C/EBPα-p30 confers AML cell susceptibility to the terminal unfolded protein response and resistance to Venetoclax by activating DDIT3 transcription.

BACKGROUND: Acute myeloid leukemia (AML) with biallelic (CEBPAbi) as well as single mutations located in the bZIP region is associated with a favorable prognosis, but the underlying mechanisms are still unclear. Here, we propose that two isoforms of C/EBPα regulate DNA damage-inducible transcript 3 (DDIT3) transcription in AML cells corporately, leading to altered susceptibility to endoplasmic reticulum (ER) stress and related drugs. METHODS: Human AML cell lines and murine myeloid precursor cell line 32Dcl3 cells were infected with recombinant lentiviruses to knock down CEBPA expression or over-express the two isoforms of C/EBPα. Quantitative real-time PCR and western immunoblotting were employed to determine gene expression levels. Cell apoptosis rates were assessed by flow cytometry. CFU assays were utilized to evaluate the differentiation potential of 32Dcl3 cells. Luciferase reporter analysis, ChIP-seq and ChIP-qPCR were used to validate the transcriptional regulatory ability and affinity of each C/EBPα isoform to specific sites at DDIT3 promoter. Finally, an AML xenograft model was generated to evaluate the in vivo therapeutic effect of agents. RESULTS: We found a negative correlation between CEBPA expression and DDIT3 levels in AML cells. After knockdown of CEBPA, DDIT3 expression was upregulated, resulting in increased apoptotic rate of AML cells induced by ER stress. Cebpa knockdown in mouse 32Dcl3 cells also led to impaired cell viability due to upregulation of Ddit3, thereby preventing leukemogenesis since their differentiation was blocked. Then we discovered that the two isoforms of C/EBPα regulate DDIT3 transcription in the opposite way. C/EBPα-p30 upregulated DDIT3 transcription when C/EBPα-p42 downregulated it instead. Both isoforms directly bound to the promoter region of DDIT3. However, C/EBPα-p30 has a unique binding site with stronger affinity than C/EBPα-p42. These findings indicated that balance of two isoforms of C/EBPα maintains protein homeostasis and surveil leukemia, and at least partially explained why AML cells with disrupted C/EBPα-p42 and/or overexpressed C/EBPα-p30 exhibit better response to chemotherapy stress. Additionally, we found that a low C/EBPα p42/p30 ratio induces resistance in AML cells to the BCL2 inhibitor venetoclax since BCL2 is a major target of DDIT3. This resistance can be overcome by combining ER stress inducers, such as tunicamycin and sorafenib in vitro and in vivo. CONCLUSION: Our results indicate that AML patients with a low C/EBPα p42/p30 ratio (e.g., CEBPAbi) may not benefit from monotherapy with BCL2 inhibitors. However, this issue can be resolved by combining ER stress inducers.

IFN-ß Pretreatment Alleviates Allogeneic Renal Tubular Epithelial Cell-Induced NK Cell Responses via the IRF7/HLA-E/NKG2A Axis.

Immune checkpoint molecules are promising targets for suppressing the immune response but have received little attention in immune tolerance induction in organ transplantation. In this study, we found that IFN-ß could induce the expression of HLA-E as well as PD-L1 on human renal tubular epithelial cell line HK-2 and renal tissue of the C57BL/6 mouse. The JAK/STAT2 pathway was necessary for this process. Upregulation of both HLA-E and PD-L1 was fully abrogated by the JAK1/2 inhibitor ruxolitinib. Signaling pathway molecules, including STAT1, STAT2, mTOR, Tyk2, and p38 MAPK, were involved in HLA-E and PD-L1 upregulation. IRF7 is the key transcription factor responsible for the activation of HLA-E and PD-L1 promoters. Through screening an epigenetic regulation library, we found a natural compound, bisdemethoxycurcumin, enhanced IFN-ß-induced HLA-E and PD-L1 expression in vitro and in vivo. In PBMC-derived CD56+ NK cells, we found that NKG2A but not PD1 was constitutively expressed, indicating HLA-E/NKG2A as a more potent target to induce tolerance to innate immune cells. Pretreating HK-2 cells by IFN-ß significantly attenuated the degranulation of their coincubated NK cells and protected cells from NK-mediated lysis. In conclusion, IFN-ß pretreatment could activate HLA-E and PD-L1 transcription through the JAK/STAT/IRF7 pathway and then could protect renal tubular epithelial cells from allogeneic immune attack mediated by NK cells.

The Global Burden of Leukemia and Its Attributable Factors in 204 Countries and Territories: Findings from the Global Burden of Disease 2019 Study and Projections to 2030.

Background: Leukemia is a common malignancy that has four main subtypes and is a threat to human health. Understanding the epidemiological status of leukemia and its four main subtypes globally is important for allocating appropriate resources, guiding clinical practice, and furthering scientific research. Methods: Average annual percentage changes (AAPCs) were calculated to estimate the change trends of age-standardized rates (ASRs) from 1990 to 2019 in 204 countries and territories. The risk factors for leukemia death and disability-adjusted life-year (DALY) were also analyzed. In addition, the future trends in ASRs were projected through 2030. Results: The total number of incident cases, deaths, and DALYs from leukemia in 2019 was 0.64, 0.33, and 11.66 million, respectively. Decreasing trends in age-standardized incidence rate (ASIR), the age-standardized death rate (ASDR), and age-standardized DALY rate were detected on a global level while increasing trends in ASIR were detected in the high-sociodemographic index (SDI) regions. The leukemia burden was heavier in males than in females. By cause, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and chronic lymphocytic leukemia (CLL) were more likely to impose a burden on the elderly, while acute lymphoblastic leukemia (ALL) showed a greater impact in the younger population. A significant positive correlation was observed between SDI and AAPC in ASIR, while SDI was negatively correlated with AAPCs in both ASDR and age-standardized DALY rate. Smoking remained the most significant risk factor associated with leukemia-related death and DALY, especially in males. Similar deaths and DALYs were caused by smoking and high body mass index (BMI) in females. Future projections through 2030 estimated that ASIR and ASDR will continue to increase, while the DALY rate is predicted to decline. Conclusions: Patterns and trends of leukemia burden are correlated with SDI. The estimated contributions to leukemia deaths indicate that timely measures are needed to reduce smoking and obesity.

CEBPA mutants down-regulate AML cell susceptibility to NK-mediated lysis by disruption of the expression of NKG2D ligands, which can be restored by LSD1 inhibition.

NK group 2, member D (NKG2D) is one of the most critical activating receptors expressed by natural killer (NK) cells. There is growing evidence that acute myeloid leukemia (AML) cells may evade NK cell-mediated cell lysis by expressing low or no ligands for NKG2D (NKG2D-Ls). We hypothesized that CCAAT/enhancer-binding protein α (C/EBPα), one of the most studied lineage-specific transcription factors in hematopoiesis, might influence the expression of NKG2D-Ls. To test this hypothesis, we first examined the endogenous expression of wild-type C/EBPα (C/EBPα-p42) in human AML cell lines and demonstrated that its expression level was highly relevant to the sensitivity of AML cells to NK cell cytotoxicity. Induction of C/EBPα-p42 in the low endogenous CEBPA-expressing AML cell line increased the sensitivity to NK-induced lysis. Moreover, decreased expression of C/EBPα-p42 by RNA interference in AML cells abrogated NK-mediated cytotoxicity. We further showed that the increase in NK susceptibility caused by C/EBPα-p42 occurred through up-regulation of the NKG2D-Ls ULBP2/5/6 in AML cells. More importantly, chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing captured C/EBPα motif signatures at the enhancer regions of the ULBP 2/5/6 genes. Whilst, the AML-associated C/EBPα C-terminal mutant and N-terminal truncated mutant (C/EBPα-p30) diminished ULBP2/5/6 transcription. Finally, we identified that histone demethylase lysine-specific demethylase 1 (LSD1) inhibition can restore the expression of ULBPs via induction of CEBPA expression in AML cells, which may represent a novel therapeutic strategy for CEBPA-mutated AML. Abbreviations: C/EBPα: CCAAT/enhancer-binding protein α; TF: Transcription factor; AML: Acute myeloid leukemia; TAD: Transactivation domain; FS: Frameshift; NK: Natural killer; NKG2D: NK group 2, member D; NKG2D-Ls: Ligands for NKG2D; MHC: Major histocompatibility complex; MICA: MHC class I-related chain A; ULBP: UL16-binding protein; STAT3: Signal transducer and activator of transcription 3; LSD1: Lysine-specific demethylase 1; Ab: Antibody; PBMC: Peripheral blood mononuclear cell; PBS: Phosphate-buffered saline; CFSE: Carboxyfluorescein diacetate succinimidyl ester; PI: Propidium iodide; shRNA: Short hairpin RNA; ChIP: Chromatin immunoprecipitation; BM: Binding motif; HCNE: Highly conserved noncoding element; TSS: Transcription start site; HMA: Hypomethylating agent; AZA: Azacitidine/5-azacytidine; DAC: Decitabine/5-aza-29-deoxycytidine; 2-PCPA: Tranylcypromine; RBP: RNA-binding protein; MSI2: MUSASHI-2; HDACi: Inhibitor of histone deacetylases; VPA: Valproate; DNMTi: DNA methyl transferase inhibitor; SCLC: Small cell lung cancer.