bluuebunny/crossref_metadata_2025 huggingface dataset:
Histone Methyltranferase SETD8 Stimulates Proliferation and Anti-Apoptotic Effect in Acute Myeloid Leukemia through Epigenetically Upregulation of CXCR4
Authors: Cui Zelong , Xu Man , Gao Zhenxing , Zhou Minran , Fu Yue , Feng Huimin , Zhang Lu , Chen Chunyan
Published: December 2023

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.
Potential Significance of CTPS1 in Multiple Myeloma
Authors: Huang Han-Ying , Liang Yang
Published: December 2023

Introduction

With the increasing interest to the metabolism of multiple myeloma (MM), a number of metabolic genes and their associated regulatory networks are essential for MM progression. Our team's prior work indicated that high expression of metabolism-related gene CTPS1 (CTP synthase 1) correlated with poor prognosis of MM. However, the mechanism of CTPS1 in MM is unknown.

Methods

The difference in CTPS1 expression level in patients with plasma cell malignancies, various stages, and drug-resistant recurrence was examined using publicly available data. MM cell lines were engineered to overexpress or knockdown CTPS1. RNA-seq was used to unbiased interrogate the downstream effector genes modulated by CTPS1. Severe immunodeficient NRG-SGM mice expressing humanized myeloid cytokines were used to determine the effect of CTPS1 on MM proliferation in vivo.

Results

CTPS1 expression levels varied substantially in various plasma cell malignancies. High CTPS1 expression was associated with shorter overall survival and progression-free survival in newly diagnosed MM patients. Moreover, there was a strong correlation between CTPS1 and sex, albumin, β2 microglobulin, lactate dehydrogenase, and ISS/R-ISS stage. In vitro, CTPS1 overexpressing cells proliferated more quickly than CTPS1 knockdown cells. NRG-SGM3 mouse experiments demonstrated that tumor growth was significantly accelerated in vivo in the CTPS1 overexpression group. In the pharmacological experiment, CTPS1 overexpression group's sensitivity to 20 nM bortezomib reduced, but CTPS1 down-knocked group's sensitivity to 20 nM bortezomib rose. CTPS1 was also shown to be involved in metabolism and synthesis, notably mRNA metabolism, DNA metabolism, chromosomal combination, cell cycle, spliceosome, and cellular senescence. Furthermore, Myc was found to be tightly connected to CTPS1 in both MM cell lines and newly diagnosed MM.

Conclusions

This study found that increased CTPS1 expression is associated with a poor prognosis and reduced sensitivity to bortezomib in MM. Additionally, CTPS1 upregulated Myc-signaling genes, which promoted progression of MM.
NLRP3/Caspase 1/Gsdmd Mediated Pyroptosis Exerts a Crucial Role in Blood-Brain Barrier Pathological Injury Induced By Cryptococcus Neoformans
Authors: Zhang Zile , Zou Jinhu , Luo Zifeng , Luo Jiamin , Cao Hong
Published: December 2023

Background:

Cryptococcus neoformans (Cn) traversal of the blood-brain barrier (BBB), composed of human brain microvascular endothelial cells (HBMECs), is a critical process in central nervous system infection. Previous studies have achieved significant progress in pathogenic mechanisms how Cn penetrates BBB. However, it is still largely elusive about the cellular and molecular mechanism of the abrasive interaction between Cn and HBMECs. Here, we focus on the pathogenesis of direct damage to the HBMECs by Cn.

Methods:

The immortalized HBMECs were incubated with Cryptococcus neoformans strain B-4500FO2 in different final concentrations (1×10^6/mL, 1×10^7/mL) for 3 hours at 37℃. The control group was treated with isovolumic phosphate buffered saline. Then, total soluble proteins of HBMECs in each group were extracted with RIPA lysis buffer for immunoblotting analysis. ImageJ and SPSS software were used for grayscale and statistical analysis respectively.

Results:

We found that the expression levels of pyroptosis-related protein, including NLRP3, Caspase 1, N-terminal of GSDMD especially, were significantly upregulated in HBMECs treated with higher concentrations (1×10^7/mL) of Cn compared to control group ( P < 0.05), in spite of the fact that there was no statistically significant difference in the expression of GSDMD ( P > 0.05).

Conclusion:

These data support that Cn might impair BBB through facilitating pyroptosis of HBMECs via NLRP3/Caspase 1/GSDMD signaling. More intensive investigations are still required to elucidate the underlying mechanism how pyroptosis is triggered and transmitted in the interaction between Cn and HBMECs.

( Acknowledgements: Undergraduate Training Program for Innovation and Entrepreneurship of SMU, China, No. 202312121330; Corresponding author: Hong Cao, gzhcao@smu.edu.cn )
Efficacy and Safety of Olverembatinib-Based Therapies in Patients with Ph/BCR-ABL1-Positive Acute Lymphoblastic Leukemia
Authors: Zhao Yanhong , Zhai Weihua , Pang Aiming , Yang Donglin , He Yi , Wei Jialin , MA Qiaoling , Chen Xin , Han Mingzhe , Feng Sizhou , Zhang Rongli , Jiang Erlie
Published: December 2023

Background Olverembatinib is an oral, third-generation BCR-ABL1 tyrosine kinase inhibitor (TKI) developed in China that could effectively target wild-type and mutant BCR-ABL1 kinase. It is effective and well-tolerated for patients with chronic myeloid leukemia. However, its efficacy and safety in patients with Ph/BCR-ABL1-Positive acute lymphoblastic leukemia are not entirely clear. This study aims to analyze the efficacy and safety of Olverembatinib-based therapies in patients with Ph/BCR-ABL1-Positive acute lymphoblastic leukemia.

Methods Patients with Ph/BCR-ABL1-positive acute lymphoblastic leukemia who received the Olverembatinib-based therapies in our center were included to conduct this retrospective study.

Results Totally 33 patients with Ph/BCR-ABL1-positive acute lymphoblastic leukemia were included in this study with a median age of 41 years, of whom 21 (63.63%) were female. Among them, 19 subjects who had been previously treated with standard chemotherapy and three newly diagnosed patients received Olverembatinib-based therapies, while 11 patients received Olverembatinib-based therapies after hematopoietic stem cell transplantation. After the first course of the Olverembatinib-based regimen, 33 (100%) patients experienced remission (CR/CRi/ Morphologic leukemia-free state), and 22 (66.67%) achieved the negative minimal residual disease (MRD) of BCR-ABL1/ ABL. Followed by the first course of the Olverembatinib-based regimen, five patients with negative MRD were treated with HSCT successfully and 27 received the second course of Olverembatinib-based therapy, of whom 21 (77.78%) acquired negative MRD. For six patients with negative MRD of BCR-ABL1/ ABL1, Olverembatinib-based therapies could remain the negative MRD. These patients with negative MRD will receive allogeneic hematopoietic stem cell transplantation after Olverembatinib-based therapy. Olverembatinib-based therapies were generally well tolerated. Common hematologic adverse events included leukopenia, neutropenia, anemia, and thrombocytopenia. Common nonhematologic adverse events included hepatotoxicity, nephrotoxicity, myalgia, arthralgia, rash, and edema, of which most were mild.

Conclusion Olverembatinib-based therapies are generally well tolerated and efficacious for patients with Ph/BCR-ABL1-positive acute lymphoblastic leukemia.
Immune Checkpoint Blockade Promotes Thrombosis Via T-Cell and Neutrophil Activation, and Tumor-Cell Associated Tissue Factor (TF) in a Murine Model of Colorectal Cancer
Authors: Shim Young Jun , Sharma Bal Krishan , Hisada Yohei , Mackman Nigel , Palumbo Joseph S , Diaz-Montero Claudia , Khorana Alok A. , McCrae Keith R.
Published: December 2023

Background:

Immune checkpoint blockade (ICB) is a paradigm shift in cancer therapy. Despite clinical benefit, various adverse events are associated with ICB using immune checkpoint inhibitors (ICI). Venous thrombosis is an emerging clinical problem in cancer patients receiving ICI. Little is known about how ICI promotes thrombosis in cancer. To define the underlying mechanisms of ICI-associated thrombosis (IAT), we assessed the ICI impact on blood hypercoagulability and tumor TF expression using a mouse thrombosis model of colorectal cancer.

Methods:

We developed a CT26 mouse thrombosis model with ICI (aPD-1+aCTLA4) administration. Systemic hypercoagulability was assessed by measurement of circulating nucleosomes (Nu.Q H3.1 Immunoassay), neutrophil extracellular traps (NETs) (CitH3 immunofluorescence), circulating neutrophil-platelet aggregates (immunophenotyping), and levels of thrombin-antithrombin (TAT) complexes in ICI-treated mice. Tumor-associated TF and TF activity in circulating extracellular vesicles (EV) were assessed by western blotting and Factor Xa generation assays. To gain mechanistic insights into TF induction by ICI, tumor cytokines were also profiled (proteome profiler array). The role of cancer cell-derived TF in IAT was determined using mice implanted with CRISPR/Cas9-mediated TF knockout (TFKO) CT26 tumor cells.

Results:

ICI treated tumor-bearing mice developed larger thrombi than control mice and demonstrated elevated levels of circulating nucleosomes (114 vs 82 ng/ml), NET (15.8 vs 6.8%), platelet-neutrophil aggregates (44.8 vs 22.3%), and TAT complexes (12.6 vs 7.9 ng/ml) compared to IgG-treated mice. Tumor TF expression was increased in tumor extracts. Cytokine profiling suggested enhanced production of IFNɣ (2-fold) and TNFα (5-fold), as well as CXCL11 (6.8-fold), an effector T cell-recruiting chemokine. IFNɣ-increased TF expression in cultured CT26 cells, suggesting that T-cell derived IFNɣ may play a role in enhancing tumor TF expression in vivo. Circulating EV TF antigen and procoagulant activity were increased by ICI. CRISPR/Cas9-mediated TF KO in CT26 cells resulted in complete loss of TF expression, and mice bearing these cells showed reduced tumor and EV-associated TF (Fig. A), associated with a significant decrease in thrombus formation after ICI treatment (24.2 vs 20.5 mg, Fig. B). There was no significant difference in growth of wild-type and TF KO CT26 tumors.

Conclusions:

Our findings provide insights into cellular and molecular mechanisms associated with ICI-induced thrombosis, and suggest a role for neutrophil and granulocyte activation, and tumor-associated TF. Additional studies are ongoing to further define these pathways in the murine model and in patients.
Thrombosis Caused By a Novel Coagulation Factor IX Mutation (FIX Shanghai II)
Authors: WU Wenman , Wang Rui , Xu Qin , Wang Guanzheng , Jiang Shifeng , Lou Can , Wu Xi , Shao Yanyan , Lu Yeling , Liu Yu , Xu Guanquan , Ding Qiulan , Hu Xiaobo , Wang Xuefeng , Dai Jing
Published: December 2023

Most thrombophilia patients are caused by insufficient suppression of coagulation reactions, which could be either mediated by deficiencies of protein C, protein S,antithrombin, or inhibition-resistant coagulation factor mutants, such as Factor V Leiden and prothrombin Yukuhashi. The thrombosis caused by coagulation factor mutants with increased procoagulant activity is very rare and so far only two Factor IX (FIX) variants involving residue Arg384, FIX Padua (p.Arg384Leu) and Shanghai (p.Arg384Gln), which increased FIX activity by up to 8 folds, had been reported to be associated with the incidence of thrombosis.

In a young male venous thrombosis patient we had identified a novel F9 gene missense mutation c.1018G>A, resulting in p. Glu340Lys substitution (designated as FIX Shanghai II). The patient's APTT time was 28.9 seconds (normal reference 22.3~38.7s), and his PT was also within the normal reference range (11.5 seconds, normal reference 10.0~16.0s). Although the patient had the FIX: Ag comparable to the normal reference, 128.20±6.07%, the his FIX:C was around 201.69±10.38% of normal control (normal reference 50~150%). Thrombin generation assay (TGA) showed that compared with normal control, the patient had a shorter Lag time (2.67 vs 3.33 min), elevated ETP (1649.43 vs 1311.62 nM*min), and higher Peak value (387.34 vs 200.86 nM), indicating increased global coagulation activity of the patient. The activity of protein C, protein S, and antithrombin were all within the normal reference ranges and the patient was negative for antiphospholipid antibodies.

Consistent with the patient's coagulation laboratory manifestations, the purified recombinant FIX Glu340Lys mutant showed 1.82 folds higher procoagulant activity than wild type FIX as determined by FIX:C/FIX:Ag ratio. The recombinant FIX Shanghai II mutant can be converted into FIXa by activated factor XI (FXIa) in a similar fashion as FIX WT. The kinetic study of FIXa-catalyzed FX activation showed that p. Glu340Lys mutation led to accelerated FX activation by FIXa.

The p.Glu340Lys mutation also enhanced the procoagulant activity of previously reported thrombosis-causing FIX mutants, FIX Shanghai and Padua, in a similar fashion as it did on FIX WT. The FIX:C/FIX:Ag ratio of double mutants containing Glu340Lys and Arg384Gln (FIX Shanghai) or Arg384Leu (FIX Padua) was about twice of FIX Shanghai or Padua alone, 11.48- and 22.53-folds vs 6.16- and 8.13- folds higher than FIX WT respectively.
Chromatin Remodeler DEK Is a Sensor to Replication Stress and Contributes to HSC Defects in Fanconi Anemia
Authors: Chen Zhe , Wu Feng , Li Lei , Li Yan , Zhao Xueya , Hou Yu
Published: December 2023

Hematopoietic stem cells (HSCs) have self-renewal capacity and response for life-long hematopoiesis. Any interference with normal DNA replication leads to replication stress, which drives function decline in HSCs. However, the response of HSCs to replication stress remains largely unknown. Here, draw a transcriptional and epigenetic landscape of HSCs under replication stress. We found that HSCs required opened chromatin to counteract replication stress, along with altered H3K27 acetylation and H3K4 trimethylation. The increased chromatin accessibility was mainly attributed to the reduced chromatin remodelers DEK, while DEK overexpressed HSCs confronted with a strong replication challenge, shown by increased γ-H2AX. Excessive DEK protein impeded proliferation and impaired HSC maintenance (>50% reduction), along with reduced oxidative phosphorylation and protein synthesis. Notably, we found that DEK protein was abnormally accumulated in BM CD34 + cells in Fanconi anemia, which arises from mutations in FA genes essential for replication stress tolerance. Inhibition of DEK substantially recovered HSC proliferation capacity in vitro and engraftment in vivo. At the molecular level, we identified that ATF2 directly promoted the transcription of DEK, especially relying on phosphorylated ATF2 (ser69/71) rather than phosphorylated ATF2 (ser490/498). Rad3-related (ATR) induced the phosphorylation of ATF2 at ser490/498 and mediated the reduction of DEK under replication stress. Nonetheless, knock-down of FA genes (FANCD2) in HSCs resulted in increased phosphorylated ATF2 (ser69/71), which might contribute to DEK accumulation in FA. Collectively, our findings uncover the landscape of HSC response to replication stress, reveal a novel ATR-ATF2-DEK axis helping HSCs counteract replication stress, and suggest inhibition of DEK as a therapeutic utility in FA.
The RNA Helicase DHX16 Is Required for the Maintenance of Hematopoietic Stem Cells
Authors: Li Zhigang , Fan Jiankun , Zhou Yuanze , Hou Yu
Published: December 2023

Lifelong hematopoiesis relies on the homeostasis of hematopoietic stem cells(HSCs), but the underlying mechanisms remain incompletely understood. Here, we identified a critical role for DHX16, a member of DEAH-box RNA helicase, in the maintenance of HSCs. After inducing deletion of Dhx16 gene in mice, the knockout animals developed pancytopenia within 5-7 days and died within two weeks. Flow cytometric analyses demonstrated that Dhx16-deficient mice had significantly reduced hematopoietic stem and progenitor cells, altered cell cycle dynamics with decreased G0 phase and increased G2/M phase, and increased apoptosis of HSCs. Competitive BM transplantation assays convinced the impaired self-renewal ability of Dhx16 knockout mice. To identify Dhx16-dependent genes and molecular pathways involved in the regulation of HSC function, we performed the RNA-Seq and long-read Iso-Seq analysis to compare the transcriptome between the Dhx16-deficient HSCs and Dhx16 fl/fl HSCs. RNA-Seq results showed that signaling pathways in Dhx16-deficient hematopoietic stem cells, including ribosome and spliceosome assembly, G2/M checkpoint, and p53 pathway were significantly affected. While long-read Iso-Seq revealed 210 genes with splicing alterations. Through further study, we confirmed that Dhx16-deficiency led to the retention of intron 4 in Emg1, a gene essential for ribosome biogenesis, resulting in a premature termination and nonsense-mediated mRNA decay. Therewith, decreased Emg1 expression in HSCs impaired ribosome biogenesis, activated the p53 pathway, and resulted in the rapid exhaustion of HSCs. In conclusion, these findings reveal a critical role for DHX16 in HSC survival and function and uncover an unsuspected link between RNA helicases, alternative splicing, ribosome biogenesis, and HSC homeostasis.
Analysis of Methylation Level and Clinical Characteristics of Juvenile Myelomonocytic Leukemia
Authors: Yang Wenyu , Chen Yunlong , Zhang Jingliao , Zhu Xiaofan
Published: December 2023

Objective: juvenile myelomonocytic leukemia (Juvenile myelomonocytic leukemia JMML) is a rare clonal disease of hematopoietic stem cells in young children. The response to chemotherapy is poor, the prognosis is poor, and a few patients can be relieved spontaneously. The level of methylation is closely related to the prognosis of JMML. In this study, the distribution characteristics and clinical indexes of JMML methylation were analyzed retrospectively. Methods: the newly diagnosed JMML from 2008.1 to 2022.11 in our center was selected as the research object. The samples were detected for DNA methylation and the clinical characteristics were analyzed. Results: methylation level was detected in 35 of 96 children with newly diagnosed JMML. The median age of onset was 27 months (3-82 months). There were 27 males and 8 females. The median platelet count was 70.6 × 109 × 10 ~ 9 × 10 ~ 9 × 10 ~ 9 / L, and the median absolute value of monocytes was 5.0 × 10 ~ 9 × 10 ~ 9 × 10 ~ 9 × 10 ~ 9 ml. All patients underwent JMML mutation and oral mucosal verification, including PTPN11 somatic mutation in 18 cases (51.4%), NRAS mutation in 10 cases (28.6%), KRAS mutation in 4 cases (11.4%), NF1 mutation in 7 cases (20%) and CBL mutation in 1 case (2.9%). Among them, 9 cases (25.7%) were compound mutations and 5 cases were recurrent mutations of PTPN11 and NF1. The DNA methylation level was divided into three subgroups: 4 cases (11.4%) hypomethylation (LM) group and 14 cases (40%) methylation group (IM) 17 cases (48.6%) hypermethylation (HM) group. Among the 17 patients in HM group, there were 7 cases of PTPN11 single mutation (41.2%) and 9 cases of compound mutation (52.9%), which were significantly higher than 3 cases of PTPN11 in IM group (21.4%). In IM group, simple NRAS or KRAS mutations accounted for 50%. The mutation distribution in LM group was 1 case of KRAS mutation, 2 cases of RANSBP2-ALK fusion gene and 1 case of no mutation. There were significant differences in age, platelet, fetal hemoglobin (HbF), PTPN11 mutation and ≥ 2 somatic mutation among different methylation subgroups (P < 0.05). The correlation analysis showed that methylation was positively correlated with 24 months of age, ≥ 33 × 10 9 mmol L, HbF, PTPN11 mutation and ≥ 2 somatic mutation (P < 0.05). Conclusion: high risk factors such as age at first diagnosis, PTPN11 mutation and compound mutation are significantly correlated with methylation in patients.
The Oncoprotein SKI As a Driver in KMT2A-MLLT3/NRasG12D-Positive Acute Myeloid Leukemia
Authors: Frech Miriam , Schaprian Janine , Ems Miriam , Teichler Sabine , Bauer Uta-Maria , Neubauer Andreas
Published: December 2023

KMT2A-rearranged acute myeloid leukemias (AML) have an intermediate to adverse prognosis. It was shown in a mouse model that the oncogene Myb contributes to AML maintenance in the KMT2A-MLLT3/NRasG12D genetic background (Zuber et al., 2011). Interestingly, the Myb-regulated genetic network mostly overlaps with KMT2A-MLLT3 target genes, especially with leukemic stem cell-associated gene sets. However, canonical KMT2A target genes are not included. Hence, MYB seems not to act as a co-factor for KMT2A complexes. SKI is a nuclear co-regulator and an oncoprotein, which is overexpressed in AML and several solid tumors (Bonnon & Atanasoski, 2012). Overexpression (OE) of SKI is prognostic in AML and correlates with shorter overall survival. Our group showed that SKI OE in AML is partially regulated by MYB (Frech et al., 2018). As a MYB target gene, we wanted to know if SKI can mediate MYB oncogenic activity in KMT2A-MLLT3/NRasG12D-positive AML.

We adopted the Zuber mouse model and performed an shRNA-mediated Ski knockdown (KD) in murine KMT2A-MLLT3/NRasG12D-positive AML cells (RN2). Mice were transplanted with RN2 cells carrying two different anti-Ski shRNAs or an anti-Renilla shRNA as control. The expansion of RN2 cells in mice was monitored by luciferase-based bioimaging. Upon disease onset, shRNA expression was induced by doxycycline treatment. The course of the disease was further monitored by bioimaging. Bone marrow cells were isolated and analyzed via flow cytometry. The expression of KMT2A target genes was analyzed by RT-qPCR in doxycycline-induced RN2 cells.

Ski KD induced a decreased viability of the RN2 cells in vitro. Moreover, SKI KD led to the eradication of RN2 cells in the mice in vivo and was associated with longer overall survival (log-rank p<0.0005). In line with the SKI OE mouse model of Singbrant et al. (2014), Ski KD led vice versa in our model to a decrease of myeloid cells and an increase of B cells. In contrast to Myb KD in the RN2 cells, Ski KD led to a lower expression of canonical KMT2A target genes, implicating that Ski may be part of KMT2A complexes in AML.

In summary, SKI seems to be crucial for KMT2A-MLLT3/NRasG12D-driven AML and may be a potential target for new therapeutic strategies.
Clinical Implication of Minimal Residual Disease By Multiparameter Flow Cytometry in Light Chain Amyloidosis
Authors: Xu Tianhong , Li Jing , Yang Yang , Wang Wenjing , Wang Pu , Yu Chenqi , Liu Peng
Published: December 2023

We evaluated bone marrow minimal residual disease (MRD) by multiparameter flow cytometry (sensitivity 10 -5) in 91 patients with light chain amyloidosis (AL). The overall MRD negative rate was 42.9% (n=39). The MRD negative rate among patients in complete response, very good partial response, and less than PR was 61.5% (24/39), 35.7% (10/28), and 0% (0/11), respectively. There were no significant differences in baseline characteristics regarding age, light chain burden, and organ involvement, while lower proportion of male sex and cytogenetics t(11;14) by FISH was observed in MRD negative patients. Patients with MRD negativity had a numerically higher proportion of cardiac response (54.8% vs 40.5%, P=0.224) and renal response (52.6% vs 41.7%, P=0.474). After a median follow-up time of 29.0 months, patients with MRD negativity had a near significantly longer event-free survival (EFS, defined as hematological progression/organ dysfunction/initiation of next-line therapy and death) from treatment initiation than patients with MRD positivity (NR vs 38.8 months, P=0.078). The 5-year overall survival (OS) for patients with MRD negativity and MRD positivity were 82.7% and 67.8%, respectively (P=0.229).
Clinical Significance of Serum High-Density Lipoprotein Cholesterol and Its Dynamic Change in Patients with Lymphoma-Associated Hemophagocytic Lymphohistiocytosis
Authors: Shi Wenyu , Miao Yi , Zhang Jing , Li Jianyong
Published: December 2023

Background: The lipid profile in serum is frequently dysregulated in lymphoma-associated hemophagocytic lymphohistiocytosis (HLH). We aimed to explore the clinical significance of serum lipoprotein cholesterol in lymphoma-associated HLH.

Methods: Patients with lymphoma-associated HLH were included. Serum levels of baseline low-density lipoprotein cholesterol (LDL-c) and high-density lipoprotein cholesterol (HDL-c) were collected. Serum levels of LDL-c and HDL-c after treatment (first test after treatment, time interval ≥ 14 days) were also collected.

Results: A total of 169 lymphoma-associated HLH were included. T/NK-cell lymphomas (n=88, 52.1%) account for over half of cases. Low HDL-c (< 1.03 mmol/L) was observed in 161 (95.3%) patients and low LDL-c (< 2.6 mmol/L) was presented in 145 (85.8%) patients. The best cutoff of 0.50 mmol/L for HDL-c was determined to predict the 60-day survival. Patients with HDL-c ≥ 0.50 mmol/L showed a significantly higher 60-day survival rate than patients with HDL-c <0.50 mmol/L (70.9% vs 42.2%, p < 0.001.). Patients with HDL-c ≥ 0.50 mmol/L also showed longer overall survival (OS) (median OS: 346 days vs 34 days, p < 0.001). LDL-c showed no significant impacts on 60-day survival and OS. Dynamic data of HDL-c were available in 69.2% (117) of the patients. The median interval between the two tests was 18 (IQR: 15-25) days. The level of HDL-c after treatment increased in 80.3% (94/117) of the patients. The ratio of post-treatment HDL-c/baseline HDL-c was calculated and the median number was 1.67 (IQR: 1.05-2.48). The ratio was significantly lower in the patients with T/NK-cell malignancies than those with B-cell lymphomas (median 1.40 versus 1.97, p < 0.001). A ratio of 0.78 was determined as the best cutoff to predict the 60-day survival. Patients with a ratio<0.78 had significantly reduced 60-day survival (42.4% vs 76.6%, P=0.0035, Figure 1A) and OS (median 36 days vs 407 days, P=0.0001, Figure 1B). Then we divided these patients with available data into two groups based on baseline HDL-c and the ratio of post-treatment HDL-c/baseline HDL-c: Group 1 included patients with baseline HDL-c ≥ 0.5 mmol/L and Ratio ≥ 0.78, while others were classified as Group 2. Patients in Group 2 had significantly reduced 60-day survival (57.5% vs 87.6%, P=0.0003, Figure 1C) and OS (median 107 days vs not reached, P=0.0001, Figure 1D) than Group 1.

Conclusion: Our study demonstrated serum HDL-c and its dynamic Change were robust and clinically feasible markers for predicting the outcomes of patients with lymphoma-associated HLH.
Associations and Hospital Outcomes of Splanchnic Vein Thrombosis in Patients with Polycythemia Vera
Authors: Olusoji Rahman , Abbey Enoch , Mohamed Salih Rajaa , Chaity Monjila , Ahluwalia Meena , Omore Ibrahim
Published: December 2023

Background: Thrombosis means the presence of a blood clot in a blood vessel. While thrombosis is a relatively common condition in deep veins of the limbs and the lungs, its incidence in splanchnic veins is at least 25 times lower compared to the former. Splanchnic Vein Thrombosis (SVT) involves portal, hepatic, splenic or mesenteric veins. Polycythemia Vera (PV) is one of the significant myeloproliferative neoplasms (MPN) that cause SVT.

Objectives: This study examines the relationship between PV and SVT and its impact on hospital outcomes, explicitly focusing on mortality rates in affected patients.

Methods: This retrospective cohort study uses the 2018 to 2020 National Inpatient Sample (NIS) database. Our inclusion criteria were for patients 18 years or older with Splanchnic Vein thrombosis and Polycythemia Vera diagnosis during hospitalization. We defined splanchnic vein thrombosis as patients with portal and hepatic vein thrombosis. We examined these patients' demographics and their relationship with hospital outcomes, such as length of hospital stay, cost of hospitalization and mortality. Using univariate and multivariate logistics regression models, we measured mortality as the primary outcome.

Result: We identified 160,570 patients with SVT, among whom 1,120 (0.69%) had PV. PV patients were primarily female and white, with a mean age of 58.9. The PV cohort exhibited a lower mortality rate of 4.5% compared to the SVT from other etiology. Being black, increasing age, and increasing comorbidity indices were associated with increasing mortality. Though not statistically significant, there was a trend of decreasing mortality in both unadjusted and adjusted models for PV [0.68, 95% CI (0.26-1.05)] and [0.55, 95% CI (0.26-1.16)], likely underpowered by the rarity of diagnosis.

Conclusion: Our study shows that the mortality of SVT from PV was 45% lower compared to other causes of SVT. PV did not pose an increased mortality risk compared to other etiologies, and it was not statistically significant. There is a need for further studies to understand better and ultimately help in management guidelines for SVT in PV compared to SVT due to other causes.
Clinical Characteristics and Prognosis of Acute Myeloid Leukemia Patients with Protein Tyrosine Phosphatase Non-Receptor Type 11 Gene Mutation
Authors: Zhao Weihua , Huang Rui , Zhang Yiting , Lin Yu , Pang Ruli , Yang Zhi
Published: December 2023

Objective: The purpose of our study was to investigate the clinical characteristics, molecular biological characteristics and prognosis of patients with acute myeloid leukemia (AML) harboring protein tyrosine phosphatase non-receptor type 11 (PTPN11) gene mutation.

Methods: The clinical data of 30 newly diagnosed adult AML patients with PTPN11 gene mutation were analyzed retrospectively. Prognostic analysis and screening of prognostic factors were conducted using the Kaplan-Meier method and Cox proportional hazards regression model.

Results: High frequency mutation sites of PTPN11 gene were located in exon 3 of chromosome 12, which are D61 and A72（16.7%）, followed by E76 (13.3%). The median variant allele frequency (VAF) of PTPN11 mutant gene was determined to be 18.4%. The patients were divided into two groups according to PTPN11 VAF 35.3% (upper quartile). We observed that the peripheral blood leukocyte count in patients with VAF ≥35.3% was significantly higher than patients with VAF < 35.3% (p=0.019).Moreover, the higher VAF was closely related to M5 subtype AML (p=0.016) and internal tandem duplication (ITD) of FMS-like tyrosine kinase 3 (FLT3) (FLT3-ITD) mutation (p= 0.048). Taking PTPN11 VAF 20% and 35.3% as the cutoff value, the patients were divided into two groups, and the overall survival(OS) and event free survival (EFS) of these two groups were not significant. Multivariate analysis using the COX proportional hazards model identified that white blood cell count and Eastern Cooperative Oncology Group (ECOG) physical status score were independent risk factors affecting the prognosis of EFS.

Conclusion: Our study suggests that PTPN11 VAF may not serve as a prognostic factor in patients with PTPN11 mut AML. However, newly diagnosed patients with high white blood cell count and poor performance status were identified as independent risk factors for EFS in PTPN11 mut AML.

Keywords: Acute myeloid leukemia, PTPN11 mutation, clinical characteristics
Increased Hydroxyurea Adherence Associated with Pediatric, Adolescent and Young Adult Sickle Cell Anemia Care at US Military Healthcare Facilities: A Cross-Sectional Study
Authors: Smith Benjamin Russell , Marshal Amanda K , Ahmed Anwar , Zanetti Richard C
Published: December 2023

Sickle Cell Anemia (SCA) causes significant acute complications, chronic complications, and premature mortality. The foundational treatment for SCA is hydroxyurea (HU) which reduces acute and chronic complications. Several barriers prevent patient HU adherence and thus treatment benefit.

We performed a cross-sectional study of 206 pediatric, adolescent, and young adult (AYA) patients with HgbSS or HgbS-beta-thalassemia-zero in the United State Military Health System who were prescribed HU to evaluate for to factors associated with HU adherence. These patients have access to military or civilian pediatric hematology care at no or low cost.

We categorised patient's adherence dichotomously according to the American Society of Hematology quality metric for HU adherence. The adherence group, 26% of the study population, had a younger mean age, 11.7 versus 14.9 (p = 0.0003), and was more likely to receive hematology care in the MHS.

Logistic regression analysis of HU adherence, adjusted for age and gender demonstrated the patients receiving pediatric hematology care at a military healthcare facility had 2.96 (1.47-5.99, 95% CI) the odds of adherence compared to patients receiving pediatric hematology care at civilian healthcare facilities. The monthly base pay of the patient's sponsor, a strong indicator of socioeconomic status was not associated with HU adherence odds ratio 1.00 (0.99-1.01, 95% CI).

Among Pediatric and AYA patients with SCA in the MHS: HU adherence appears equitable throughout the range of sponsor rank; and hematology care at military facilities is associated with increased HU adherence.
Multiomic Single-Cell Analysis Identifies Von Willebrand Factor and TIM3-Expressing BCR-ABL1+ Chronic Myeloid Leukemia Stem Cells
Authors: Nilsson Malin S. , Komic Hana , Gustafsson Johan , Sheybani Zahra , Paul Sanchari , Rolfson Ola , Hellstrand Kristoffer , Wennström Lovisa , Martner Anna , Thorén Fredrik B.
Published: December 2023

Characterization of the leukemic stem cell (LSC) population in chronic myeloid leukemia (CML) may identify therapeutic targets for sustained elimination of leukemic cells. High-throughput single-cell RNAseq (scRNAseq) has paved the way for detailed transcriptional assessment of CML LSC. Building on this technique, multiomic CITE-seq approaches carry additional benefit in that the paired protein expression information may enable identification of associated cell surface marker profiles, subsequent FACS-based isolation and functional characterization. However, detection of BCR-ABL1 transcripts, which is arguably the only unequivocal marker to distinguish CML LSC from healthy hematopoietic stem cells (HSC), remains challenging using current high-throughput 3' end capture-based scRNAseq methods.

For the present study, we performed single-cell CITE-seq analysis of the expression of 597 genes and 51 proteins in >70,000 stem and progenitor cells from 16 chronic phase CML patients and five healthy donors. Furthermore, we developed and employed a strategy allowing parallel detection of BCR-ABL1 transcripts at the single-cell level.

In bone marrow samples from CML patients, we observed pronounced expansion of erythroid and myeloid progenitors within the CD14 -CD34 + compartment, and substantial heterogeneity within the traditionally defined CD34 +CD38 -/low LSC compartment. In-depth analysis of the latter identified a group of immature BCR-ABL1 +cells displaying a CD45RA -cKIT -CD26 + TKI resistance phenotype sitting atop a hierarchy of myeloid progenitors. Within this group of potential LSC, expression of both previously reported ( e.g. CD25 and CD26) and unreported gene and protein markers was found to distinguish these cells from their healthy counterparts. Unlike HSC, the immature LSC showed high surface expression of TIM3 and high transcription of the von Willebrand factor gene ( VWF). While overexpression of VWF within the stem cell population may be linked to the aberrant myeloid-biased hematopoiesis characterizing the disease, the cell surface upregulation of TIM3 suggests that its targeting may have merit in CML.

In conclusion, we here performed detailed multiomic characterization of CML stem and progenitor cells, paired with BCR-ABL1 detection at the single-cell level. Our findings revealed LSC-specific expression patterns that may have implications for the phenotypic definition of CML LSC. Additionally, the results identify TIM3 as a conceivable target for sustained elimination of immature LSC in CML.
Non-Viral RNA-Lipid Nanoparticles for High Efficiency Genome Editing of CD34+ Hematopoietic Stem and Progenitor Cells for Advanced Cell and Gene Therapies
Authors: Geczy Reka , Park Stella , Zhao Rita , Swaminathan Meenakshi , Jain Nikita , Thomas Anitha , Zhang Angela , Clarke Samuel
Published: December 2023

Background and Aims: The application of CRISPR-Cas9 gene editing in hematopoietic stem (HSC) and progenitor cells (HSPCs) has the potential to revolutionize the treatment of genetic blood disorders by correcting disease-causing mutations. Previously, we demonstrated the utility of a novel lipid nanoparticle (LNP) reagent for the multiplexed engineering of gene-edited CAR T cells, showing high cell viabilities, rapid onset editing, and potent CAR-mediated killing. In this current work, we demonstrate the capability of LNPs for the efficient and gentle delivery of genetic material to CD34+ HSPCs. The benefits of a non-viral LNP-mediated approach paves the way for the development of next generation gene therapy products in the HSC field.

Methods: LNPs encapsulating S.p. Cas9 mRNA and CD33 or CD45 targeted guide RNA (sgRNA) were produced using the scalable NanoAssemblr® NxGen™ microfluidic platform technology ( Fig. 1A). The LNP composition was optimized for the gentle and efficient cargo delivery to HSC/HSPCs. Purified human CD34+ cells were cultured, stimulated, and treated by the direct addition of the RNA-LNPs. Various commercial cell sources were tested including mobilized peripheral blood and cord blood. Gene knockout and viability were assessed using flow cytometry, cell proliferation using an automated cell counter, and differentiation capacity by colony-forming unit (CFU) assays.

Results: One-step addition of LNPs to CD34+ HSPCs resulted in 84 ± 6% CD33 and 81 ± 2% CD45 knockout efficiencies (n=6 CD34+ donors, Fig. 1B). After treatment, cells maintained on average 95 ± 3% absolute cell viability, compared to 99% viability of the untreated cells, Fig. 1C. Furthermore, LNP treated HSCs maintained excellent cell proliferation, with over >90% relative cell proliferation to the untreated controls. The CFU assays showed no significant change in relative lineage formation for both the RNA-LNPs and the empty LNP vehicle control ( Fig. 1D). Finally, LNP production was successfully scaled-up using microfluidics from discovery to pre-clinical scales.

Conclusions: LNP-mediated CRISPR-Cas9 mRNA delivery is a promising approach for gene editing in HSPCs. The simple and gentle nature of LNP cell treatment allows for multiple genetic engineering steps for simultaneous expression and deletion of proteins for novel gene therapies. Furthermore, LNPs can be easily manufactured using microfluidics, enabling small-scale screening of RNA libraries and rapid scale-up for clinical translation.
Predictive Value of Pre-Infusion FDG-PET/CT Parameters for Clinical Outcomes and Toxicities in Relapsed/Refractory (R/R) Large B-Cell Lymphoma (LBCL) Following CAR-T Cell Therapy
Authors: Belbachir Safia , Alexander Sweeka , Dhamija Mayank , Al-Ogaili Zeyad , Purtill Duncan , Barraclough Allison A
Published: December 2023

Introduction:

CD19-targeting chimeric antigen receptor (CAR) T-cell therapy has demonstrated significant efficacy in patients with relapsed/refractory (R/R) non-Hodgkin lymphoma but is associated with serious adverse effects such CRS and ICANS. Pre-infusion disease burden has been investigated as a potential predictor of clinical outcome and toxicity. FDG-PET/CT derived metabolic tumor volume (MTV) and total lesion glycolysis (TLG) are quantitative indicators of this baseline tumor load. We aim to assess the predictive value of pre-CART PET metrics on outcome and toxicity in R/R large B-Cell Lymphoma (LBCL) following CAR-T cell therapy.

Method:

We conducted a retrospective single site study of 25 consecutive patients with R/R LBCL treated with CAR-T therapy in our institution between May 2020 and October 2022, All patients underwent FDG-PET/CT imaging pre-CAR-T infusion (PET-0) and at day 30 post infusion (PET-1). The images were reviewed by two expert lymphoma PET physicians. Metabolic parameters including MTV, TLG (both calculated using a relative threshold of 40%), SUVmax and Deauville score were calculated. PET-1 was classified as complete metabolic response (CMR, Deauville score 1-3) or non-CMR (Deauville score 4-5) as per the Lugano classification.

Results:

25 patients were analyzed , Based on the median MTV (74,6 mL), TLG (379,4), SUVmax (11,8) values, patients were divided into two groups “low” and “high”.

The median follow-up was 12 months. Patients with low pre-infusion MTV and TLG had significantly superior day 30 CMR rates (84,6% vs 25%, p = 0.001 & 80% vs 20%, p = 0.001 respectively). Pre-infusion SUVmax ( p = 0.29 ) and Deauville score ( p = 0.103), however, did not predict response to treatment. In our population, pre-infusion PET parameters were not associated with CRS or ICANS .

Patients with low pre-infusion MTV and TLG had significantly superior PFS (MTV: p=0.02; TLG: p=0.019) and OS (MTV: p=0.017; TLG: p=0.05), Pre-infusion SUVmax had no statistical significant impact on OS ( p = 0.61) or PFS ( p = 0.37)

Conclusion:

In our study, pre-infusion quantitative PET metrics, TLG and MTV, predict response and outcomes of CART therapy in R/R LBCL. Our results highlight the importance of adequate disease control before CAR-T administration and the value of pre-infusion FDG-PET/CT as a predictor of outcomes in R/R LBCL patients treated by CAR-T-therapy.
Lipid Nanoparticle-Mediated Gene Editing of Human Primary T Cells and Off-Target Analysis of the CRISPR-Cas9 Indels
Authors: Geczy Reka , Thommandru Bernice , Swaminathan Meenakshi , Zhao Rita , Tan Krystal , Low Kevin , Balgi Aruna , Park Stella , Watt Ethan , Thada Vaughn , Rettig Garrett , Zhang Angela , Turk Rolf , Clarke Samuel
Published: December 2023

Background and Aims: The CRISPR/Cas9 system has emerged as a powerful tool for gene editing of primary cells. In our previous work, we demonstrated a novel lipid nanoparticle (LNP) reagent for the multi-step engineering of gene-edited CAR T cells. We showed high cell viabilities and potent CAR-T mediated killing, even after multiple genetic manipulations. Here, we extend this work by assessing potential off-target editing effects in both LNP-treated T cells and T-cells where the CRISPR reagents were delivered by electroporation. Further, we evaluated multiple Cas9 variants and guide RNA targets.

Methods: LNPs encapsulating wild type or high fidelity S.p. Cas9 mRNA and various TRAC and CD52 targeted guide RNAs (sgRNAs) were produced using our scalable NanoAssemblrTM microfluidics platform. Concurrently, electroporation was performed to deliver equivalent cargoes. Purified primary T cells were cultured, activated, and expanded in serum-free media in plates, flasks, or small bioreactors. The LNPs were added to cells by direct addition for gene editing. Gene expression and cell viability were measured using flow cytometry or colorimetric assays. Multi-target performance of CRISPR-Cas9 editing was evaluated through rhAmpSeq-based targeted next-generation sequencing (NGS) and indels analyzed for with CRISPRAltRations.

Results: TCR or CD52 targeted Cas9 mRNA-LNP addition or electroporation yielded high single and double knockout efficiencies. High-throughput NGS analysis showed strong agreeance to flow cytometry for on-target analysis. We tested a range of sgRNA targets, wild-type and high-fidelity Cas9 mRNAs, and determined off-target editing for all targets and variants investigated. Similar results were obtained when comparing different LNP batch sizes (micro to milligram RNA) and cell culture vessels (0.1 to 45 million cells), demonstrating scalability of both LNP production and cell treatment.

Conclusions: The results from this study further support the utility RNA-LNPs for the genetic engineering of primary T cells. The simple and gentle nature of LNP cell treatment allows for multiple genetic engineering steps for simultaneous expression and deletion of proteins for the next cell therapies. These LNPs can be easily manufactured from small-scale screening of RNA libraries to rapid scale-up for clinical translation.