Effects In Acute Myeloid Leukemia Research Articles

Development of a Competitive Nutrient-Based T-Cell Immunotherapy Designed to Block the Adaptive Warburg Effect in Acute Myeloid Leukemia

Development of a Competitive Nutrient-Based T-Cell Immunotherapy Designed to Block the Adaptive Warburg Effect in Acute Myeloid Leukemia

Gossypol acetic acid regulates leukemia stem cells by degrading LRPPRC via inhibiting IL-6/JAK1/STAT3 signaling or resulting mitochondrial dysfunction.

Leukemia stem cells (LSCs) are found to be one of the main factors contributing to poor therapeutic effects in acute myeloid leukemia (AML), as they are protected by the bone marrow microenvironment (BMM) against conventional therapies. Gossypol acetic acid (GAA), which is extracted from the seeds of cotton plants, exerts anti-tumor roles in several types of cancer and has been reported to induce apoptosis of LSCs by inhibiting Bcl2. To investigate the exact roles of GAA in regulating LSCs under different microenvironments and the exact mechanism. In this study, LSCs were magnetically sorted from AML cell lines and the CD34+CD38- population was obtained. The expression of leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) and forkhead box M1 (FOXM1) was evaluated in LSCs, and the effects of GAA on malignancies and mitochondrial function were measured. LRPPRC was found to be upregulated, and GAA inhibited cell proliferation by degrading LRPPRC. GAA induced LRPPRC degradation and inhibited the activation of interleukin 6 (IL-6)/janus kinase (JAK) 1/signal transducer and activator of transcription (STAT) 3 signaling, enhancing chemosensitivity in LSCs against conventional chemotherapies, including L-Asparaginase, Dexamethasone, and cytarabine. GAA was also found to downregulate FOXM1 indirectly by regulating LRPPRC. Furthermore, GAA induced reactive oxygen species accumulation, disturbed mitochondrial homeostasis, and caused mitochondrial dysfunction. By inhibiting IL-6/JAK1/STAT3 signaling via degrading LRPPRC, GAA resulted in the elimination of LSCs. Meanwhile, GAA induced oxidative stress and subsequent cell damage by causing mitochondrial damage. Taken together, the results indicate that GAA might overcome the BMM protective effect and be considered as a novel and effective combination therapy for AML.

Preparation of a dual-specific antibody targeting human CD123 and exploration of its anti-acute myeloid leukemia effects

Objective: To construct a novel dual-specific antibody targeting human CD123 (CD123 DuAb) and study its effects in acute myeloid leukemia (AML) . Methods: Based on the variable region of the CD123 monoclonal antibody independently developed at our institution, the CD123 DuAb expression plasmid was constructed by molecular cloning and transfected into ExpiCHO-S cells to prepare the antibody protein. Through a series of in vitro experiments, its activation and proliferation effect on T cells, as well as the effect of promoting T-cell killing of AML cells, were verified. Results: ① A novel CD123 DuAb plasmid targeting CD123 was successfully constructed and expressed in the Expi-CHO eukaryotic system. ②The CD123 DuAb could bind both CD3 on T cells and CD123 on CD123(+) tumor cells. ③When T cells were co-cultured with MV4-11 cells with addition of the CD123 DuAb at a concentration of 1 nmol/L, the positive expression rates of CD69 and CD25 on T cells were 68.0% and 44.3%, respectively, which were significantly higher than those of the control group (P<0.05). ④Co-culture with CD123 DuAb at 1 nmol/L promoted T-cell proliferation, and the absolute T-cell count increased from 5×10(5)/ml to 3.2×10(6)/ml on day 9, and CFSE fluorescence intensity decreased significantly. ⑤ With the increase in CD123 DuAb concentration in the culture system, T-cell exhaustion and apoptosis increased. When the CD123 DuAb was added at a concentration of 1 nmol/L to the culture system, the proportion of CD8(+) PD-1(+) LAG-3(+) T cells was 10.90%, and the proportion of propidium iodide (PI) (-) Annexin Ⅴ(+) T cells and PI(+) Annexin Ⅴ(+) T cells was 18.27% and 11.43%, respectively, which were significantly higher than those in the control group (P<0.05). ⑥ The CD123 DuAb significantly activated T cells, and the activation intensity was positively correlated with its concentration. The expression rate of CD107a on T cells reached 16.05% with 1 nmol/L CD123 DuAb, which was significantly higher than that of the control group (P<0.05). ⑦The CD123 DuAb promoted cytokine secretion by T cells at a concentration of 1 nmol/L, and the concentration of IFN-γ and TNF-α in the supernatant of the co-culture system reached 193.8 pg/ml and 169.8 pg/ml, respectively, which was significantly higher than that of the control group (P<0.05). ⑧When CD123 DuAb was added at a concentration of 1 nmol/L to the co-culture system of T cells and CD123(+) tumor cells, the killing intensity of T cells significantly increased, and the residual rates of CD123(+) MV4-11 cells, CD123(+) Molm13 cells, and CD123(+) THP-1 cells were 7.4%, 6.7%, and 14.6% on day 3, respectively, which were significantly lower than those in the control group (P<0.05) . Conclusion: In this study, a novel CD123 DuAb was constructed and expressed. In vitro experiments verified that the DuAb binds to CD123(+) tumor cells and T cells simultaneously, promotes T-cell activation and proliferation, and facilitates their anti-leukemia effect, which provides a basis for further clinical research.

Histone Methyltranferase SETD8 Stimulates Proliferation and Anti-Apoptotic Effect in Acute Myeloid Leukemia through Epigenetically Upregulation of CXCR4

Acute myeloid leukemia (AML) is the most common type of leukemia., characterized by the rapid and uncontrolled growth of abnormal cells. Epigenetic modifications control gene expression without affecting its DNA sequence. H4K20me1 is the mono-methylation occurring on lysine 20 of histone H4, acting as an epigenetic mark that promotes gene expression. SETD8 is responsible for catalyzing the mono-methylation of lysine 20 on histone H4. While SETD8's role and mechanisms have been studied in various types of cancer, its function in AML remains unclear. This study is based on the screening of a small molecule compound library, which identified the inhibitor UNC0379 targeting the histone methyltransferase SETD8, showing clear inhibitory effects on three non-M3 AML cell lines. Bioinformatical analysis revealed elevated SETD8 expression in AML patients compared to normal individuals and higher SETD8 expression corresponded with lower overall survival rates (OS). Further investigations using AML cell lines and normal peripheral blood mononuclear cells (PBMCs) demonstrated that SETD8 knockdown or UNC0379 intervention inhibited cell proliferation and increased apoptosis in AML cells, while PBMC cells remained unaffected. In an AML model constructed with C1498 cell injection in C57BL/6J mice, we observed that SETD8 knockdown and treatment with UNC0379 inhibited tumor formation and infiltration. To elucidate the molecular mechanism of SETD8's impact on AML, we conducted transcriptome sequencing on SETD8 knockdown AML cells and found significantly decreased mRNA expression of CXCR4, which was further validated at the protein level. Also, BeatAML and TCGA datasets demonstrated a positive correlation between SETD8 and CXCR4 mRNA expression. These phenomena motivated us to clarify the regulation relationship between SETD8 and CXCR4. CXCR4 is a crucial membrane receptor. After stimulation, it promotes AML cell proliferation and survival. CHIP experiments in SETD8 knockdown cells revealed reduced H4K20me1 enrichment in the CXCR4 promoter region. Subsequent rescue experiments demonstrated that overexpressing CXCR4 or using its corresponding agonist SDF-1alpha in SETD8 knockdown cells moderately restored cell proliferation and reduced apoptosis. In summary, we discovered that SETD8 promotes CXCR4 transcriptional activation through catalyzing the H4K20me1 mark, thereby influencing AML cell proliferation and apoptosis. SETD8 presents a promising therapeutic target through genetic or pharmacological intervention.

Azacitidine Combined with Novel Flavonoid Derivative GL-V9 Demonstrated Synergistic Anti-Leukemia Effect in Acute Myeloid Leukemia By Targeting DDIT4/mTOR Signaling

Background Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by the clonal expansion of malignant progenitor cells and impaired cell differentiation. GL-V9, a nature compound derived from wogonin, has shown superior anti-tumor potential by inhibiting cancer cell growth. Azacitidine (AZA), a DNA hypomethylation agent, has demonstrated therapeutic efficacy against AML. In this study, we investigated the anti-leukemia effect and potential mechanism of AZA combined with GL-V9 in AML cells, aiming to provide evidence for future clinical treatment. Methods We employed Cell Counting Kit-8 (CCK-8) to assess cell viability, Annexin-V/PI staining followed by flow cytometry analysis to measure apoptosis. CalcuSyn analysis was used to evaluate the synergistic effect. RNA-seq was performed in U937 cells and differentially expressed genes (DEGs) were identified and subjected to KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. RT-qPCR and Western Blot were used to examine gene expression. Results Our results demonstrated significant cell proliferation arrest in U937 and MV4-11 cells treated with the combination of AZA and GL-V9, compared to the single drug ( Fig. 1A&amp;B). CalcuSyn analysis showed a strong synergistic effect for the combination ( Fig. 1A&amp;B). Similar results were also found in primary AML cells derived from 2 AML patients [patient 1 exhibited t(6;11) (q27;q23) chromosomal translocation, while Patient 2 presented with secondary AML, transformed from CMML (Chronic Myelomonocytic Leukemia) and featured ASXL1 and SRSF2 mutations] ( Fig. 1C&amp;D). Moreover, AZA plus GL-V9 induced a substantial increase in cell apoptosis in U937 and MV4-11 cells compared to the single drug ( Fig. 1E&amp;F). Consistently, the AZA+GL-V9 combination led to a notable increase in the protein level of Bax, BAD, BIM, and a decrease in BCL2 ( Fig. 1G). These data indicated the combination of AZA with GL-V9 has synergistic anti-leukemia effects in AML. To understand the underlying mechanisms of this synergy, we performed RNA-seq analysis in U937 cells, identifying 1385 and 586 DEGs (|log 2FC|≥2.0, P&amp;lt;0.05) upon AZA or GL-V9 treatment, respectively ( Fig. 2A). The mTOR signaling pathway exhibits prominent enrichment in the KEGG analysis of the overlapping DEGs between AZA and GL-V9 ( Fig. 2B). DDIT4, a negative regulator of mTOR emerged as one of the top DEGs in response to both AZA and GL-V9 treatment. Importantly, the AZA+GL-V9 treatment exhibited a higher expression level of DDIT4 compared to either drug alone and suppressed the phosphorylation of mTOR ( Fig. 2C). Additionally, the DDIT4 expression level was significantly decreased in AML patients, which derived from a public database (TCGA-AML, GSE13159)] ( Fig. 2D) and Zhongda Hospital (Nanjing, China) compared to the healthy controls ( Fig. 2E). To be noticed, high DDIT4 expression was associated with extended overall survival (P=0.021) ( Fig. 2F) and relapse-free survival (P=0.015) ( Fig. 2G), indicating DDIT4 may act as a tumor suppressor in AML. These findings suggested that the combination may exert the anti-leukemia effect by targeting the DDIT4/mTOR signaling pathway, as summarized in Fig. 2H. Conclusions Our study provides novel evidence of the synergistic effect on cell growth arrest and apoptosis treated with a new nature compound derived from wogonin combined with AZA in AML. Furthermore, we identified the mechanism underlying the synergy through targeting of DDIT4/mTOR signaling. These results offer preliminary support for the potential application of this combination therapy in treating AML patients.

Developing a novel SARS-CoV-2 risk index to predict the prognostic and therapeutic effects in acute myeloid leukemia

There is growing evidence of a strong association between SARS-CoV-2 and cancer prognosis and treatment outcome. However, there are no reliable SARS-CoV-2 assessment models to accurately predict prognostic and therapeutic effects in acute myeloid leukemia (AML). Here, differentially expressed genes associated with SARS-CoV-2 were detected, and multiple Cox regression methods were used to construct a SARS-CoV-2 risk index (SC2RI). Then, RT-qPCR was used to validate the gene expression levels in the AML samples. Finally, we explored how the SC2RI affected prognosis, immune infiltration, immunotherapy, and drug sensitivity in AML. We found that CYB5R3 and CLIP4 had been confirmed as hub genes in AML and were used to generate the SC2RI. The datasets indicated that the SC2RI had a superior predictive impact on the prognosis of AML. In addition, high expression of immune checkpoints and numerous immunological infiltrations were substantially correlated with a high SC2RI. However, it responded poorly to immune checkpoint blockade, which may be related to T-cell dysfunction, lack of effective antigens, and deficiency of synaptic capacity. Moreover, a high SC2RI was less susceptible to mTOR-related pathway medications but more sensitive to cell cycle suppressors. Therefore, categorization based on SC2RI could enhance the prognostic prediction of AML and help identify novel therapeutic approaches.

Linoleic acid derivatives target miR-361-3p/BTG2 to confer anticancer effects in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a deadly hematologic malignancy. In this study, miR-361-3p and BTG2 gene expression in AML blood and healthy specimens were analyzed using quantitative real-time reverse transcription polymerase chain reaction. A significant negative correlation between miR-361-3p and BTG2 was observed. The cell viability and apoptosis were measured by CCK-8 assay, EdU incorporation assay and flow cytometry. A dual-luciferase reporter gene assay was performed to confirm the binding sequence between miR-361-3p and BTG2 messenger RNA 3'-untranslated region. 9s-Hydroxyoctadecadienoic acid (9s-HODE), a major active derivative of linoleic acid, reduced the viability and induced cell apoptosis of HL-60 cells. Furthermore, the miR-361-3p mimics and siBTG2 reversed the above effects of 9s-HODE. 9s-HODE exerted an anti-AML effect through, at least partly, regulating the miR-361-3p/BTG2 axis.

Downregulation of Polo-like kinase 4 induces cell apoptosis and G2/M arrest in acute myeloid leukemia.

Polo-like kinase 4 (PLK4) is a crucial regulator for centriole replication and is reported to be aberrantly expressed in various cancers, where it participates to tumorigenesis. However, PLK4 effect in acute myeloid leukemia (AML), is still uncertain. This study investigates the function of PLK4 in AML. Quantitative real-time PCR was used to measure the level of PLK4. Centrinone, a selective PLK4 small molecule inhibitor, was used for PLK4 inhibition and explore its effect in AML cells. The cell growth was detected by the CCK8, while the cell cycle and apoptosis were assessed by flow cytometry. The level of proteins associated with apoptosis, cell cycle and endoplasmic reticulum (ER) stress were analyzed by western blotting. PLK4 was overexpressed in AML cells. PLK4 knockdown or its specific inhibition by centrinone induced G2/M phase arrest via suppressing the expression of cyclin B1 and Cdc2 and promoting the level of proapoptotic proteins. Moreover, PLK4 targeting enhanced the level of proteins related to ER stress, such as GRP78, ATF4, ATF6, and CHOP. These findings demonstrated that targeting PLK4 can induce apoptosis, G2/M and ER stress in AML cells.

Heterogeneity of Patient-Derived Acute Myeloid Leukemia Cells Subjected to SYK In Vitro Inhibition

Acute myeloid leukemia (AML) is an aggressive hematological malignancy with a dismal prognosis. The cytoplasmic spleen tyrosine kinase (SYK) is highly expressed by hematopoietic cells and has emerged as a potential therapeutic target. In this study, we evaluated the in vitro antileukemic effects of five SYK inhibitors, fostamatinib, entospletinib, cerdulatinib, TAK-659, and RO9021, in a consecutive AML patient cohort. All inhibitors demonstrated a concentration-dependent antiproliferative effect, although there was considerable heterogeneity among patients. For fostamatinib and TAK-659, the antiproliferative effects were significantly higher in FLT3 mutated patients compared to nonmutated patients. Fostamatinib, entospletinib, TAK-659, and RO9021 induced significant apoptosis in primary AML cells, although the proapoptotic effects of the SYK inhibitors were less pronounced than the antiproliferative effects. Finally, most of the SYK inhibitors caused a significant decrease in the release of cytokines and chemokines from primary AML cells, indicating a potent inhibitory effect on the release of these leukemic signaling molecules. We concluded that the SYK inhibitors had antileukemic effects in AML, although larger studies are strongly needed to identify which patient subsets will benefit most from such a treatment.

Identification of TSC22D3 As a Novel Immune-Related Prognostic Biomarker of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common type of hematological neoplasms in adults. TSC22 Domain Family Member 3 (TSC22D3) is one of glucocorticoid-induced genes and has an important regulatory role in immunosuppressive and cell proliferation. However, its prognostic value and immune infiltration effect in acute myeloid leukemia are remain elusive. Here, we investigated the association of TSC22D3 with immune infiltrates and its prognostic value in AML patients. We first analyzed 173 patients from the TCGA and 70 healthy controls from GTEx databases and found that TSC22D3 was highly expressed in AML (Figure A)and closely associated with various biological processes and signaling pathways, including stress-activated MAPK cascade and proteoglycans in cancer and cytokine-cytokine receptor interaction (Figure B). In addition, GESA analysis demonstrated that TSC22D3 was closely corelated with several critical signaling pathways including Toll-like receptor 4 (TLR4) pathway, EGFR pathway, NOTCH pathway, TNFR2 non-canonical NF-κB pathway (Figure C), which further highlighted the important role of TSC22D3 in the pathogenesis of AML. Immunogen analysis found positive associations with various immune cells, especially macrophages (Figure D), and TSC22D3 was also significantly associated with PD-1 and CTLA-4 expression in AML (Figure E). Our results suggest that TSC22D3 is closely associated with immune injectors and promotes immune escape in AML patients in the tumor microenvironment. High level of TSC22D3 was related with poor prognosis of AML in age, BM blasts and several gene mutation (IDH1 R132, FLT3) subgroups (Figure F), indicated that TSC22D3 can be identified as a prognostic molecule in these clinical feature subgroups. Subsequently, a calibration nomogram based on the age and genetic risk level of the clinical patient was conducted. There was a favorable consistency between the actual observed and predicted values for 1-, 3-, 5-year OS based on the calibration chart (Figure G). Based on the complementary perspective for respective tumors, the constructed model provided a personalized score for individual patients. Taken together, this study firstly reported that the high expression of TSC22D3 was significantly associated with the poor survival rate and immune infiltration in AML patients, which may promote the tumorigenesis of AML through enhancing the abnormal inflammation and immune escape. In particular, the immunomodulatory effects of TSC22D3 may play a crucial role in predicting the prognosis and have the potential to become a new biomarker of AML. Thus, this study identified that TSC22D3 may be useful as an immunomodulatory agent for AML clinical management and provided a new insight for further investigation on the clinicopathological significance and molecular pathogenesis of AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

BRD4 PROTAC degrader MZ1 exerts anticancer effects in acute myeloid leukemia by targeting c-Myc and ANP32B genes

ABSTRACT Acute myeloid leukemia (AML) is a highly cancerous and aggressive hematologic disease with elevated levels of drug resistance and relapse resulting in high mortality. Recently, bromodomains and extra-terminal (BET) protein inhibitors have been extensively researched in hematological tumors as potential anticancer agents. MZ1 is a novel BET inhibitor that mediates selective proteins degradation and suppression of tumor growth through proteolysis-targeting chimeras (PROTAC) technology. Accordingly, this study aimed to investigate the role and therapeutic potential of MZ1 in AML. In this study, we first identified that AML patients with high BRD4 expression had poor overall survival than those with low expression group. MZ1 inhibited AML cell growth and induced apoptosis and cycle arrest in vitro. MZ1 induced degradation of BRD4, BRD3 and BRD2 in AML cell strains. Additionally, MZ1 also initiated the cleavage of poly-ADP-ribose polymerase (PARP), which showed cytotoxic effects on NB4 (PML-RARa), K562 (BCR-ABL), Kasumi-1 (AML1-ETO), and MV4-11 (MLL-AF4) cell lines representing different molecular subtypes of AML. In AML mouse leukemia model, MZ1 significantly decreased leukemia cell growth and increased the mouse survival time. According to the RNA-sequencing analysis, MZ1 led to c-Myc and ANP32B genes significant downregulation in AML cell lines. Knockdown of ANP32B promoted AML cell apoptosis and inhibited cell growth. Overall, our data indicated that MZ1 had broad anti-cancer effects on AML cell lines with different molecular lesions, which might be exploited as a novel therapeutic strategy for AML patients.

A novel organic arsenic derivative MZ2 remodels metabolism and triggers mtROS-mediated apoptosis in acute myeloid leukemia.

Acute myeloid leukemia (AML) is one of the most common neoplasms in adults, and it is difficult to achieve satisfactory results with conventional drugs. Here, we synthesized a novel organic arsenic derivative MZ2 and evaluated its ability to remodel energy metabolism to achieve anti-leukemia. MZ2 was characterized by the average 1-min full mass spectra analysis. Biological methods such as Western blot, qPCR, flow cytometry and confocal microscopy were used to assess the mode and mechanism of MZ2-induced death. The in vivo efficacy of MZ2 was assessed by constructing a patient-derived xenograft (PDX) AML model. Unlike the precursor organic arsenical Z2, MZ2 can effectively reduce the level of aerobic glycolysis. Our in-depth found that MZ2 inhibited the expression of PDK2 in a dose-dependent manner and did not affect the expression of LDHA, another key enzyme of the glycolytic pathway. MZ2 reconstituted energy metabolism to induce the generation of mitochondrial ROS (mtROS) and then triggerd intrinsic apoptosis pathway. We also assessed whether MZ2 generates autophagy and results showed that MZ2 can induce autophagy of AML cells, which may be associated with the precursor organic arsenic drug. In vivo, MZ2 effectively attenuated leukemia progression in mice, and immunohistochemical results suggested its PDK2 inhibitory effect. In summary, the novel organic arsine derivative MZ2 exhibited excellent anti-tumor effects in acute myeloid leukemia, which may provide a potential strategy for the treatment of acute myeloid leukemia.

Pediatric and young adult leukemia adoptive therapy (PLAT)-08: A phase 1 study of SC-DARIC33 in pediatric and young adults with relapsed or refractory CD33+ AML.

TPS7078 Background: The acute and late effects of acute myeloid leukemia (AML) therapy in children can be devastating, and relapse rates remain high. Targeted therapies are needed to decrease toxicity and improve cure rates. Most AML patients express CD33 on their leukemic blasts. CD33 is a viable target with CD33 targeted therapies approved for AML. CD33 targeted CAR T cell therapies are being developed, but safety and efficacy remain a challenge. Safety concerns include cytokine release syndrome, neurotoxicity and aplasia. Efficacy is limited, with antigen escape and T cell exhaustion proposed as mechanisms of treatment failure. Dimerizing Agent Regulated ImmunoReceptor Complex (DARIC) is a next-generation technology that utilizes a split receptor design separating the antigen binding and intracellular signaling subunits (Leung, 2019). These subunits remain inactive until the administration of rapamycin which allows for dimerization of the subunits, enabling T cell activation when antigen is present. Modulating CAR T cell activation may aid hematopoietic recovery, enhance CAR T cell function and persistence, and mitigate toxicity. We developed a DARIC T cell product, SC-DARIC33, that couples the DARIC technology with a lentiviral construct that incorporates the use of a novel humanized camelid nanobody for targeting the C2 splice isoform within the membrane proximal domain of CD33. Methods: Up to 18 patients will be enrolled in this phase 1 study (NCT05105152). Patients must have early first relapse of AML (≤ 6 months from diagnosis), first relapse refractory to at least one reinduction attempt, second or greater relapse, or refractory de novo AML. The first 3 patients must be ≥18 years, all patients who meet eligibility criteria must be ≤28 years. The primary objectives of this Phase I study (NCT05105152) are assessment of safety/toxicity and feasibility of manufacturing of SC-DARIC33. Secondary objectives include assessment of efficacy, on/off rate of DARIC expression, engraftment, expansion and persistence of SC-DARIC33. Also, the effect of cessation of rapamycin on activation state of SC-DARIC 33 and on hematopoietic recovery will be evaluated. Subjects receive lymphodepletion prior to a single dose of SC-DARIC33 T cells on Day 0 followed by initiation of rapamycin on Day +2 with the purpose of activating the T cells. Rapamycin continues until Day 21 and bone marrow evaluation occurs on Day 28. Subsequent cycles of rapamycin are allowed for patients who respond. The trial will implement a BOIN design with three possible dose levels (1 x 106, 5 x 106, and 10 x 106 DARIC+ cells/kg) to determine the maximum tolerated dose (MTD). The trial is open for enrollment at Seattle Children’s Hospital. Clinical trial information: NCT05105152.

Targeting PI3K/AKT/mTOR pathway to enhance the anti-leukemia efficacy of venetoclax

BackgroundThe treatment of acute myeloid leukemia (AML) is developing towards “targeted therapy”, which faces challenges such as low sensitivity and drug resistance. Therefore, targeted drugs need to be used in combination with other drugs to overcome clinical problems. ObjectiveAML cells and animal models were used to determine the synergistic anti-leukemic effect of Dactolisib (BEZ235) and Venetoclax (ABT199) and explore its mechanism. MethodsIn vitro experiments, we used cell counting kit-8 (CCK8), flow cytometry, real-time quantitative PCR (qPCR), and Western blot to detect the anti-leukemic effects of ABT199 and BEZ235. In vivo experiments, female nude mice were injected subcutaneously with THP-1 cells to form tumors, evaluate the combined effect of ABT199 and BEZ235 by indicators such as tumor size, tumor weight, Ki67 and cleaved-Caspase3 staining. The mice's body weight and HE staining were used to evaluate the liver injury and adverse drug reactions. ResultsThe combination of BEZ235 and ABT199 has a synergistic effect through promoting apoptosis and inhibiting proliferation. The BEZ235 increased the drug sensitivity of ABT199 by reducing the MCL-1 protein synthesis and promoted the degradation of MCL-1 protein, which is considered as the mechanism of reversing ABT199 resistance. Furthermore, the BEZ235 and ABT199 can synergistically enhance the inhibition of PI3K/AKT/mTOR pathway. ConclusionThe combination of BEZ235 and ABT199 exhibits a synergistic anti-tumor effect in AML by down-regulating MCL-1 protein.

Discovery of Dual CDK6/PIM1 Inhibitors with a Novel Structure, High Potency, and Favorable Druggability for the Treatment of Acute Myeloid Leukemia.

Nowadays, the simultaneous inhibition of two or more pathways plays an increasingly important role in cancer treatment due to the complex and diverse pathogenesis of cancer, and the combination of the cyclin-dependent kinase 6 (CDK6) inhibitor and PIM1 inhibitor was found to generate synergistic effects in acute myeloid leukemia (AML) treatment. Therefore, we discovered a novel lead 1 targeting CDK6/PIM1 via pharmacophore-based and structure-based virtual screening, synthesized five different series of new derivates, and obtained a potent and balanced dual CDK6/PIM1 inhibitor 51, which showed high kinase selectivity. Meanwhile, 51 displayed an excellent safety profile and great pharmacokinetic properties. Furthermore, 51 displayed stronger potency in reducing the burden of AML than palbociclib and SMI-4a in vivo. In summary, we offered a new direction for AML treatment and provided a great lead compound for AML preclinical studies.

Maintenance with Hypomethylating Agents after Allogeneic Stem Cell Transplantation in Acute Myeloid Leukemia and Myelodysplastic Syndrome: A Systematic Meta-Analysis

▪Background: After allogeneic hematopoietic stem cell transplantation (allo-SCT), 35%-45% of patients suffer disease relapse, resulting in dismal outcomes. Several strategies have been adopted to prolong disease-free intervals. Hypomethylating agents (HMA) seem to have a range of properties favorable to post-allo-SCT maintenance. They mediate a direct anti-leukemic effect in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), regardless of their molecular mutation profile. Moreover, their abilities to induce a CD8+ tumor-specific T cell response, together with the expansion of regulatory T cells, leads to an epigenetically enhanced graft-versus-leukemia effect that is not counterbalanced by an increased risk of graft-versus-host disease (GVHD).Materials and Methods: Two investigators independently searched the Embase and MEDLINE databases for relevant studies published from inception to 31 January 2021. The inclusion criteria were as follows: (1) the studies had to be either randomized controlled trials or cohort studies; (2) the patients underwent allo-SCT for AML or MDS; (3) the studies compared 2 patient groups: one receiving HMA maintenance post-allo-SCT, and the other being an observational group; and (4) the studies needed to report at least one of our primary outcomes of interest; overall survival (OS), relapse-free survival (RFS), grade II-IV acute GVHD, and chronic GVHD.Results: Eleven eligible studies compared HMA maintenance to observation following allo-SCT for AML or MDS. The OS of the HMA maintenance group was superior to the observation group, with a pooled odds ratio (OR) of 2.52 (95% CI, 1.60-3.94; I 2, 51%; Figure 1A). Similarly, a pooled meta-analysis found that the RFS was significantly better for patients receiving HMA maintenance, with a pooled OR of 2.64 (95% CI, 1.79-3.90; I 2, 0%; Figure 1B). Moreover, the cumulative incidence of relapse (CIR) was significantly lower in patients who received HMAs. The HMA group also had lower non-relapse mortality (NRM) compared with the observation group. Overall, the incidences of grade II-IV acute GVHD and chronic GVHD did not differ in both groups. However, when looking specifically at those receiving decitabine maintenance, the rate of chronic GVHD seemed to be lower compared with observation alone.Conclusions: The current systematic review and meta-analysis illustrated that AML and MDS patients receiving HMA maintenance after allo-SCT had better outcomes in regards to OS, RFS, NRM, CIR as well as a reduced incidence of chronic GVHD.Figure 1. Forest plots of the meta-analysis of HMA maintenance compared with observation. (A) OS rate. (B) RFS rate. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Synergistic effect of chidamide and venetoclax on apoptosis in acute myeloid leukemia cells and its mechanism.

BackgroundAcute myeloid leukemia (AML) is a hematological malignancy with a low remission rate and high recurrence rate. Overexpression of the antiapoptotic protein Bcl-2 is associated with a lower overall survival rate in AML patients. Venetoclax (ABT199) is a selective inhibitor of Bcl-2 that has a significant effect in AML, but single-drug resistance often occurs due to the high expression of Mcl-1 protein. Studies have confirmed that chidamide can downregulate the expression levels of Bcl-2 and Mcl-1 and induce apoptosis.MethodsThis study aimed to use AML cell lines and primary cells to study the effects of venetoclax and chidamide combination therapy on AML cell apoptosis, the cell cycle, and changes in related signaling pathways in vitro; establish an AML mouse model to observe the efficacy and survival time of combination therapy in vivo; and analyze the drug effects with multi-omics sequencing technology. The changes in gene and protein expression before and after treatment were examined to clarify the molecular mechanism driving the synergistic effect of the two drugs.Results(I) Both venetoclax and chidamide promoted apoptosis in AML cell lines and primary cells in a time- and concentration-dependent manner. The effect was further enhanced when the two drugs were combined, and a synergistic effect was observed (combination index <1). (II) At both the mRNA and protein levels, the expression of Mcl-1 was upregulated by venetoclax and downregulated by chidamide, and the expression of Mcl-1 decreased further after combination treatment. (III) Transcriptome sequencing showed that differentially expressed genes in the combination group compared with the venetoclax monotherapy group were mainly enriched in the PI3K-AKT pathway and JAK2/STAT3 pathway. Moreover, qRT-PCR and Western blot confirmed these results. (IV) The combination therapy group exhibited significantly inhibited disease progression and a prolonged survival time among AML mice.ConclusionsChidamide combined with venetoclax synergistically promoted apoptosis in AML cell lines and primary cells by inhibiting activation of the PI3K/AKT pathway and JAK2/STAT3 pathway.

Hsa-circ_0003420 induces apoptosis in acute myeloid leukemia stem cells and impairs stem cell properties

Objective To explore the effect of circular RNA-0003420 (circ_0003420) in acute myeloid leukemia (AML) relapse and leukemia stem cells (LSCs) properties. Materials and methods TRIzol reagent was used to extract total RNA from AML tissues or cells. Cell viability was assessed by Cell Counting Kit-8 and EdU staining assays. Cell apoptosis was determined by flow cytometry. Results Compared with normal hematopoietic stem cells, circular RNA hsa-circ_0003420 expression was considerably decreased in non-m3 AML stem cells. Furthermore, the lack of hsa-circ_0003420 is correlated with poor clinical results and impaired therapeutic effects in AML. Overexpression of hsa-circ_0003420 via transfection caused LSC death and inhibited the characteristics of leukemia tumor stem cells, including expression of Homeobox B4 (HOXB4), MYB, and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) axis. Furthermore, hsa-circ_0003420 targets the mRNA of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) and hsa-circ_0003420 expression markedly repressed IGF2BP1 levels in LSCs. Restoration of IGF2BP1 eliminated the effect of hsa-circ_0003420 on the replication, apoptosis, and LSC phenotype of KG-1a cells. Discussion and conclusions Up-regulation of hsa-circ_0003420 expression in LSCs caused redox disorder, inflammation and apoptosis, suggesting that this protein could be used as a target for the treatment AML.

Can the New and Old Drugs Exert an Immunomodulatory Effect in Acute Myeloid Leukemia?

Simple SummaryThe advent of novel immunotherapeutic strategies has revealed the importance of immune dysregulation and of a tolerogenic microenvironment for acute myeloid leukemia (AML) fitness. We reviewed the “off-target” effects on the immune system of different drugs used in the treatment of AML to explore the advantages of this unexpected interaction.Acute myeloid leukemia (AML) is considered an immune-suppressive neoplasm capable of evading immune surveillance through cellular and environmental players. Increasing knowledge of the immune system (IS) status at diagnosis seems to suggest ever more attention of the crosstalk between the leukemic clone and its immunologic counterpart. During the last years, the advent of novel immunotherapeutic strategies has revealed the importance of immune dysregulation and suppression for leukemia fitness. Considering all these premises, we reviewed the “off-target” effects on the IS of different drugs used in the treatment of AML, focusing on the main advantages of this interaction. The data reported support the idea that a successful therapeutic strategy should consider tailored approaches for performing leukemia eradication by both direct blasts killing and the engagement of the IS.

Abstract 2017: Dual targeting of glutathione-GPX4 nexus and mitochondrial protease CLPP causes synergistic anti-leukemia effects in acute myeloid leukemia (AML)

Abstract Cancer cells depend on aberrant antioxidant mechanisms to survive increased oxidative stress, which is particularly critical for cancer stem cell maintenance and its therapy resistance phenotype. Mitochondria is the primary site of oxygen consumption and reactive oxygen species (ROS) production, thus is essential for regulating cellular redox homeostasis. We have recently reported that hyperactivation of mitochondrial (mito-) protease CLPP by imipridones (ONC201/212) exerts cancer selective lethality through degradation of mito-proteins including respiratory chain complexes and induction of oxidative stress (Ishizawa et al., Cancer Cell 2019). However, the cell death mechanisms remain to be fully determined. We hypothesized that deregulation of mito-redox homeostasis induces a distinct mode of cell death in CLPP-hyperactivated cells and that concomitant inhibition of both mito- and cytosolic antioxidant mechanisms could enhance the anti-leukemia effects. We first tested if inhibition of glutathione (GSH) synthesis enhances the anti-leukemia effects by CLPP hyperactivation by using buthionine sulfoximine (BSO), a glutamate-cysteine ligase inhibitor. As hypothesized, BSO highly sensitized AML cells to genetic or pharmacologic hyperactivation of CLPP by overexpressing CLPP-Y118A (an activating mutation) or ONC201 treatment. We next investigated if a downstream target of GSH depletion is causing the synergy, and re-analysis of recently published CRISPR-knockout screens using leukemic cells treated with imipridones identified GPX4 as one of the 15 top-hit genes whose knockout sensitized cells to CLPP hyperactivation. GPX4 is a selenoprotein that is known for its GSH-dependent reduction of lipid hydroperoxides to protect cells from oxidative cell death including ferroptosis. We found that CLPP hyperactivation increased GPX4 protein levels in AML cells, prominently in mitochondria. Consistently, the GPX4 inhibitors ML210 and JKE1674 sensitized cells to ONC201 in multiple AML cell lines. Overexpression of the activating CLPP-Y118A was also synthetically lethal to GPX4 inhibition, confirming the on-target effect of ONC201. Mechanistically, GPX4 inhibition induced iron-dependent lipid peroxidation and AML cell killing, thus defining ferroptosis as the operational mechanism of cell death. Dual targeting of CLPP and GPX4 induced enhanced lipid peroxidation and mito-ROS but ferroptosis inhibitors only partially blocked cell death, indicating that other modes of oxidative cell death are also involved in the combinatorial synergism. Collectively, our data indicate therapeutic potential of targeting GPX4 and CLPP in AML and potential identification of a novel mode of cancer cell death. Investigations are ongoing to elucidate the underlying molecular mechanisms and to assess in vivo efficacy of the combinatorial treatment. Citation Format: Hiroki Akiyama, Ran Zhao, Lauren B. Ostermann, Jo Ishizawa, Michael Andreeff. Dual targeting of glutathione-GPX4 nexus and mitochondrial protease CLPP causes synergistic anti-leukemia effects in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2017.