Conserved methylation signatures associate with the tumor immune microenvironment and immunotherapy response.

BACKGROUND: Aberrant DNA methylation is a major characteristic of cancer genomes. It remains unclear which biological processes determine epigenetic reprogramming and how these processes influence the variants in the cancer methylome, which can further impact cancer phenotypes. METHODS: We performed pairwise permutations of 381,900 loci in 569 paired DNA methylation profiles of cancer tissue and matched normal tissue from The Cancer Genome Atlas (TCGA) and defined conserved differentially methylated positions (DMPs) based on the resulting null distribution. Then, we derived independent methylation signatures from 2,465 cancer-only methylation profiles from the TCGA and 241 cell line-based methylation profiles from the Genomics of Drug Sensitivity in Cancer (GDSC) cohort using nonnegative matrix factorization (NMF). We correlated DNA methylation signatures with various clinical and biological features, including age, survival, cancer stage, tumor immune microenvironment factors, and immunotherapy response. We inferred the determinant genes of these methylation signatures by integrating genomic and transcriptomic data and evaluated the impact of these signatures on cancer phenotypes in independent bulk and single-cell RNA/methylome cohorts. RESULTS: We identified 7,364 differentially methylated positions (2,969 Hyper-DMPs and 4,395 Hypo-DMPs) in nine cancer types from the TCGA. We subsequently retrieved three highly conserved, independent methylation signatures (Hyper-MS1, Hypo-MS1, and Hypo-MS4) from cancer tissues and cell lines based on these Hyper and Hypo-DMPs. Our data suggested that Hypo-MS4 activity predicts poor survival and is associated with immunotherapy response and distant tumor metastasis, and Hypo-MS4 activity is related to TP53 mutation and FOXA1 binding specificity. In addition, we demonstrated a correlation between the activities of Hypo-MS4 in cancer cells and the fractions of regulatory CD4 + T cells with the expression levels of immunological genes in the tumor immune microenvironment. CONCLUSIONS: Our findings demonstrated that the methylation signatures of distinct biological processes are associated with immune activity in the cancer microenvironment and predict immunotherapy response.

Epigenome-augmented eQTL-hotspots reveal genome-wide transcriptional programs in 36 human tissues.

Expression quantitative trait loci (eQTLs) are used to inform the mechanisms of transcriptional regulation in eukaryotic cells. However, the specificity of genome-wide eQTL identification is limited by stringent control for false discoveries. Here, we described a method based on the non-homogeneous Poisson process to identify 125 489 regions with highly frequent, multiple eQTL associations, or 'eQTL-hotspots', from the public database of 59 human tissues or cell types. We stratified the eQTL-hotspots into two classes with their distinct sequence and epigenomic characteristics. Based on these classifications, we developed a machine-learning model, E-SpotFinder, for augmented discovery of tissue- or cell-type-specific eQTL-hotspots. We applied this model to 36 tissues or cell types. Using augmented eQTL-hotspots, we recovered 655 402 eSNPs and reconstructed a comprehensive regulatory network of 2 725 380 cis-interactions among eQTL-hotspots. We further identified 52 012 modules representing transcriptional programs with unique functional backgrounds. In summary, our study provided a framework of epigenome-augmented eQTL analysis and thereby constructed comprehensive genome-wide networks of cis-regulations across diverse human tissues or cell types.

Simvastatin Preferentially Targets FLT3/ITD Acute Myeloid Leukemia by Inhibiting MEK/ERK and p38-MAPK Signaling Pathways.

BACKGROUND: The FLT3/ITD mutation exists in many acute myeloid leukemia (AML) patients and is related to the poor prognosis of patients. In this study, we attempted to evaluate the antitumor activity of simvastatin, a member of the statin class of drugs, in vitro and in vivo models of FLT3/ITD AML and to identify the potential mechanisms. METHODS: Cell Counting Kit-8 (CCK-8) and Annexin V/propidium iodide (PI) staining kits were used to detect cell viability and apoptosis, respectively. Subsequently, Western blot and rescue experiment were applied to explore the potential molecular mechanism. In vivo anti-leukemia activity of simvastatin was evaluated in xenograft mouse models. RESULTS: In vitro experiments revealed that simvastatin inhibited AML progression in a dose- and time-dependent manner, while in vivo experiments showed that simvastatin significantly reduced tumor burden in FLT3/ITD xenograft mouse models. After simvastatin treatment of FLT3/ITD AML cells, intracellular Rap1 was downregulated and the phosphorylation levels of its downstream targets MEK, ERK and p38 were significantly inhibited. The rescue experiment showed that mevalonate, an intermediate product of the metabolic pathway of mevalonate, and its downstream geranylgeranyl pyrophosphate (GGPP) played a key role in this process. Finally, we demonstrate that simvastatin can induce apoptosis of primary AML cells, while having no effect on peripheral blood mononuclear cells from normal donors. CONCLUSIONS: Simvastatin can selectively and effectively eradicate FLT3/ITD AML cells in vitro and in vivo, and its mechanism may be related to the disruption of the HMG-CoA reductase pathway and the downregulation of the MEK/ERK and p38-MAPK signaling pathways.

Differences in clinical characteristics and outcomes between patients with grade 3a and grades 1-2 follicular lymphoma: a real-world multicenter study.

BACKGROUND: The difference between clinical characteristics and outcomes between follicular lymphoma grade 1-2 (FL1-2) and FL3a defined pathologically remains unclear, resulting in uncertainty how to treat FL3a. However, it may be crucial for clinicians to discriminate grade 3a and grade 1-2 for predicting prognosis and thus making treatment decisions. METHODS: We compared 1403 patients with FL1-2 and 765 patients with FL3a diagnosed between January 2000 and December 2020 from fifteen centers nationwide in China to describe differences in clinical characteristics and outcomes. RESULTS: Compared with FL1-2 patients, FL3a subgroup had a higher percentage of elderly patients (P = 0.003), and relatively more FL3a patients presented with increased levels of LDH (P < 0.0001) and higher Ki-67 indexs greater than 30% (P < 0.001). More FL3a patients were treated with CHOP ± R (P < 0.0001), and fewer were treated with the watchful-waiting approach (P < 0.0001). The results showed a higher incidence of relapse among FL3a patients, in which more patients underwent histological transformation (HT) when compared to FL1-2 (P = 0.003). 1470 (76.2%) patients of the entire cohort received R-CHOP therapy; survival analysis revealed that FL3a patients had a worse progression-free survival (PFS) rate than FL1-2 patients. Survival of FL3a patients with respect to FLIPI showed an inferior PFS in the intermediate and high-risk groups than FL1-2 patients. FL3a patients had a much worse prognosis than FL1-2 with or without progression of disease within 24 months (POD24). FL3a patients had higher likelihood of lymphoma-related death (LRD, P < 0.05), whereas the rates for non-LRD were comparable. CONCLUSION: In conclusion, this study demonstrates a marked difference in clinical features and outcomes in FL3a patients compared with FL1-2 patients. The results highlight the need for applying therapeutic approaches distinct from FL1-2 when treating FL3a patients.

Stratification and therapeutic potential of ELL in cytogenetic normal acute myeloid leukemia.

Optimizing prognostic stratification of patients with cytogenetic normal acute myeloid leukemia (CN-AML), a highly heterogeneous subgroup in AML, appears to be important to improve its treatment and clinical outcome. Here, we report a potential role of ELL, a gene associated with leukemogenesis in AML, in prognostic stratification of CN-AML patients. By analyzing public available databases, we found that ELL was highly expressed in AML patients compared with healthy donors. Kaplan-Meier analysis revealed that ELL expression markedly correlated with short overall survival (OS) of CN-AML patients. In COX multivariable regression analysis, higher ELL expression was an independent prognostic factor for OS in CN-AML. Knockdown of ELL by shRNAs sensitized KG-1α cells to anti-leukemic agents such as idarubicin (IDA) and chidamide (CS055), supporting its role in therapeutic response and outcome in AML. To understand its function in CN-AML, we further analyzed the ELL-driving gene signature. ELL-related genes were particularly enriched in cell adhesion molecules, cell differentiation, pathways in cancer, sequence-specific DNA binding, and extracellular matrix (ECM)-receptor interaction. Analysis of the PPI network identified 25 hub genes, including the stem cell gene BMP4. While BMP4 expression was significantly associated with ELL in CN-AML, knockdown of ELL markedly down-regulated BMP4 expression, suggesting that ELL might function via regulating BMP4 in AML. Together, these observations suggest a novel mechanism underlying pro-leukemogenic role of ELL via BMP4 up-regulation in AML and its potential value to serve as a predictive biomarker for therapeutic response and outcome of CN-AML patients.

Anlotinib exerts potent antileukemic activities in Ph chromosome negative and positive B-cell acute lymphoblastic leukemia via perturbation of PI3K/AKT/mTOR pathway.

OBJECTIVES: Despite advances in the development of novel targeted therapies, the need for B-ALL alternative treatments has not been met. Anlotinib could blunt the proangiogenic activity of VEGFR, PDGFR, and FGFR, and has shown strong antitumor activities across multiple tumors. However, anlotinib cytotoxicity against B-ALL has not ever been evaluated, thus prompting us to initiate this study. METHODS: Expression2Kinases program was used to identify potential treatment targets. Cell viability and apoptosis were determined by CCK-8 and Annexin V/PI staining kit, respectively. qRT-PCR and Western blotting were utilized to investigate the molecular mechanisms. In vivo antileukemia activity of Anlotinib was evaluated in a Ph+ B-ALL patient-Derived Xenograft (PDX) model. RESULTS: Compared with treatment-naive B-ALL cases, RR B-ALL patients had higher activities in the VEGF/VEGFR signaling and the PI3K/AKT/mTOR pathway. Exposure of Ph- and Ph+ B-ALL cells to anlotinib resulted in significant cell viability reduction, apoptosis enhancement, and cell cycle arrest at G2/M phase. Importantly, anlotinib treatment led to remarkably decreased leukemia burdens and extended the survival period in a Ph+ B-ALL PDX model. Blockade of the role of the proangiogenic mediators, comprising VEGFR2, PDGFR-beta, and FGFR3, played a critical role in the cytotoxicity of anlotinib against Ph- and Ph+ B-ALL. Moreover, anlotinib dampened the activity of PI3K/AKT/mTOR pathway that resides in the convergence of the three mentioned proangiogenic signals. CONCLUSION: This work provides impressive preclinical evidence of anlotinib against Ph- and Ph+ B-ALL and raises a rationale for future clinical evaluation of this drug in the management of Ph- and Ph+ B-ALL.

The COVID-19 Vaccination and Vaccine Inequity Worldwide: An Empirical Study Based on Global Data.

Vaccination is critical for controlling the COVID-19 pandemic. However, the progress of COVID-19 vaccination varies from different countries, and global vaccine inequity has been a worldwide public health issue. This study collected data from the Our World in Data COVID-19 vaccination data set between 13 December 2020 and 1 January 2022. The measurement reflecting the pandemic situation included New cases, New deaths, Hospital patients, ICU patients, and the Reproduction rate. Indicators for measuring the vaccination coverage included Total vaccinations per hundred and People vaccinated per hundred. The Human Development Index (HDI) measured the country's development level. Findings indicated that countries with higher HDI have more adequate vaccine resources, and global vaccine inequity exists. The study also found that vaccination significantly mitigates the pandemic, and reaching 70% immunization coverage can further control the epidemic. In addition, the emergence of Omicron variants makes the COVID-19 epidemic situation even worse, suggesting the importance and necessity of addressing vaccine inequity. The globe will face a greater challenge in controlling the pandemic if lower-vaccinated countries do not increase their vaccination coverage. Addressing the issue of vaccine inequity needs the cooperation of HIC, LMIC, public health departments, and vaccine producers. Moreover, the media has to contribute to effective public health communication by raising public perceptions of the COVID-19 pandemic, vaccination, and vaccine inequity.

Therapeutic synergy of Triptolide and MDM2 inhibitor against acute myeloid leukemia through modulation of p53-dependent and -independent pathways.

Dysregulation of MDM2, a p53 negative regulator, frequently occurs in acute myeloid leukemia (AML) and is associated with unfavorable prognoses, rendering the p53-MDM2 axis an attractive target for the development of small-molecule inhibitors. MDM2 antagonists have been intensely developed but only lead to limited clinical activity, suggesting combination with additional drugs is an unmet medical need. In this study, we reported that Triptolide synergized with MDM2 inhibitor Nutlin-3a to suppress cell proliferation and induce mitochondrial-mediated apoptosis in p53 wt AML in vitro and ex vivo. More importantly, Triptolide cooperated with Nutlin-3a to delay tumor growth and abrogate leukemia burden in an AML xenograft model. In addition, we observed that Triptolide and Nutlin-3a were also cooperative in part of p53 deficient cases. Mechanistically, Nutlin-3a upregulated the transcriptional expressions of the p53 downstream targets PUMA and p21, while Triptolide declined the mRNA levels of two anti-apoptotic factors, XIAP and Mcl-1, in p53 wt cells. These effects were more notable when Triptolide and Nutlin-3a were combined. Our results revealed that Triptolide monotherapy exerted its antileukemia effect via both p53-dependent and independent ways, with the latter through perturbation of the MYC-ATF4 axis-mediated ER stress. Collectively, these data suggested that the Triptolide-Nutlin-3a combination might be a novel potential therapeutic intervention for patients with AML and it warrants further clinical evaluations.

Blood urea nitrogen to serum albumin ratio as a new prognostic indicator in critical patients with chronic heart failure.

AIMS: Chronic heart failure (CHF) is often a common comorbidity in critically ill patients admitted to the intensive care unit (ICU) and carries an extremely poor prognosis. The study aimed to investigate the relationship between the blood urea nitrogen to serum albumin ratio (BAR) and the prognosis of patients with CHF admitted to the ICU. METHODS AND RESULTS: This retrospective cohort study included 1545 critically ill patients with CHF as a diagnosed comorbidity admitted to the ICU deposited in the MIMIC-III database, of whom 90 day all-cause mortality was 27.6% (n = 427) and in-hospital mortality was 17.3% (n = 267). The results of multiple logistic regression analysis indicated that BAR is an independent risk factor for in-hospital mortality in critically ill patients with CHF [compared with BAR ≤ 0.83; 0.83 < BAR ≤ 1.24: odds ratio (OR) 2.647, 95% confidence interval (CI) 1.797-3.900, P < 0.001; BAR ≥ 1.24: OR 3.628, 95% CI 2.604-5.057, P < 0.001]. Multiple COX regression analysis found a relationship between BAR and all-cause mortality at 90 day follow-up (0.83 < BAR ≤ 1.24: OR 1.948, 95% CI 1.259-3.014, P < 0.003; BAR ≥ 1.24: OR 1.807, 95% CI 1.154-2.830, P < 0.01; BAR ≤ 0.83 as a reference). Kaplan-Meier curves also showed similar results as well (P < 0.001). The areas under the receiver operating characteristic curves for predicting in-hospital mortality and 90 day all-cause mortality were 0.622 and 0.647, respectively. CONCLUSIONS: BAR is an independent risk factor for in-hospital mortality and 90 day mortality in critically ill patients with CHF admitted to the ICU.

Themis is indispensable for IL-2 and IL-15 signaling in T cells.

To perform their antiviral and antitumor functions, T cells must integrate signals both from the T cell receptor (TCR), which instruct the cell to remain quiescent or become activated, and from cytokines that guide cellular proliferation and differentiation. In mature CD8+ T cells, Themis has been implicated in integrating TCR and cytokine signals. We investigated whether Themis plays a direct role in cytokine signaling in mature T cells. Themis was required for IL-2- and IL-15-driven CD8+ T cell proliferation both in mice and in vitro. Mechanistically, we found that Themis promoted the activation of the transcription factor Stat and mechanistic target of rapamycin signaling downstream of cytokine receptors. Metabolomics and stable isotope tracing analyses revealed that Themis deficiency reduced glycolysis and serine and nucleotide biosynthesis, demonstrating a receptor-proximal requirement for Themis in triggering the metabolic changes that enable T cell proliferation. The cellular, metabolic, and biochemical defects caused by Themis deficiency were corrected in mice lacking both Themis and the phosphatase Shp1, suggesting that Themis mediates IL-2 and IL-15 receptor-proximal signaling by restraining the activity of Shp1. Together, these results not only shed light on the mechanisms of cytokine signaling but also provide new clues on manipulating T cells for clinical applications.

Preclinical Evaluation of the HDAC Inhibitor Chidamide in Transformed Follicular Lymphoma.

The key factors leading to transformed follicular lymphoma (t-FL) include the aberrations of epigenetic modifiers as early and driving events, especially mutations in the gene encoding for histone acetyltransferase. Therefore, reversal of this phenomenon by histone deacetylase (HDAC) inhibitors is essential for the development of new treatment strategies in t-FL. Several t-FL cell lines were treated with various doses of chidamide and subjected to cell proliferation, apoptosis and cell cycle analyses with CCK-8 assay, Annexin V/PI assay and flow cytometry, respectively. Chidamide dose-dependently inhibited cell proliferation, caused G0/G1 cycle arrest and triggered apoptosis in t-FL cells. In addition, the effects of chidamide on tumor growth were evaluated in vivo in xenograft models. RNA-seq analysis revealed gene expression alterations involving the PI3K-AKT signaling pathway might account for the mechanism underlying the antitumor activity of chidamide as a single agent in t-FL. These findings provide a basis for further clinical exploration of chidamide as a promising treatment for FL.

Simvastatin potentiates the cell-killing activity of imatinib in imatinib-resistant chronic myeloid leukemia cells mainly through PI3K/AKT pathway attenuation and Myc downregulation.

Constitutively activated BCR-ABL kinase is considered the driver event responsible in the initiation and development of chronic myeloid leukemia (CML). The advent of the first BCR-ABL inhibitor imatinib has significantly improved the clinical outcome of CML cases. However, resistance to imatinib occurs in 25-30% of CML patients. Due to the lack of effective therapeutic strategies, novel treatment approaches are urgently required for imatinib-resistant CML. Simvastatin, a well-known HMG-CoA reductase inhibitor that confers tremendous clinical benefits in cardiovascular diseases, has attracted mounting attentions for its potent antitumor effects on multiple tumor types. In this study, we demonstrated that simvastatin monotherapy was effective in diminishing cell viability in both imatinib-sensitive and imatinib-resistant CML cells, including T351I mutated cells, with the latter being less vulnerable to the simvastatin than the former. Notably, we found that simvastatin acted as a robust cytotoxic sensitizer of imatinib to kill imatinib-resistant and T315I mutated CML cells in vitro and in vivo. Mechanistically, the cooperative interaction of simvastatin and imatinib was associated with the inactivation of the PI3K/Akt signaling pathway, which was a classical downstream pro-survival cascade of the BCR-ABL kinase. In addition, this drug combination obviously decreased Myc expression through attenuation of canonical Wnt/ß-catenin signaling and increased H3K27 trimethylation. Taken together, we provide attractive preclinical results for the combinatorial regimen of simvastatin and imatinib against imatinib-resistant and T315I mutated CML cells. This combined regimens warrants further clinical investigations in patients with imatinib-resistant CML.

Therapeutic Interaction of Apatinib and Chidamide in T-Cell Acute Lymphoblastic Leukemia through Interference with Mitochondria Associated Biogenesis and Intrinsic Apoptosis.

T-cell acute lymphoblastic leukemia (T-ALL) shows poor clinical outcome and has limited therapeutic options, indicating that new treatment approaches for this disease are urgently required. Our previous study demonstrated that apatinib, an orally selective VEGFR-2 antagonist, is highly effective in T-ALL. Additionally, chidamide, a histone deacetylase inhibitor, has proven to be cytotoxic against T-ALL in preclinical and clinical settings. However, whether the therapeutic interaction of apatinib and chidamide in T-ALL remains unknown. In this study, apatinib and chidamide acted additively to decrease cell viability and induce apoptosis in T-ALL in vitro. Notably, compared with apatinib or chidamide alone, the combinational regimen was more efficient in abrogating the leukemia burden in the spleen and bone marrow of T-ALL patient-derived xenograft (PDX) models. Mechanistically, the additive antileukemia effect of apatinib and chidamide was associated with suppression of mitochondrial respiration and downregulation of the abundance levels of several rate-limiting enzymes that are involved in the citric acid cycle and oxidative phosphorylation (OXPHOS). In addition, apatinib enhanced the antileukemia effect of chidamide on T-ALL via activation of the mitochondria-mediated apoptosis pathway and impediment of mitochondrial biogenesis. Taken together, the study provides a potential role for apatinib in combination with chidamide in the management of T-ALL and warrants further clinical evaluations of this combination in patients with T-ALL.

Genetic Determinants of Somatic Selection of Mutational Processes in 3,566 Human Cancers.

The somatic landscape of the cancer genome results from different mutational processes represented by distinct "mutational signatures." Although several mutagenic mechanisms are known to cause specific mutational signatures in cell lines, the variation of somatic mutational activities in patients, which is mostly attributed to somatic selection, is still poorly explained. Here, we introduce a quantitative trait, mutational propensity (MP), and describe an integrated method to infer genetic determinants of variations in the mutational processes in 3,566 cancers with specific underlying mechanisms. As a result, we report 2,314 candidate determinants with both significant germline and somatic effects on somatic selection of mutational processes, of which, 485 act via cancer gene expression and 1,427 act through the tumor-immune microenvironment. These data demonstrate that the genetic determinants of MPs provide complementary information to known cancer driver genes, clonal evolution, and clinical biomarkers. SIGNIFICANCE: The genetic determinants of the somatic mutational processes in cancer elucidate the biology underlying somatic selection and evolution of cancers and demonstrate complementary predictive power across cancer types.

MiR-3130-5p is an intermediate modulator of 2q33 and influences the invasiveness of lung adenocarcinoma by targeting NDUFS1.

Genome-wide association studies (GWAS) have reported a handful of loci associated with lung cancer risk, of which the pathogenic pathways are largely unknown. We performed cis-expression quantitative trait loci (eQTL) mapping for 376 lung cancer related GWAS loci in 227 TCGA lung adenocarcinoma (LUAD) and reported two risk loci as eQTL of miRNA. Among the miRNAs in association with lung cancer risk, we further predicted and validated miR-3130-5p as an intermediate modulator of risk loci 2q33 and the tumor suppressor NDUFS1. We assessed the phenotypic impacts of the interaction between miR-3130-5p and NDUFS1 in both lung cancer cell lines and mice xenograft models. As a result, miR-3130-5p directly regulates the expression of NDUFS1 and the corresponding tumor invasiveness, migration and epithelial-mesenchymal transition (EMT). Our findings provide important clues for the pathogenic mechanism of 2q33 in lung carcinogenesis which informs clinical diagnosis and prognosis of LUAD. We performed a cis-eQTL analysis for 376 lung cancer risk loci based on the expression profiles of 251 miRNAs in a cohort of 227 TCGA lung adenocarcinoma. We report a novel pathogenic pathway of 2q33 via miR-3130-5p and NDUFS1.

Anlotinib suppresses MLL-rearranged acute myeloid leukemia cell growth by inhibiting SETD1A/AKT-mediated DNA damage response.

Leukemias driven by chromosomal translocation of the mixed-lineage leukemia (MLL) gene are highly prevalent in hematological malignancy. The poor survival rate and lack of effective targeted therapy for patients with MLL-rearranged (MLL-r) leukemias emphasize an urgent need for improved knowledge and novel therapeutic approaches for these malignancies. The present study aimed to investigate the potential effectiveness and mechanism of Anlotinib, a novel receptor tyrosine kinase inhibitor, in MLL-r acute myeloid leukemia (AML). The findings revealed that Anlotinib significantly inhibited the growth of MLL-r AML cells in both in vivo and a murine xenograft model. RNA sequencing identified that multiple genes involved in DNA damage response were responsible for Anlotinib activity. To further elucidate the correlation between the DNA damage response induced by Anlotinib and MLL fusion, Gene Expression Profiling Interactive Analysis (GEPIA) was conducted. It revealed that Anlotinib impaired DNA damage response via inhibiting SETD1A and AKT. In conclusion, Anlotinib exerts anti-leukemia function by inhibiting SETD1A/AKT-mediated DNA damage response and highlights a novel mechanism underlying Anlotinib in the treatment of MLL-r AML.

Disruption of CTCF Boundary at HOXA Locus Promote BET Inhibitors' Therapeutic Sensitivity in Acute Myeloid Leukemia.

Both HOX gene expression and CTCF regulation have been well demonstrated to play a critical role in regulating maintenance of leukemic stem cells (LSCs) that are known to be resistant to BET inhibitor (BETi). To investigate the regulatory role of CTCF boundary in aberrant HOX gene expression and the therapeutic sensitivity of BETi in AML, we employed CRISPR-Cas9 genome editing technology to delete 47 base pairs of the CTCF binding motif which is located between HOXA7 and HOXA9 genes (CBS7/9) in different subtypes of AML with either MLL-rearrangement or NPM1 mutation. Our results revealed that HOXA9 is significantly downregulated in response to the CBS7/9 deletion. Moreover, CBS7/9 boundary deletion sensitized the BETi treatment reaction in both MOLM-13 and OCI-AML3 cells. To further examine whether BETi therapeutic sensitivity in AML is depended on the expression level of the HOXA9 gene, we overexpressed the HOXA9 in the CBS7/9 deleted AML cell lines, which can rescue and restore the resistance to BETi treatment of the CBS7/9 KO cells by activating MAPK signaling pathway. Deletion of CBS7/9 specifically decreased the recruitment of BRD4 and RNA pol II to the posterior HOXA genes, in which, a transcription elongation factor ELL3 is the key factor in regulating HOXA gene transcription monitored by CBS7/9 chromatin boundary. Thus, disruption of CBS7/9 boundary perturbs HOXA9 transcription and regulates BETi sensitivity in AML treatment. Moreover, alteration of CTCF boundaries in the oncogene loci may provide a novel strategy to overcome the drug resistance of LSCs. Graphical abstract.

Low-frequency TP53 hotspot mutation contributes to chemoresistance through clonal expansion in acute myeloid leukemia.

TP53 mutations (TP53mut) in AML patients associate with poor prognosis that may affect therapy and outcome. In addition to TP53 mut patients, TCGA AML patient sequencing data show that there are around 3% of patients have detectable low-frequency TP53mut reads. Importantly, these patients showed worse outcome as compared with the TP53 wild type (TP53wt) patients. We have studied the effect of low-frequency TP53mut in two AML cell lines, OCI-AML2 and MV4-11. Both cells have low-frequency single hotspot TP53mut. Interestingly, the resistant cells derived from both lines have homogeneous TP53mut. TP53mut clones isolated from the parental cells also show increased chemoresistance potential and have higher population of leukemia stem cell (LSC) maker positive cells, a characteristic of chemoresistant cells. When mixed with TP53wt cells, the TP53mut cells show survival advantage suggesting its potential to develop chemoresistance. We previously showed that histone deacetylase inhibitor Romidepsin can re-sensitize chemoresistant cells by eradicating LSC marker positive cells. Here we further show that Romidepsin can reactivate p53 targeted genes which are dysregulated in TP53mut cells and preferentially targets TP53mut subpopulation. Therefore, our study shows that low-frequency TP53mut is linked to chemoresistance and sheds light on therapeutic strategies for treatments on chemoresistance.

Co-inhibition of HDAC and MLL-menin interaction targets MLL-rearranged acute myeloid leukemia cells via disruption of DNA damage checkpoint and DNA repair.

While the aberrant translocation of the mixed-lineage leukemia (MLL) gene drives pathogenesis of acute myeloid leukemia (AML), it represents an independent predictor for poor prognosis of adult AML patients. Thus, small molecule inhibitors targeting menin-MLL fusion protein interaction have been emerging for the treatment of MLL-rearranged AML. As both inhibitors of histone deacetylase (HDAC) and menin-MLL interaction target the transcription-regulatory machinery involving epigenetic regulation of chromatin remodeling that governs the expression of genes involved in tumorigenesis, we hypothesized that these two classes of agents might interact to kill MLL-rearranged (MLL-r) AML cells. Here, we report that the combination treatment with subtoxic doses of the HDAC inhibitor chidamide and the menin-MLL interaction inhibitor MI-3 displayed a highly synergistic anti-tumor activity against human MLL-r AML cells in vitro and in vivo, but not those without this genetic aberration. Mechanistically, co-exposure to chidamide and MI-3 led to robust apoptosis in MLL-r AML cells, in association with loss of mitochondrial membrane potential and a sharp increase in ROS generation. Combined treatment also disrupted DNA damage checkpoint at the level of CHK1 and CHK2 kinases, rather than their upstream kinases (ATR and ATM), as well as DNA repair likely via homologous recombination (HR), but not non-homologous end joining (NHEJ). Genome-wide RNAseq revealed gene expression alterations involving several potential signaling pathways (e.g., cell cycle, DNA repair, MAPK, NF-κB) that might account for or contribute to the mechanisms of action underlying anti-leukemia activity of chidamide and MI-3 as a single agent and particularly in combination in MLL-r AML. Collectively, these findings provide a preclinical basis for further clinical investigation of this novel targeted strategy combining HDAC and Menin-MLL interaction inhibitors to improve therapeutic outcomes in a subset of patients with poor-prognostic MLL-r leukemia.

CS2164 suppresses acute myeloid leukemia cell growth via inhibiting VEGFR2 signaling in preclinical models.

Acute myeloid leukemia (AML) arises from neoplastic transformation of hematopoietic stem and progenitor cells, and resistance to conventional chemotherapy remains one of the greatest challenges in treating the disease. Extensive data have demonstrated that angiogenesis is associated with AML progression and chemotherapy resistance. Thus, targeting angiogenesis may be a promising approach for AML treatment. In this study, we investigated the effectiveness of CS2164 (named as Chiauranib), a novel receptor tyrosine kinase inhibitor, in AML cells. Our results illustrated that CS2164 significantly suppressed cell proliferation and abolished clonogenicity in AML cells in a dose- and time-dependent manner. Meanwhile, CS2164 markedly induced apoptosis of AML cell lines and primary AML cells from 42 adult patients. Furthermore, we found that CS2164 has a comprehensive activity against AML irrespective of disease status and genetic mutations. Also, CS2164 suppressed AML growth in xenograft models in vivo. Mechanistically, CS2164-induced cytotoxicity was closely associated with inhibition of VEGFR2 and its downstream signaling cascades, including Src/Fyn/p38 and Erk/MEK. In conclusion, our study indicates that CS2164 exerts anti-leukemia effect by inducing apoptosis through suppressing the VEGFR2 pathway, supporting a potential role for CS2164 in the treatment of AML.