The Role of Genes Related to the Apoptosis and Neddylation Pathway in Patients With Acute Myeloid Leukemia

Evaluation of Neddylation and Apoptosis-Related Gene Expression in Patients with Acute Myeloid Leukemia

Molecular Interactions between Mitochondrial Ferroptosis and Apoptosis Pathways in Acute Myeloid Leukemia

Treatment of acute myeloid leukemia (AML) remains challenging and novel targets and synergistic therapies still need to be discovered. We performed a high-throughput RNAi screen in three different AML cell lines and primary human leukemic blasts to identify genes that synergize with common antileukemic therapies. We used a pooled shRNA library that covered 5043 different genes and combined transfection with exposure to either azacytidine or cytarabine analog to the concept of synthetic lethality. Suppression of the chemokine CXCL12 ranked highly among the candidates of the cytarabine group. Azacytidine in combination with suppression of genes within the neddylation pathway led to synergistic results. NEDD8 and RBX1 inhibition by the small molecule inhibitor pevonedistat inhibited leukemia cell growth. These findings establish an in vitro synergism between NEDD8 inhibition and azacytidine in AML. Taken together, neddylation constitutes a suitable target pathway for azacytidine combination strategies.

Background: One third of MDS cases transforms into AML. MDS and AML are considered one spectrum of myeloid stem cell disease. We have previously reported MDS and AML specific proteomic signatures associated with different survival outcomes. However, there exists no study that integrated both MDS and AML cases to explore common proteomic signatures that harbor prognostic implications. Methods: We collected bone marrow and peripheral blood CD34+ samples from 116 MDS patients and 511 AML (non-APL) patients newly diagnosed at the MD Anderson Cancer Center from 1998 till 2006. Reverse phase protein array (RPPA) platform was used for functional proteomic profiling on 119 proteins and phospho-proteins for cell-cycle, apoptosis, and several signal transduction pathways known to play a role in hematologic malignancies. Each antibody was validated with Western blotting. We integrated the RPPA data between MDS and AML patients via normalization. Kaplan-Meier method was used for survival analysis performed on 90 MDS patients and 415 AML patients who received treatments. Results: Among 627 patients with MDS or AML, six distinct functional proteomic signatures were identified; signature 1 (21.0% of total patients), 2 (15.6%), 3 (15.6%), 4 (16.0%), 5 (24.0%), and 6 (7.7%). Most of patients in signature group 6 had MDS compared with patients in other groups (chi-square test p<0.001, proportion of MDS patients in group 1 (7.5%), 2 (0%), 3 (15.2%), 4 (9.9%), 5 (28.9%), and 6 (69.2%); thus, group 1 to 5: AML-like signature vs. group 6: MDS-like signature). Among MDS patients, 10.3% of patients in group 6 later transformed into AML while 1.8% of patients in other groups did so. Among AML patients, 1.0% of patients in group 6 had history of MDS while 4.4% of patients in other groups did so. Six groups demonstrated differences in complete remission (CR) rate (chi-square test p=0.03). Among patients with MDS or AML who received treatment, CR rate in patients in group 6 was 61.5% while that in patients in other groups was 51.1%. However, six groups did not differ in CR duration. Among MDS patients, there were no statistically significant differences in overall survival between group 6 and other groups (log-rank test p=0.63, HR=0.87). However, among AML patients, patients in group 6 was associated with superior overall survival compared with patients in other groups (log-rank test p=0.02, HR=0.39). Conclusion: Integrated functional proteomic profiling revealed six common distinct signature groups that are associated with CR rate and overall survival outcome. In AML patients, MDS-like signature (group 6) was linked with favorable survival outcome and possibly higher CR rate. Citation Format: Young Kwang Chae, Suk Young Yoo, Kevin R. Coombes, Steven M. Kornblau. Integrated functional proteomic profiling in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML): MDS-like signature is associated with favorable outcome in patients with AML. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5310. doi:10.1158/1538-7445.AM2014-5310

AML-069 The Role of Genes Related to the Apoptosis and Neddylation Pathway in Patients With Acute Myeloid Leukemia

BackgroundDysregulation or abnormality of the programmed cell death (PCD) pathway is closely related to the occurrence and development of many tumors, including acute myeloid leukemia (AML). Studying the abnormal characteristics of PCD pathway-related molecular markers can provide a basis for prognosis prediction and targeted drug design in AML patients.MethodsA total of 1394 genes representing 13 different PCD pathways were examined in AML patients and healthy donors. The upregulated genes were analyzed for their ability to predict overall survival (OS) individually, and these prognostic genes were subsequently combined to construct a PCD-related prognostic signature via an integrated approach consisting of 101 models based on ten machine learning algorithms. RNA transcriptome and clinical data from multiple AML cohorts (TCGA-AML, GSE106291, GSE146173 and Beat AML) were obtained to develop and validate the AML prognostic model.ResultsA total of 214 upregulated PCD-related genes were identified in AML patients, 39 of which were proven to be prognostic genes in the training cohort. On the basis of the average C-index and number of model genes identified from the machine learning combinations, a PCD index was developed and validated for predicting AML OS. A prognostic nomogram was then generated and validated on the basis of the PCD index, age and ELN risk stratification in the Beat AML cohort and the GSE146173 cohort, revealing satisfactory predictive power (AUC values ≥ 0.7). With different mutation patterns, a higher PCD index was associated with a worse OS. The PCD index was significantly related to higher scores for immunosuppressive cells and mature leukemia cell subtypes. As the gene most closely related to the PCD index, the expression of SMAD3 was further validated in vitro. AML cells harboring KMT2A rearrangements were more sensitive to the SMAD3 inhibitor SIS3, and the expression of the autophagy-related molecular marker LC3 was increased in KMT2A-rearranged cell lines after SIS3 monotherapy and combined treatment.ConclusionThe PCD index and SMAD3 gene expression levels have potential prognostic value and can be used in targeted therapy for AML, and these findings can lead to the development of effective strategies for the combined treatment of high-risk AML patients.

Background: Despite the recent advances in AML with the emergence of multiple targeted therapies, patient outcomes remain poor, particularly in the relapsed/refractory setting underscoring the need for novel therapeutic strategies. Proteasome inhibitors (PIs) have antitumor activity in AML through inhibition of the nuclear factor κB pathway and induction of apoptosis with multiple clinical trials showing promising activity of bortezomib particularly when combined with other cytotoxic agents (Csizmar 2016). CFZ, a second-generation PI, has preferential preclinical activity in AML compared to bortezomib and is therefore an agent of interest in AML therapy (van der Helm 2015). Here we assessed the activity of CFZ alone and in combination with cytarabine and the autophagy inhibitor CHQ in AML cell lines. Methods: After screening 18 AML cell lines, 3 sensitive: KASUMI1, ML2, and MV411, and 3 resistant cell lines: AML193, THP1, and NOMO1, were selected for further analysis using an IC50 cutoff for CFZ of 10 nM. Apoptosis, cell cycle, and cell senescence analysis were performed after 72 hours of CFZ exposure at 10 nM. Combination assays using CFZ 10 nM, cytarabine 200 nM, and CHQ 20 µM were performed to evaluate for potential interaction in the form of antagonism or potentiation. Proteomic analysis was performed at baseline using reverse phase protein assay (RPPA) and confirmed with western blot analysis. Results: Single-agent CFZ induced apoptosis in sensitive cell lines with apoptotic rates increasing by 85% to 95%; while apoptotic rates remained unchanged in resistant cell lines. Similarly, CFZ resulted in G0/G1 cell cycle arrest in sensitive but not resistant AML cell lines. Lack of difference in cellular senescence confirmed apoptosis and cell cycle arrest as the major mechanisms of CFZ-induced growth inhibition in AML cell lines. No antagonism was noted when CFZ 10 nM was combined with cytarabine (200 nM). RPPA revealed that AML cell lines with low expression of autophagy related proteins are more sensitive to proteasome inhibition with CFZ treatment compared to resistant cell lines. This was confirmed with western blot which showed upregulation of the autophagy related proteins in resistant cell lines upon exposure to CFZ. However, sensitive cell lines did not upregulate autophagy pathways which may lead to cell cycle arrest and apoptosis. Inhibiting autophagy with CHQ sensitized CFZ resistant lines to CFZ treatment. Conclusion: CFZ demonstrated single agent activity in human AML cell lines and upregulation of autophagy may contribute to resistance to CFZ which can be overcome using autophagy inhibitors such as CHQ. These results support the development of CFZ and autophagy inhibitor-based combinations for AML. Citation Format: Mao Yu Peng, Yasmin Abaza, Dylan Conklin, Erika Von Euw, Monica Mead, John Timmerman, Dennis Slamon, Sarah Larson. Activity of carfilzomib (CFZ) alone and in combination with cytarabine and chloroquine diphosphate (CHQ) in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 297.

The Role and Mechanism of NDUFS2 in FLT3-Mutated Acute Myeloid Leukemia

Targeting PLZF Enhances Glucocorticoid Sensitivity in Acute Myeloid Leukemia

The Role of VEGFA in Malignant Progression in AML

Single-Cell Multiomics Unveils Venetoclax-Resistant Monocytic Differentiation and Immune Evasion in TP53 Mutant AML Clones

Synergistic Activity of BMS-986397, a First-in-Class a Cereblon (CRBN) E3 Ligase Modulator (CELMoD) Targeting Casein Kinase 1α (CK1α), in Combination with Venetoclax and/or Azacitidine in Preclinical Models of Acute Myeloid Leukemia (AML)

Synergistic Anti-Tumor Effect on Novel Combination of Selinexor with Azacitidine in AML By Targeting XPO1/eIF4E/C-MYC Signaling

BackgroundMost patients with acute myeloid leukemia (AML) remain uncurable and require novel therapeutic methods. Gain-of-function FMS-like tyrosine kinase 3 (FLT3) mutations are present in 30–40% of AML patients and serve as an attractive therapeutic target. In addition, FLT3 is aberrantly expressed on blasts in > 90% of patients with AML, making the FLT3 ligand-based drug conjugate a promising therapeutic strategy for the treatment of patients with AML. Here, E. coli was used as a host to express recombinant human FLT3 ligand (rhFL), which was used as a specific vehicle to deliver cytotoxic drugs to FLT3 + AML cells.MethodsRecombinant hFL was expressed and purified from induced recombinant BL21 (DE3) E. coli. Purified rhFL and emtansine (DM1) were conjugated by an N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) linker. We evaluated the potency of the conjugation product FL-DM1 against FLT3-expressing AML cells by examining viability, apoptosis and the cell cycle. The activation of proteins related to the activation of FLT3 signaling and apoptosis pathways was detected by immunoblotting. The selectivity of FL-DM1 was assessed in our unique HCD-57 cell line, which was transformed with the FLT3 internal tandem duplication mutant (FLT3-ITD).ResultsSoluble rhFL was successfully expressed in the periplasm of recombinant E. coli. The purified rhFL was bioactive in stimulating FLT3 signaling in AML cells, and the drug conjugate FL-DM1 showed activity in cell signaling and internalization. FL-DM1 was effective in inhibiting the survival of FLT3-expressing THP-1 and MV-4-11 AML cells, with half maximal inhibitory concentration (IC50) of 12.9 nM and 1.1 nM. Additionally, FL-DM1 induced caspase-3-dependent apoptosis and arrested the cell cycle at the G2/M phase. Moreover, FL-DM1 selectively targeted HCD-57 cells transformed by FLT3-ITD but not parental HCD-57 cells without FLT3 expression. FL-DM1 can also induce obvious apoptosis in primary FLT3-positive AML cells ex vivo.ConclusionsOur data demonstrated that soluble rhFL can be produced in a bioactive form in the periplasm of recombinant E. coli. FL can be used as a specific vehicle to deliver DM1 into FLT3-expressing AML cells. FL-DM1 exhibited cytotoxicity in FLT3-expressing AML cell lines and primary AML cells. FL-DM1 may have potential clinical applications in treating patients with FLT3-positive AML.

Acute myeloid leukaemia (AML) is a heterogeneous disease with dismal outcome. Sunitinib is an orally active inhibitor of multiple tyrosine kinase receptors approved for renal cell carcinoma and gastrointestinal stromal tumour that has also been studied for AML in several clinical trials. However, the precise mechanism of sunitinib action against AML remains unclear and requires further investigation. For this purpose, this study was conducted using human AML cell lines (HL60 and KG-1) and AML patients' mononucleated cells. Sunitinib induced G1 phase arrest associated with decreased cyclin D1, cyclin D3, and cyclin-dependent kinase (Cdk)2 and increased p27(Kip1), pRb1, and p130/Rb2 expression and phosphorylated activation of protein kinase C alpha and beta (PKCα/β). Selective PKCα/β inhibitor treatment abolished sunitinib-elicited AML differentiation, suggesting that PKCα/β may underlie sunitinib-induced monocytic differentiation. Furthermore, sunitinib increased pro-apoptotic molecule expression (Bax, Bak, PUMA, Fas, FasL, DR4, and DR5) and decreased anti-apoptotic molecule expression (Bcl-2 and Mcl-1), resulting in caspase-2, caspase-3, caspase-8, and caspase-9 activation and both death receptor and mitochondria-dependent apoptosis. Taken together, these findings provide evidence that sunitinib targets AML cells through both differentiation and apoptosis pathways. More clinical studies are urgently needed to demonstrate its optimal clinical applications in AML.