catenin in signaling pathway were down-regulated. Drug sensitivity of MV411 cells to doxorubicin, quizartinib and midostaurin can be enhanced significantly after miR-155 knockout, which is related to the inhibition of multiple signaling pathways including mTOR and Wnt signaling pathways.

Abstract 3129: BYON4413, an in vivo active CD123-targeting antibody-drug conjugate, combines effectively with azacitidine and venetoclax in acute myeloid leukemia cell lines

Abstract Background: CD123, the alpha chain of the interleukin-3 receptor (IL-3Rα), is overexpressed in multiple hematologic malignancies and found particularly highly expressed on the surface of acute myeloid leukemia (AML) cells. Furthermore, increased CD123 expression on leukemic stem cells relative to non-neoplastic hematopoietic stem cells makes CD123 an attractive tumor-associated target for an antibody-drug conjugate (ADC). BYON4413 is an ADC that consists of a novel humanized IgG1 anti-CD123 antibody site-specifically conjugated with Byondis' proprietary duocarmazine linker-drug technology, ByonZine® and ByonShieLD®. The efficacy of BYON4413 is studied as monotherapy and in combination with azacitidine and venetoclax (AZA/VEN), since the AZA/VEN regimen is the standard of care (SOC) for AML patients who are ineligible for intensive chemotherapy. Method: A panel of CD123+ and CD123- hematological cell lines and AML patient-derived bone marrow mononuclear cells (BMMCs)/peripheral blood mononuclear cells (PBMCs) were utilized in in vitro studies to determine the cytotoxic effects of BYON4413. The induction of apoptosis and cell killing by BYON4413 in a triple combination with AZA/VEN was studied in vitro using the CD123+ AML cell lines MOLM-13 and MV4-11. AML patient and cell line-derived xenograft models were used to investigate the in vivo efficacy of BYON4413. Results: In vitro studies with human AML cell lines show that BYON4413 is highly effective in eradicating CD123+ cells while having little impact on CD123- cells. These results were confirmed ex vivo with AML patient-derived BMMCs/PBMCs. BYON4413 effectively killed AML patient-derived blasts while largely sparing healthy hematopoietic cells. The triple combination of BYON4413 with AZA/VEN enhanced both apoptosis and cell killing of AML cell lines compared to the singular BYON4413 treatment. In vivo studies demonstrate that BYON4413 is remarkably efficient at reducing the tumor burden in AML patient- and cell line-derived xenograft models. Conclusions: Preclinical studies show that BYON4413 has potential to be an effective targeted therapy against CD123+ hematological malignancies such as AML. The enhanced efficacy of BYON4413 together with AZA/VEN suggests that a clinical benefit from the triple combination may be achievable and is worth further exploration. A first-in-human dose-escalation and expansion trial with BYON4413, designed to enroll R/R AML and high-risk MDS patients, is scheduled to start in Q2 2024. Citation Format: Miranda M.C. van der Lee, Tanja van Achterberg, Kaylee Hersmus, Annet Brouwers-Vos, Monique van der Vleuten, Maurien Pruis, Wendy Kappers, Gijs Verheijden, Gerwin Huls, Glenn van Wigcheren, Ruud Ubink, Aloys Sesink, Alyson MacInnes, Jan Jacob Schuringa, Wim Dokter. BYON4413, an in vivo active CD123-targeting antibody-drug conjugate, combines effectively with azacitidine and venetoclax in acute myeloid leukemia cell lines [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 3129.

The UBE2J2/UBE2K-MARCH5 Ubiquitination Machinery Regulates Apoptosis in Response to Venetoclax in Acute Myeloid Leukemia

Evasion of apoptosis is crucial for the growth, survival and chemoresistance of many cancer types, including acute myeloid leukemia (AML); thus, the reactivation of apoptosis can be exploited as a therapeutic approach. Apoptosis induction is mainly controlled by the balance between anti-apoptotic and pro-apoptotic BCL2 family proteins on the mitochondrial membrane. Venetoclax, a selective inhibitor antagonizing the anti-apoptotic protein BCL2, has emerged as a promising therapy in AML. Despite high response rates in combination with hypomethylating agents, however, some patients display upfront resistance, and most patients will ultimately relapse. Therefore, identification of synergistic targets for combination therapies with venetoclax is important for improving the clinical application of this drug. To systematically identify key genes that can modulate the venetoclax effect, we performed a genome-scale CRISPR-Cas9 screen in the AML cell line MV4-11. Consistent with previous reports, loss of the apoptosis effector BAX or the pro-apoptotic gene NOXA ( PMAIP1) caused venetoclax resistance, while sgRNAs targeting the anti-apoptotic genes BCLXL ( BCL2L1), BCL2L2 and BCL2A1 were significantly more depleted in venetoclax-treated cells. Furthermore, depletion of the E2 ubiquitin-conjugating enzymes, UBE2J2 and UBE2K, ranked highly among the venetoclax sensitizers. We validated that deletion of either UBE2J2 or UBE2K increased induction of apoptosis in multiple AML cell lines and patient-derived xenograft (PDX) cells upon venetoclax treatment and was deleterious as single gene perturbation in some models. Exploiting the Broad Institute Cancer DepMap dataset, which includes genome-scale CRISPR-Cas9 screens in over 1000 cancer cell lines, revealed that dependency on UBE2J2 or UBE2K significantly correlated with a dependency on the E3 ligase MARCH5. We previously identified the repression of MARCH5 as a strong inducer of apoptosis in AML. Since E2s coordinate with ubiquitin E3 ligases to execute ubiquitination, this DepMap result suggested that UBE2J2 and UBE2K serve as MARCH5 E2 partners. To test this hypothesis, we utilized NanoBiT technology, a structural complementation reporter system, to detect protein interactions between MARCH5 and the E2 candidates. LgBIT and SmBIT, two split subunits of luciferase, were fused with MARCH5 and the E2 proteins, respectively. The luminescent signal was activated upon the co-expression of LgBIT-MARCH5 with either of the SmBIT-tagged E2 proteins but not an empty vector, suggesting that MARCH5 and UBE2J2/UBE2K constitute ubiquitination machinery that regulates apoptosis in AML. We next tested the possible redundancy between the two E2s and showed that the double knockout of UBE2J2/ UBE2K further enhanced the venetoclax sensitivity. MARCH5 depletion results in increased NOXA expression, an important node in dictating venetoclax response. Several reports indicated that NOXA is also a critical downstream mediator of MARCH5. In contrast, we previously showed that MARCH5 can regulate apoptosis independently of NOXA in AML. To reassess the role of NOXA and other pro-apoptotic proteins in MARCH5-mediated apoptosis, we conducted an unbiased CRISPR rescue screen in a MARCH5-dTAG degradation system derived from PDX17-14, a complex karyotype, MLL-AF10 PDX model. BAX was the top rescuing target, emphasizing that apoptosis induction is the main mechanism accounting for the growth inhibition of MARCH5-depleted cells. However, depletion of other pro-apoptotic BCL2 members, including NOXA and BIM, did not rescue MARCH5 depletion in this screen consistent with our previously published data. Here, we confirmed that NOXA KO does not rescue MARCH5 depletion in additional AML models. Similarly, KO of UBE2J2 or UBE2K can repress AML cell growth and increase venetoclax sensitivity even in the context of NOXA KO. Our study highlights that UBE2J2 and UBE2K are two important functional partners of MARCH5 in regulating apoptosis in AML cells and can serve as additional targets for enhancing venetoclax efficacy. Unbiased screening and low-throughput target validation further emphasize that the MARCH5 ubiquitination machinery regulates apoptosis in AML cells largely in a NOXA-independent manner. Additional studies are needed to dissect the MARCH5/UBE2J2/UBE2K complex-mediated apoptosis regulation in AML.

Inhibition of FLT3-ITD Kinase in Acute Myeloid Leukemia by New Imidazo[1,2-b]pyridazine Derivatives Identified by Scaffold Hopping.

FLT3 kinase is a potential drug target in acute myeloid leukemia (AML). Patients with FLT3 mutations typically have higher relapse rates and worse outcomes than patients without FLT3 mutations. In this study, we investigated the suitability of various heterocycles as central cores of FLT3 inhibitors, including thieno[3,2-d]pyrimidine, pyrazolo[1,5-a]pyrimidine, imidazo[4,5-b]pyridine, pyrido[4,3-d]pyrimidine, and imidazo[1,2-b]pyridazine. Our assays revealed a series of imidazo[1,2-b]pyridazines with high potency against FLT3. Compound 34f showed nanomolar inhibitory activity against recombinant FLT3-ITD and FLT3-D835Y (IC50 values 4 and 1 nM, respectively) as well as in the FLT3-ITD-positive AML cell lines MV4-11, MOLM-13, and MOLM-13 expressing the FLT3-ITD-D835Y mutant (GI50 values of 7, 9, and 4 nM, respectively). In contrast, FLT3-independent cell lines were much less sensitive. In vitro experiments confirmed suppression of FLT3 downstream signaling pathways. Finally, the treatment of MV4-11 xenograft-bearing mice with 34f at doses of 5 and 10 mg/kg markedly blocked tumor growth without any adverse effects.

Shotgun Lipidomics Elucidates the Lipidome Alterations of the Mcl-1 Inhibitor S63845 in AML Cell Lines with a Focus on Sphingolipids

Objective: Acute myeloid leukemia (AML) is a vigorous type of leukemia requiring effective treatment. Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic molecule that is upregulated in AML and is studied as a target for treatment. The specific Mcl-1 inhibitor, S63845, has antiproliferative effects on AML cells. Bioactive sphingolipids have crucial roles in cells and regulate Mcl-1 stability. This study aimed to elucidate the changes in lipid profiles of AML cell lines in response to Mcl-1 inhibitor S63845 treatment, with a special focus on sphingolipids. Materials and Methods: The cytotoxic effects of S63845 were identified in the AML cell lines MV4-11, HL60, and KG1 using the MTT cell proliferation assay. Lipidome analysis was conducted by quantitative shotgun lipidomics covering 378 individual lipid species in 26 classes within the major lipid categories. Results: The IC50 values of S63845 have been calculated as 7 nM for MV4-11, 53 nM for HL60, and 479 nM for KG1. The lipidome results reveal the S63845 treatment to increase ceramide (Cer) levels in the MV4-11 and KG1 cell lines at the expense of downstream sphingolipids while increasing the hexosylceramide (HexCer) levels in the HL60 cell line at the expense of the Cer and sphingomyelin (SM). Conclusion: This study showed S63845 to be able to suppress cell proliferation by altering lipid compositions in AML cell lines. More importantly, the study suggested S63845 to differentially affect the lipid profiles of AML cell lines.

The Role of Integrin Beta-1 in the Chemoresistance of Acute Myeloid Leukemia

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive disease that has a poor prognosis with a survival rate of 30-40%. The bone marrow microenvironment provides protection to AML cells from chemotherapy and thus confers cell adhesion-mediated drug resistance (CAM-DR) and promotes relapse. Integrins are key anchor molecules in CAM-DR of leukemia cells; they are heterodimeric adhesion molecules on the cell surface consisting of an alpha and a beta unit. Upregulated integrin alpha 4 and 6 expression is associated with poor survival in leukemias. We hypothesize that the targeting of integrin beta 1 (ITGB1, β1, or CD29) will also affect the integrin alpha units as they are heterodimer partners. This could be a superior approach to overcome CAM-DR in AML than just targeting the individual alpha units. METHODS: ITGB1 gene was knocked out via CRISPR-Cas9 using 2 guide RNAs in AML cell lines and expression of integrin subunits was profiled by flow cytometry. AML cell lines were treated in vitro with the anti-human ITGB1 monoclonal antibody OS2966 in combination with Ara-C chemotherapy and we determined effects on cell viability by flow cytometry and on cell signaling using RNA-sequencing. Using human patient-derived AML xenograft mouse models, we tested if blockade of ITGB1 combined with chemotherapy can overcome CAM-DR in vivo. Mice were treated with control total Ig (10 mg/kg), OS2966 (10 mg/kg), total Ig + Ara-C (600 mg/kg), or OS2966 + Ara-C twice a week for two weeks. RESULTS: Analysis of patient-derived AML cells at diagnosis and relapse, and of AML cell lines, showed high expression of ITGB1 and varying levels of other alpha and beta units. Loss-of-function studies of ITGB1 using CRISPR/Cas9-mediated knockout of ITGB1 in MLL-rearranged and FLT3-ITD+ AML cell lines MV4-11 and MOLM-13, and in AML cell line U937, revealed that surface expression of integrin alphas 3, 4, 5 and 6 were markedly decreased as determined by flow cytometry analysis. ITGB2 expression was high in all 3 AML cell lines and not affected by ITGB1 deletion. Remarkably, ITGB3 was downregulated upon ITGB1 deletion in 2 out of 3 AML cell lines, whereas the 3rd AML cell line did not express any ITGB3. As a result of the ITGB1 deletion, AML cells showed less adhesion to murine bone marrow stroma (OP9) cells and to extracellular matrix proteins. We found that inhibition of surface ITGB1 on human AML using OS2966 de-adhered the AML cells from OP9 cells. Moreover, ITGB1 blockade using OS2966 synergized with Ara-C in vitro and significantly decreased cell viability compared to single agent controls in human AML cell lines U937 and MV4-11. RNA sequencing data suggest that the NFkB pathway is significantly dysregulated in the combination treatment Ara-C and OS2966 compared to Ara-C alone. To test ITGB1 inhibition in vivo, a patient-derived AML sample, HEM279 (Fig. 1A), and AML cell line, U937 (Fig. 1B), were engrafted into NSG-SGM3 mice. Leukemic mice were treated with single agents including control total Ig, OS2966, or Ara-C; and with total Ig in combination with Ara-C or OS2966. In both xenograft models, the combination treatment of OS2966 with Ara-C significantly prolonged survival of leukemic mice compared to Ara-C only: HEM279: MST 115 +/- 15.4 days vs 73.1 +/- 5.2 days post-AML injection; p=0.0005; U937: MST 170 days vs 32 days post-AML injection; p=0.0004; 50% of the mice engrafted with U937 and treated with OS2966 with Ara-C survived until day 290 post-AML injection, which is when the experiment was terminated. CONCLUSIONS: Integrin alpha subunit expression is dependent on ITGB1 expression, supporting our hypothesis that targeting integrin beta 1 affects alpha integrin units. ITGB1 inhibition with OS2966 combined with chemotherapy significantly decreases cell viability of AML cell lines in vitro and prolongs survival of xenograft mice in vivo. Our findings will allow for better understanding of the role of ITGB1 in drug resistance of AML and of ITGB1 as a preclinical target in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mesenchymal Stromal Cells Protect Acute Myeloid Leukemia Cells from Venetoclax-Induced Apoptosis through Contact-Dependent Mechanisms

Acute myeloid leukemia (AML) patients have a poor prognosis with standard treatments due to a high relapse rate. Recently, Venetoclax (VEN), the first B-cell lymphoma 2 (BCL-2) selective inhibitor to enter the clinic, has shown a breaking-through effect in combination with demethylating agents in older patients with treatment-naïve AML. However, resistance mechanisms were described, hampering the complete remission of VEN-treated patients. It has been proposed that the bone marrow microenvironment in general and Mesenchymal stromal cells (MSCs), in particular, favor a protective niche that sustains leukemic cell viability and plays a crucial role indetermining drug sensitivity in several hematological disorders, including AML. A previous study demonstrated that VEN, as well as other drugs commonly used in AML therapy, showed a reduced effect in the presence of an MSC-conditioned medium, suggesting that MSC-dependent release of inflammatory signals and other soluble factors could be involved in drug resistance. Our study aims to further investigate the mechanisms of MSC-mediated VEN resistance in AML. We found that primary AML cells isolated from patients at diagnosis and treated with VEN showed different apoptotic rates, indicating an AML cell-specific resistance/sensitivity to the drug. In co-culture experiments, primary MSCs, isolated from healthy donors or AML patients, protected VEN-sensitive AML cells from apoptosis, confirming the critical role of MSCs in drug resistance. To deeply investigate this issue, we performed co-culture experiments using the AML cell line MV4-11, which we demonstrated as sensitive to VEN in a dose-dependent manner. We found that MSCs significantly reduced MV4-11 apoptosis after VEN exposure. The addiction of transwells to the co-culture system abrogated the MSC-protective effect on VEN-induced apoptosis, suggesting the establishment in our setting of a complex interplay between AML cells and MSCs, not only mediated by soluble factors as already demonstrated but also involving direct cell-contact. Furthermore, when we cultured MV4-11 cells in direct contact with MSCs, we were able to distinguish, among leukemic cells, a cell population that remained floating in the supernatant and a cell population that kept adherent to MSCs and could be collected by trypsin detachment only. Interestingly, MSCs protected the adherent but not the floating cells from VEN-induced apoptosis, suggesting a central role of a close adhesion in the MSC-driven resistance to VEN. To gain insight into this mechanism, we sorted adherent vs. floating cells and analyzed and compared Gene Expression Profiling (GEP). Interestingly, the principal component analysis suggested that the floating cell GEP was more similar to the GEP of MV4-11 cells cultured without MSCs compared to adherent cell GEP. We found 159 differential expressed genes (DEGs) in adherent vs. floating MV4-11 cells (FC| ≥ 2 and P ≤ 0.05, according to Transcriptome Analysis Console software guidelines). Enrichment analysis revealed that DEGs were involved in cell adhesion migration and proliferation pathways. Among DEGs, we found that CD90/Thy-1, an adhesion molecule whose expression correlated with unfavorable karyotypes and shorter survival in AML, was significantly up-regulated in adherent MV4-11 cells. Flow cytometry analysis confirmed that the adherent compared to the floating MV4-11 cells, showed a higher expression of CD90 as well as CXCR4 as expected. Our study demonstrated that MSCs protect AML cells from VEN-induced apoptosis through a complex mechanism involving tight adhesion-mediated pathways. Further clarification of these interactions can shed light on putative targets to counteract MSC-mediated drug resistance to VEN treatment.

Type 3 Innate Lymphoid Cells Protect Leukemic Blasts from Natural Killer Cell Cytotoxicity in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive form of leukemia originating in the bone marrow (BM) with poor survival outcomes that has been largely resistant to modern cellular and targeted therapies. To improve therapeutic responses, a better understanding of how AML interacts with or evades immune cells is needed. Innate lymphoid cells (ILCs) are a recently discovered class of immune cells that exert their effector functions in an antigen-independent manner. This sentinel function of ILCs likely makes them among the first immune cell types to interact with leukemic blasts. Under homeostatic conditions, type 3 innate lymphoid cells (ILC3s) play important roles in regulating mucosal immunity through production of GM-CSF and IL-22, among others. In the setting of disease, ILC3s are not confined to mucosal tissues, as recent reports have demonstrated expansion of ILC3 populations in solid tumors (Carrega et al., 2015, Nat. Commun). However, the role of ILC3s in AML is unknown. Therefore, we sought to further study and characterize the role of ILC3s in AML biology. We began by utilizing a previously generated Mll-PTD;Flt3-ITD double knock-in murine model of AML that recapitulates human disease (Zorko et al., 2012, Blood). We conducted scRNA-seq analysis of the BM of leukemic mice compared to non-leukemic controls and observed clustering of a Lin-Eomes-Rorc+Il7r+ ILC3 population unique to the AML cohort that was absent in the control group (Figure 1). Furthermore, gene set enrichment analysis showed strong enrichment of an ILC3 gene signature in this cluster. We then validated this observation via flow cytometry and observed an 18.7-fold increase of ILC3s (Lin-Eomes-RORγt+CD127+) in the BM of leukemic mice relative to controls (1.31% +/- 0.20% AML vs 0.07% +/- 0.02% WT, n=5, p<0.001). Taken together, these data demonstrate ILC3 expansion in the leukemic marrow. We next sought to determine the mechanism(s) leading to expansion of this ILC3 population in AML. We have previously implicated skewed differentiation of a common ILC precursor (ILCP) as a mechanism of NK cell dysregulation in AML (Lordo et al., 2021, J Immunol). To determine whether AML cells are capable of skewing differentiation towards ILC3s, we isolated human ILCPs from healthy donor peripheral blood and cultured them under differentiating conditions with or without the AML cell lines U937 or MV411. We observed a significant increase in ILC3 frequency when co-cultured with AML cells (59.78% +/- 3.06% with U937, 57.84% +/- 2.87% with MV411 vs 28.02% +/- 3.89% alone, n=15, p <0.001). This observation was also recapitulated in the BM of a PDX model (23.91% +/- 3.89% with MV411 vs 3.22% +/- 1.01% without MV411, n=10; p<0.0001) and further validated with single cell clonal assays. Overall, these findings suggest ILC3 expansion in AML is due at least in part to skewing of ILC development. Finally, we wanted to characterize the functional consequences of ILC3s on AML cells. To begin to address this, we co-cultured human ILC3s (Lin-CD94-NKp44+) with the AML cell line U937 in transwells for 48 hours before subjecting the AML cells to a natural killer (NK) cell killing assay. We observed significantly reduced NK cell killing of AML cells when co-cultured with ILC3s compared to controls, suggesting ILC3s protect leukemic cells from NK-mediated killing (24.3% killing +/- 3.81% AML Alone vs 14.57% +/- 1.35% AML with ILC3, n=18, p<0.01). To determine what soluble factor(s) mediate this effect, we performed qRT-PCR on ILC3s following incubation with AML cells and found increases in GM-CSF and IL-22 expression in ILC3s relative to ILC3s cultured alone, which was also validated in our murine AML model. Furthermore, colony forming assays using the murine ILC3-like cell line MNK3 with LSKs (Lin-Sca1+Kit+) isolated from leukemic mice showed increased expression of myeloid markers (CD11b, CD11c, and Gr1) on LSK-derived cells when co-incubated with MNK3 cells relative to LSKs cultured alone. This finding suggests ILC3s may promote myeloid skewing of leukemic precursors. Our data create a novel working model of immune evasion in AML in which ILC3 expansion occurs via AML-mediated skewing of ILC development towards ILC3s which then protect AML blasts from NK cell immune surveillance. Current studies are continuing to study the relationship between ILC3s and AML and how to target these interactions to restore AML susceptibility to immune-mediated therapeutic targeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

VCP inhibition induces an unfolded protein response and apoptosis in human acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is a heterogeneous malignancy characterized by the accumulation of undifferentiated white blood cells (blasts) in the bone marrow. Valosin-containing protein (VCP) is an abundant molecular chaperone that extracts ubiquitylated substrates from protein complexes and cellular compartments prior to their degradation by the proteasome. We found that treatment of AML cell lines with the VCP inhibitor CB-5083 leads to an accumulation of ubiquitylated proteins, activation of unfolded protein response (UPR) and apoptosis. Using quantitative mass spectrometry-based proteomics we assessed the effects of VCP inhibition on the cellular ubiquitin-modified proteome. We could further show that CB-5083 decreases the survival of the AML cell lines THP-1 and MV4-11 in a concentration-dependent manner, and acts synergistically with the antimetabolite cytarabine and the BH3-mimetic venetoclax. Finally, we showed that prolonged treatment of AML cells with CB-5083 leads to development of resistance mediated by mutations in VCP. Taken together, inhibition of VCP leads to a lethal unfolded protein response in AML cells and might be a relevant therapeutic strategy for treatment of AML, particularly when combined with other drugs. The toxicity and development of resistance possibly limit the utility of VCP inhibitors and have to be further explored in animal models and clinical trials.

Treatment with the Recombinant SLIT2 Protein Delays AML Leukemogenesis In Vivo

Accumulating evidence suggest that the axon guidance molecules SLIT and ROBO are not only implicated in physiological process but also in cancer progression. Depending on the type of cancer the SLIT-ROBO axis can either act as a tumor suppressor gene, in which case the SLIT2 promoter site is frequently hypermethylated or as an oncogene, whereby high expression is often associated with poor prognosis. In the context of acute myeloid leukemia (AML), low expression of SLIT2 has been associated with low overall survival (OS) (Golos et al., 2019), while the functional role of SLIT2 remains largely unknown. Recently, we showed that the knockdown of SLIT2 increased cell proliferation of acute promyelocytic leukemia (APL) cells resulting in a more aggressive course of disease progression in vivo using the murine transgenic APL model (Weinhäuser et al., 2020). Here, we aimed to study the functional role of SLIT2 in a more heterogeneous disease, such as AML.Using different publicly available datasets. (GSE58477, normal karyotype blasts: 62, healthy CD34 +: 10; GSE63409, LSC: 14, HSC: 5) we detected increased methylation at the SLIT2 promoter site of AML leukemic cells compared to healthy CD34 + cells suggesting SLIT2 tumor suppressive functions. In addition, we measured decreased levels of SLIT2 in the bone marrow (BM) plasma of AML patients compared to healthy donors. To assess the biological role of SLIT2, we treated AML cell lines (KASUMI1, MV411, and MOLM13) with recombinant SLIT2 (50 ng/mL) in vitro. Administration of SLIT2 reduced AML cell growth, colony formation and induced cell cycle arrest in the G1 phase for all AML cell lines. Conversely, the knockdown of SLIT2 promoted increased THP-1 and OCI-AML3 cell proliferation. Next, we determined whether the treatment with SLIT2 could delay leukemogenesis in vivo using the AML cell line MV4-11. Engraftment was monitored by luciferase bioluminescent signal and NSGS mice were either treated with recombinant SLIT2 using a dose of 25 ng/g of body weight or vehicle (control group). SLIT2 therapy resulted in a lower disease burden, decreased leukemic infiltration in the BM and spleen, reduced spleen size, and increased OS compared to the control group (p<0.05). In conclusion, we showed that SLIT2 methylation is recurrent in AML patients and that the level of SLIT2 in the plasma of AML patients is reduced. Moreover, SLIT2 treatment appears to have a cytostatic effect on different AML cell lines delaying leukemogenesis in vivo. Overall, our study reveals the therapeutic potential of SLIT2 in hematological malignancies, which could be used as an adjuvant in the clinic. DisclosuresNo relevant conflicts of interest to declare.

Deubiquitinase Inhibitor WP1130 Blocks FLT3-ITD to Induce Apoptosis in Leukemic Cells

FMS-like tyrosine kinase 3 with internal tandem duplication (FLT3-ITD) is the most frequent kinase mutation seen in acute myeloid leukemia (AML). The ITD mutation promotes ligand-independent constitutive activation of FLT3 kinase, resulting in altered downstream signal pathway activation to enhance cell proliferation and prevent cell death. AML with FLT3-ITD is associated with poor prognosis, which neither high-dose chemotherapy nor allogeneic hematopoietic stem cell transplantation can overcome. Several tyrosine kinase inhibitors targeting FLT3 have been developed and tested in clinical trials, but their efficacies are limited so far partly due to resistance to kinase inhibition. Therefore, development of novel treatment strategies with distinct mechanisms to block FLT3 is warranted.Deubiquitinating enzymes (DUBs) are proteases that remove ubiquitin from target proteins, resulting in changes in protein stability and signaling. WP1130 is an inhibitor for DUBs, which has been reported to inhibit BCR/ABL not through blocking the kinase activity but through promoting ubiquitination and translocation of BCR/ABL protein into perinuclear aggresomes. WP1130 also downregulates JAK2 tyrosine kinase in the same manner. However, the effect of WP1130 against FLT3-ITD has not been reported.Here we demonstrate that WP1130 exerts anti-leukemic activity against leukemic cell lines and primary AML cells harboring FLT3-ITD. WP1130 efficiently downregulated activated form of FLT3-ITD through translocation of this mutant into aggresomes and activated the mitochondria-mediated intrinsic apoptotic pathway at least partly through accumulation of intracellular reactive oxygen species (ROS).First, we examined the anti-proliferative effect of WP1130 and found that proliferation of the FLT3-ITD-harboring AML cell line MV4-11 was suppressed in a dose dependent manner with IC50 of 1.3 μM. It was notable that MV4-11 was more susceptible to WP1130 compared with K562 (IC50 = 3.3 μM) and HEL (IC50 = 3.4 μM), leukemic cell lines harboring BCR/ABL and JAK2-V617F aberrant kinases, respectively.Next, we assessed the effect of WP1130 against FLT3-ITD in MV4-11 cells. WP1130 rapidly downregulated FLT3-ITD to suppress downstream signaling pathways. The aggresomal translocation of FLT3-ITD was confirmed by confocal microscopy and western blot analysis of detergent-insoluble fraction of cellular proteins. Importantly, the downregulation was prominently observed for the activated form of FLT3-ITD autophosphorylated on Y591. Consistent with this, inhibition of FLT3 autophosphorylation by AC220 (quizartinib) resulted in attenuation of WP1130-induced FLT3-ITD downregulation.Importantly, WP1130 induced apoptosis in MV4-11 cells much more efficiently than in other leukemic cell lines that we examined, including K562 and HEL. WP1130 activated the mitochondria-mediated intrinsic apoptotic pathway as demonstrated by Bax conformation change and loss of mitochondrial membrane potential (MMP). Forced overexpression of Bcl-xL, a member of pro-survival Bcl-2 family proteins that preserve MMP, blocked WP1130-induced apoptosis. Moreover, the apoptosis was synergistically enhanced by inhibition of Bcl-xL using ABT-737 or navitoclax, further supporting the involvement of Bcl-xL in WP1130-induced apoptosis. Interestingly, the apoptosis induction was also enhanced by 17-AAG, an HSP90 inhibitor that we previously demonstrated to disrupt the chaperone association of HSP90 with FLT3-ITD resulting in enhanced ubiquitination of FLT3-ITD. FLT3-ITD down regulation and apoptosis induction by WP1130 were also confirmed using other cell lines expressing FLT3-ITD and primary cells derived from FLT3-ITD positive AML patients.Finally, since FLT3-ITD has been reported to increase intracellular ROS, we examined if ROS was involved in the anti-leukemic effect of WP1130. Treatment with WP1130 increased ROS levels in MV4-11 cells. Antioxidants N-acetyl-cysteine (NAC) and tert-butylhydroquinone (tBHQ) both attenuated WP1130-induced apoptosis, suggesting that intracellular ROS accumulation may play a role in induction of apoptosis.Further studies are in progress to elucidate the exact molecular mechanisms involved in WP1130-mediated effects and to develop novel strategies to enhance its anti-leukemic effects in synergistic manners. DisclosuresNo relevant conflicts of interest to declare.

Single Agent and Synergistic Combinatorial Efficacy of First-in-Class Small Molecule Imipridone ONC201 in Hematological Malignancies In Vitro and In Vivo through Apoptosis Via the Integrated Stress Response Pathway

▪ONC201, founding member of the imipridone class of small molecules, is currently being evaluated in advancer cancer clinical trials. We explored single agent and combinatorial efficacy of ONC201 in preclinical models of hematological malignancies.ONC201 was evaluated in 20 human leukemia cell lines (Figure 1): 13 acute myeloid leukemia (AML), 5 acute lymphoblastic leukemia (ALL) and 2 chronic myelogenous leukemia (CML) cell lines. We observed a dose-dependent decrease in cell viability in AML, ALL and CML cells (GI50 1-8 µM). ONC201 also decreased cell viability in 2 primary chronic lymphocytic leukemia (CLL) patient samples with 48-72h treatment. Dose- and time-dependent induction of apoptosis (caspase-3 cleavage), inhibition of Akt and Foxo3a phosphorylation was observed in AML cells treated with ONC201 for 24-48 hr. Sub-G1 analysis confirmed that ONC201 induced apoptosis in ALL cells and a pan-caspase inhibitor reduced ONC201-mediated apoptosis. ONC201-mediated apoptosis in ALL cells involved PARP cleavage and caspase-9 activation. Anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xl were downregulated while the pro-apoptotic Bcl-2 family member Bim was upregulated in response to ONC201 treatment in ALL cells. ONC201 also downregulates the inhibitor of apoptosis (IAP) family proteins cIAP1 and cIAP2 in ALL cells, which are known to confer a poor clinical prognosis.Low micromolar concentrations of ONC201 also reduced multiple myeloma (MM) cell viability in a time- and dose-dependent manner. ONC201 reduced cell viability and induced apoptosis in dexamethasone-resistant MM.1R cells with efficacy comparable to dexamethasone-sensitive MM.1S cells (Figure 2). ONC201 caused activation of the integrated stress response (ISR) pathway, as observed with induction of ATF/CHOP in MM cells. XIAP is highly up-regulated in MM cells and sownregulation of IAP family proteins induces apoptosis. ONC201 treatment of MM cells resulted in a dose-dependent decrease in the anti-apoptotic proteins XIAP, cIAP1 and cIAP2.Next, ONC201 was evaluated in 33 human lymphoma cell lines: 3 anaplastic large cell lymphoma (ALCL), 7 Burkitt's lymphoma, 2 cutaneous T-cell lymphoma (CTCL), 8 diffuse large B-cell lymphoma (DLBCL), 1 MCL, 2 nodular sclerosis (NS) and 10 other lymphoma cell lines. ONC201 demonstrated low micromolar range efficacy (GI50 1-6 µM) across all lymphoma subtypes. Low micromolar concentrations of ONC201 reduced cell viability of MCL and DLBCL cell lines and primary cells in a time- and dose-dependent manner (Figure 3). ONC201 caused activation of the ISR pathway, as observed with induction of ATF4 in Pfeiffer DLBCL cells. In patient-derived MCL cells, ONC201 reduced cyclin D1 expression, a diagnostic feature of MCL.We then tested the efficacy of ONC201 in combination with approved therapies in hematological malignancies in vitro and in vivo. ONC201 demonstrated synergistic reduction in cell viability in combination with cytarabine confirmed by combination indices in AML cell lines CMK, THP-1, HL60 and ALL Kasumi-2 cells. ONC201 combined with cytarabine in a Burkitt's lymphoma xenograft model exhibited superior tumor growth inhibition compared to either agent alone (Figure 4). ONC201 demonstrated synergistic reduction in cell viability in combination with 5-azacytidine that was confirmed by combination indices in AML cell lines HL-60, MOLM14 and MV411. ONC201 synergistically reduced cell viability in combination with dexamethasone or ixazomib in RPMI-8226 MM cells. ONC201 in combination with bortezomib synergistically reduced cell viability in KARPAS 299 ALCL cells and RPMI-8226 MM cells and improved apoptosis induction in UPN2 MCL cells. ONC201 combined with bortezomib was evaluated in a Burkitt's lymphoma xenograft model. Histological evaluation of tumor xenografts revealed a reduced tumor cell density at 3-4 weeks and enhanced CHOP induction at 48 hours with combination treatment compared to either agent alone. The combination treatment did not result in body weight loss.These results serve as a rationale for ongoing ONC201 single agent trials in relapsed/refractory acute leukemia (NCT02392572), non-Hodgkin's lymphoma (NCT02420795), MM (NCT02609230) and combination trial with dexamethasone in MM (NCT02863991). Our findings provide pharmacodynamic biomarkers and identify synergistic combinatorial regimens that are well tolerated and can be explored in the clinic. [Display omitted] DisclosuresPrabhu:Oncoceutics: Employment, Equity Ownership. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Other: grant; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties; Jazz Pharmaceuticals: Consultancy. Oster:Oncoceutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Allen:Oncoceutics: Employment, Equity Ownership, Patents & Royalties. El-Deiry:Oncoceutics, Inc: Equity Ownership, Patents & Royalties.

Discovery of a Potent and Selective FLT3 Inhibitor (Z)-N-(5-((5-Fluoro-2-oxoindolin-3-ylidene)methyl)-4-methyl-1H-pyrrol-3-yl)-3-(pyrrolidin-1-yl)propanamide with Improved Drug-like Properties and Superior Efficacy in FLT3-ITD-Positive Acute Myeloid Leukemia.

Overcoming the FLT3-ITD mutant has been a promising drug design strategy for treating acute myeloid leukemia (AML). Herein, we discovered a novel FLT3 inhibitor 17, which displayed potent inhibitory activity against the FLT3-ITD mutant (IC50 = 0.8 nM) and achieved good selectivity over c-KIT kinase (over 500-fold). Compound 17 selectively inhibited the proliferation of FLT3-ITD-positive AML cell lines MV4-11 (IC50 = 23.5 nM) and MOLM-13 (IC50 = 35.5 nM) and exhibited potent inhibitory effects against associated acquired resistance mutations. In cellular mechanism studies, compound 17 strongly inhibited FLT3-mediated signaling pathways and induced apoptosis by arresting the cell cycle in the sub-G1 phase. In in vivo studies, compound 17 demonstrated a good bioavailability (73.6%) and significantly suppressed tumor growth in MV4-11 (10 mg/kg, TGI 93.4%) and MOLM-13 (20 mg/kg, TGI 98.0%) xenograft models without exhibiting obvious toxicity. These results suggested that compound 17 may be a promising drug candidate for treating FLT3-ITD-positive AML.

IRF8 Is an AML-Specific Susceptibility Factor That Regulates Signaling Pathways and Proliferation of AML Cells.

Simple SummaryDespite progress, acute myeloid leukemia (AML) remains one of the deadliest cancer diseases. The identification of novel molecular targets may allow developing innovative and alternative treatment options for AML. Using public data from genome-edited cancer cells, we identified factors that are specifically essential for AML cell growth. We validated the critical role of the transcription factor IRF8 and demonstrated that it modulates the function of the cells by regulating important signaling molecules. These results support that IRF8 may be a suitable molecular target for the treatment of AML.Personalized treatment of acute myeloid leukemia (AML) that target individual aberrations strongly improved the survival of AML patients. However, AML is still one of the most lethal cancer diseases of the 21st century, demonstrating the need to find novel drug targets and to explore alternative treatment strategies. Upon investigation of public perturbation data, we identified the transcription factor IRF8 as a novel AML-specific susceptibility gene in humans. IRF8 is upregulated in a subset of AML cells and its deletion leads to impaired proliferation in those cells. Consistently, high IRF8 expression is associated with poorer patients’ prognoses. Combining gene expression changes upon IRF8 deletion and the genome-wide localization of IRF8 in the AML cell line MV4-11, we demonstrate that IRF8 directly regulates key signaling molecules, such as the kinases SRC and FAK, the transcription factors RUNX1 and IRF5, and the cell cycle regulator Cyclin D1. IRF8 loss impairs AML-driving signaling pathways, including the WNT, Chemokine, and VEGF signaling pathways. Additionally, many members of the focal adhesion pathway showed reduced expression, providing a putative link between high IRF8 expression and poor prognosis. Thus, this study suggests that IRF8 could serve as a biomarker and potential molecular target in a subset of human AMLs.

Investigation of ALLO-316: A Fratricide-Resistant Allogeneic CAR T Targeting CD70 As a Potential Therapy for the Treatment of AML

CD70, a member of the TNF superfamily, is a type II transmembrane glycoprotein that interacts with its receptor (CD27) to promote survival of primed T cells and leads to formation of effector and memory T cells. Expression of CD70 in normal tissues is restricted to activated T and B lymphocytes and mature dendritic cells. CD70 is also widely expressed in various malignancies, including renal cell carcinoma (RCC) and acute myeloid leukemia (AML). The restricted expression pattern of CD70 in normal tissues makes it an attractive target for cancer therapeutics. Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is an exciting new therapeutic modality showing great promise in hematologic malignancies. Approval of two CD19-targeting autologous CAR Ts, Kymriah® and Yescarta®, has been followed with promising results from BCMA autologous CAR T clinical trials, showing that activity can extend to other targets. We have previously described the functional screening of a library of anti-CD70 scFv-based CARs and the identification of lead CD70 allogeneic CAR T cells (AlloCAR TTM) with robust activity against RCC cell lines both in vitro and in vivo. Here, we evaluate the anti-tumor activity and safety of a lead CD70 AlloCAR T (ALLO-316) for the treatment of AML. CD70 expression was evaluated and detected on three AML cell lines and in six primary AML patient samples, with 5/6 patient samples showing expression on 24%-99% of cells. CD70 expression will be profiled in a broader subset of AML patients and preliminary data will be presented. Despite the expression of CD70 on activated T cells it was possible to generate CD70 AlloCAR T cells. No CD70 expression was observed on CAR T cells after generation, suggesting either cells are succumbing to fratricide or are being "masked" by the CAR. CD70 was also not detected on Jurkat cells expressing CARs and this data, in combination with results showing CAR expression is protective when overexpressed in RCC cells support the phenomenon of "masking". Cellectis' TALEN® gene-editing was used to inactivate the TRAC and CD52 loci with the intent to minimize the risk of graft-versus-host disease and to confer resistance to ALLO-647, an anti-CD52 antibody that can be used as part of the conditioning regimen to deplete host alloreactive immune cells potentially leading to increased persistence and efficacy of the infused allogeneic cells. ALLO-316 cells were highly effective at lysing CD70-expressing target cells and eliminated greater than 99% of cells at the high effector to target (E:T) ratio and were unable to lyse AML cells in which CD70 was knocked out. Moreover, ALLO-316 cells were able to kill primary AML blasts with CD70 expression ex vivo. An orthotopic in vivo model utilizing the AML cell line MV4-11 was developed and anti-tumor activity was observed. In addition to evaluating efficacy against AML cell lines and tumors we also explored potential toxicity liabilities related to ALLO-316 treatment. Previous studies have reported that certain AML tumor antigens can also be expressed on normal hematopoietic progenitors and such expression could potentially lead to toxicity with targeted therapeutics. No detectable CD70 was observed by flow cytometry on purified CD34+ cells from 14 healthy donors. Taken together, our results support clinical development of CD70 AlloCAR T therapy for the treatment of AML. Disclosures Srinivasan: Allogene Therapeutics: Current Employment. Tan:Allogene Therapeutics: Current Employment. Cheng:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Zhang:Allogene Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tacheva-Grigorova:Allogene Therapeutics: Current Employment, Current equity holder in publicly-traded company. Van Blarcom:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Sommer:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Nguyen:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Sasu:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Panowski:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company.

Aberrant expression of NKL homeobox genes HMX2 and HMX3 interferes with cell differentiation in acute myeloid leukemia.

The NKL-code describes normal expression patterns of NKL homeobox genes in hematopoiesis. Aberrant expression of NKL homeobox gene subclass members have been reported in several hematopoietic malignancies including acute myeloid leukemia (AML). Here, we analyzed the oncogenic role of the HMX-group of NKL homeobox genes in AML. Public expression profiling data–available for HMX1 and HMX2—indicate aberrant activity of HMX2 in circa 2% AML patients overall, rising to 31% in those with KMT2A/MLL rearrangements whereas HMX1 expression remains inconspicuous. AML cell lines EOL-1, MV4-11 and MOLM-13 expressed both, HMX2 and neighboring HMX3 genes, and harbored KMT2A aberrations, suggesting their potential functional association. Surprisingly, knockdown experiments in these cell lines demonstrated that KMT2A inhibited HMX2/3 which, in turn, did not regulate KMT2A expression. Furthermore, karyotyping and genomic profiling analysis excluded rearrangements of the HMX2/3 locus in these cell lines. However, comparative expression profiling and subsequent functional analyses revealed that IRF8, IL7- and WNT-signalling activated HMX2/3 expression while TNFa/NFkB- signalling proved inhibitory. Whole genome sequencing of EOL-1 identified two mutations in the regulatory upstream regions of HMX2/3 resulting in generation of a consensus ETS-site and transformation of a former NFkB-site into an SP1-site. Reporter-gene assays demonstrated that both mutations contributed to HMX2/3 activation, modifying ETS1/ELK1- and TNFalpha-mediated gene regulation. Moreover, DMSO-induced eosinophilic differentiation of EOL-1 cells coincided with HMX2/3 downregulation while knockdown of HMX2 induced cell differentiation, collectively supporting a fundamental role for these genes in myeloid differentiation arrest. Finally, target genes of HMX2/3 were identified in EOL-1 and included suppression of differentiation gene EPX, and activation of fusion gene FIP1L1-PDGFRA and receptor-encoding gene HTR7, both of which enhanced oncogenic ERK-signalling. Taken together, our study documents a leukemic role for deregulated NKL homeobox genes HMX2 and HMX3 in AML, revealing molecular mechanisms of myeloid differentiation arrest.

Abstract 537: Preclinical development and activity of Anti-FLT3 nanoworms in acute myeloid leukemia

Abstract Introduction: Mutations in FMS-like tyrosine kinase 3 (FLT3) occur in ~30% of acute myeloid leukemia (AML) cases causing high relapse and short survival. Acquired resistance limits the efficacy of current tyrosine kinase inhibitors against FLT3. Developing FLT3 target-specific antibodies has gained interest as a therapeutic approach for AML. Single-chain antibody fragments (scFv) are as specific as full-length monoclonal antibodies (mAbs), yet they outperform mAbs due to lower immunogenicity and a faster development process via recombinant protein engineering. However, scFvs have short circulating half-lives in humans and are difficult to purify. To overcome this, we developed anti-FLT3 scFv fused with an elastin-like polypeptide (ELP), A192, which aids in protein purifyication and extending its half life. Methods: The α-FLT3 scFv gene was fused to the amino terminus of (VPGAG)192 (A192), in the pET-25b(+) vector, purified and refolded for maximum biological activity. Binding specificity of α-FLT3-A192 was analyzed using FLT3 ITD+ AML cell lines MOLM-13 and MV4-11 and FLT3−U937 cells via flow cytometry. Viability and apoptosis assays were performed via trypan blue and Annexin-V at 72 hours, respectively. Immunoblot analyses were performed to assess the effect of α-FLT3-A192 on FLT3/STAT5/ERK signaling pathway. In vivo activity was analyzed by engrafting 106 MOLM-13 cells/ NOD scid mouse, treating with 220 uM of A192 or α-FLT3-A192 (N=8) on day 7, 10, 13 and 16, and sacrificing on day 17. Engraftment was analyzed in peripheral blood and bone marrow by HuCD45 stain and measured by flow cytometry. Kaplan Meier survival analysis was performed in MOLM-13 murine model. Results: Binding of α-FLT3-A192 (10 μM) vs. A192 (25 μM) was observed in MOLM-13 (MFI 1.0 vs 2.0; p&amp;lt;0.0001) and MV4-11 cells (MFI 1.0 vs 1.36; p=0.03). Contrarily, U937 cells had no binding. Cell viability decreased in MOLM-13 cells treated with α-FLT3-A192 vs. A192 at 10µM (p=0.0002, 23%), and 25µM (p&amp;lt;0.0001, 34%) and in MV4-11 cells at 10 µM (p&amp;lt;0.0001, 71%), and 25 µM (p&amp;lt;0.0001, 80%) vs. A192. Consistently, apoptosis increased in cells treated with 25 µM α-FLT3-A192 vs A192 (MOLM-13: p=0.007, 42.76% vs 5.27%; (MV4-11: p=0.003, 62.77% vs 25.36%). Mechanistically, α-FLT3-A192 inhibited both phospho-STAT5 and phospho-ERK in MOLM-13 and MV4-11 cells. In vivo analyses showed that spleens of mice treated with α-FLT3-A192 weighed less compared with A192 (0.48 vs 126 mg, p=0.03). Additionally, α-FLT3-A192 reduced engraftment in the peripheral blood (%huCD45- 17.5 vs 43.44, p=0.007) and bone marrow (%huCD45- 10.2 vs 26.1%, p&amp;lt;0.0001). Mice treated with α-FLT3-A192 had significantly longer OS than A192 group (median survival: 36 vs 30 Days, p=0.0015). Pharmacokinetic (PK) analysis showed that the terminal half-life of α-FLT3-A192 in NSG mice was ~ 14.9 hours. Conclusion: α-FLT3-A192 nanoworms present an effective FLT3 targeting strategy with high pharmacological activity and PK properties in cellular and xenograft murine models of AML. V.P.V. and M.P. contributed equally to the manuscript Citation Format: Vijaya Pooja Vaikari, Mincheol Park, John Andrew Mackay, Houda Alachkar. Preclinical development and activity of Anti-FLT3 nanoworms in acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 537.

An Oral Combination Study of Novel Nucleoside Analogue Sapacitabine and BCL2 Inhibitor Venetoclax to Treat Patients with Relapsed or Refractory AML or MDS

An Oral Combination Study of Novel Nucleoside Analogue Sapacitabine and BCL2 Inhibitor Venetoclax to Treat Patients with Relapsed or Refractory AML or MDS

PTPN11 Mutated Acute Myeloid Leukemia (AML) Features an Abundant Epichaperome Network and Is Sensitive to the Epichaperome Inhibitor PU-H71

PTPN11 Mutated Acute Myeloid Leukemia (AML) Features an Abundant Epichaperome Network and Is Sensitive to the Epichaperome Inhibitor PU-H71

A Novel Role for Malate Dehydrogenase 1 in Acute Myeloid Leukemia Progression

The tumor suppressor role of the metabolic genes is rarely reported. Here, we report that malate dehydrogenase 1 (MDH1) acts as an anti-oncogene in acute myeloid leukemia (AML) progression. In a screen of various leukemia cell lines, as well as peripheral blood mononuclear cells from patients with AML and healthy individuals, we observed that patients with AML had lower mRNA expression of MDH1 than healthy individuals. In addition, we found that overexpression of MDH1 in the AML cell lines MV4-11 and THP-1 in vitro promoted their differentiation, reduced their growth, induced apoptosis, and increased the proportion of cells in S phase, which were more sensitive to the chemotherapeutic drugs cytarabine and daunorubicin. Using an in vivo xenograft mouse model of AML, we found that disruption of MDH1 expression influenced tumor burden and mouse survival. These findings were consistent with an inverse relationship between MDH1 expression and overall survival in two independent cohorts of AML patients. We also identified changes in the expression of other genes, concomitant with reduced MDH1 expression in patients with AML, that largely explain the anti-oncogenic role of MDH1 in AML progression. Notably, downregulation of P53 and upregulation of the MAPK pathway in low MDH1-expressing MV4-11 cells were reversed upon overexpression of MDH1. In conclusion, we demonstrated that MDH1 plays a tumor-suppressing role in AML and can be used as a novel mimic drug therapy in the future. Disclosures No relevant conflicts of interest to declare.