bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenotype Molding of Monocytes in the Multiple Myeloma Microenvironment
Authors: Cui Jian , Wang Jingwei , Qiu Lugui , An Gang
Published: December 2023

Human monocytes are mononuclear phagocytes that comprise a major population of innate immune cells. Various monocyte-derived cells, including osteoclasts, dendritic cells, and macrophages, have been implicated in immunosuppression within the multiple myeloma (MM) tumor microenvironment (TME). Despite recent developments in understanding the link between monocyte-derived cells and immunosuppression in myeloma, there are still critical knowledge gaps in the association between monocytes themselves and immunosuppression within the context of MM. Specifically, the functional and developmental alterations of monocytes in the MM TME have not been explored based on the single-cell studies.

In the present study, we generated a comprehensive single-cell map of peripheral blood (PB) and bone marrow (BM) monocytes from both healthy donors (HDs) and MM patients. Specifically, Heparin-anticoagulated PB samples or BM aspirates were collected from seven newly diagnosed MM (NDMM) patients and healthy donors (HDs). Paired BM aspirates were obtained from 10 MM patients at the time of diagnosis and after induction therapy.

Differential expression analysis showed that type I interferon (IFN) response was upregulated in MM monocytes. And BM monocytes derived from MM patients exhibited impaired T cell attraction abilities and demonstrated an elevated capacity for suppressing T cell function. Moreover, immune checkpoint molecules, including CD274 (encoding PD-L1) and PDCD1LG2 (encoding PD-L2), were significantly upregulated in MM monocytes. Additionally, a positive correlation was observed between type I IFN response and T cell suppression capacity of MM monocytes. Trajectory analyses revealed an IFN-focused disruption of developmental pathways of both BM and PB monocytes in MM. Lastly, we included 10 MM patients at the time of diagnosis and after induction therapy to monitor transcriptional changes over time. Our results revealed that anti-tumor therapy successfully mitigated the excessive type-I interferon response of BM monocytes in MM.

We document that MM monocytes exhibited an augmented type-I IFN response compared to monocytes from HDs, while their overall heterogeneity remained unchanged. Furthermore, we extended the knowledge on immunosuppression of the MM TME that the excessive type-I IFN response in MM had a significant impact on the differentiation of both BM and PB monocytes, leading to T-cell immunosuppression, specifically in BM monocytes. In the cohort that we longitudinally followed, we observed a decrease in the type-I IFN response across nearly all monocyte subsets in MM BM monocytes after induction therapy, accompanied by a reduction in the expression of the T-cell suppression gene set. In sum, our findings indicates that monocytes in the BM promotes the immunosuppression of the MM TME and represents potential therapeutic targets for modulating the MM TME.
The Glass Wall: Gendered Authorship Disparities in in CD 19 and BCMA CAR-T Clinical Trials for Lymphoma and Myeloma
Authors: Khaliq Aroog , Wesson William , Logan Emerson , Tabak Carine , Rashid Aliya , Mansour Razan , Mushtaq Muhammad Umair , McGuirk Joseph P , Hashmi Hamza , Hamadani Mehdi , Abdallah Al-Ola , Ahmed Nausheen
Published: December 2023

Introduction: Existing literature suggests that women are significantly underrepresented in the field of hematology-oncology. Data from 2021 suggests that women make up 35.6% of hematologists, and thus the disparity continues in clinical trial research authorship. Minorities including women receive fewer awards, deliver fewer board review lectures, and hold fewer professorships, and leadership positions in clinical departments and professional organizations. To further investigate this issue, we conducted a study to assess the gender gap in authorship across 13 pivotal clinical trials that led to the approval of CD19 and BCMA Chimeric antigen receptor T cell (CAR-T) therapies.

Methods: We included the 13 pivotal trials that led to the approval of CART for Acute Lymphoblastic leukemia (ALL), lymphoma and myeloma , published between 2017-2022. These included the trials: ELIANA, ZUMA-1, JULIET, ZUMA-2, KarMMa, TRANSCEND, ZUMA-5, CARTITUDE-1, ZUMA-7, ZUMA-3, TRANSFORM, PILOT, and ELARA.

We examined the overall number of female authors, the number of lead female authors as defined as first, second, or last author, and the ratio of all authors to female authors. 367 total authors were included in the analysis, with several authors being included more than once for their work on separate studies.

Results: Table 1 summarizes the results. Of the 367 authors assessed, 109 were female, correlating to 28.9% female authorship. The only study with female authorship >50% was ELIANA, a 2017 pediatric study, while all others had >50% male authorship. 7 (54%) of the 13 studies had females as lead authors; of note, 6 out of 7 of these studies were published after 2021.

Conclusion: Gender inequities in research authorship are pervasive in many areas of medicine, including the field of immune effector cell therapy. Publications in the field broadly are overwhelmingly male dominated, possibly reflective of lower female representation in transplant and cellular therapy. Despite this, we observed improved gender equality in authorship in more recent publications since 2021, and in certain research areas such as pediatric hematology-oncology. Recognizing these seemingly invisible barriers is the first step in bringing gender equity to the field of hematopoietic stem cell transplant and cellular therapy, with gender serving as one of many domains in which underrepresented groups' ability to scale the glass wall must be supported.

Table 1.
Efficacy and Safety Analysis of Carfilzomib-Containing Regimen in the Treatment of Multiple Myeloma：a Multicenter Real-World Study
Authors: Liu Na , WANG Luqun , Liu Rentong , Zhong Yuping , Feng Xianqi , Sun Zhongliang , Li Jie , Meng Fanjun , MA Junjie , Yuan Changyong , Li Hao , Wang Ling , Xu Zhilong , Yu Jie , Zhang Hao , Chunyan Chen
Published: December 2023

Purpose

To investigate the efficacy and safety of carfilzomib-containing regimens in the treatment of multiple myeloma（MM） in different lines of treatment.

method

The clinical data of 55 multiple myeloma patients who received carfilzomib treatment in 10 medical centers in Shandong from January 2022 to March 2023 were retrospectively analyzed , and grouped according to the number of MM treatment lines, they were divided into group A : newly diagnosed multiple myeloma ( NDMM ), group B : patients with first relapse, and group C : patients treated with carfilzomib in the third line or above. Del ( 17p ), t ( 4;14 ), t ( 14;16 ) were defined as high-risk cytogenetic abnormalities

result

All patients with multiple myeloma were treated with the combined regimen based on carfilzomib, including 24 patients in group A , 9 patients in group B , and 22 patients in group C. Among all patients, there were 27 cases ( 49.1% ) of high-risk cytogenetics, and 13 cases with ECOG score ≥ 3 points ( 23.6% ), 10 cases ( 18.2 ) had eGFR <30 ml/min. The median follow-up time of patients in group A , group B and group C was 8 months, 9 months and 8 months. The overall response rate ( ORR ) was 91.7% , 88.9% and 77.3% for patients in Arms A , B and C who started carfilzomib, respectively . The ≥very good partial response ( VGPR ) rates were 75% and 66.7% for Arm A and B patients , respectively , while the ≥VGPR rate for Arm C patients was 41.0% . The negative rate of minimal residual disease ( MRD , sensitivity: 10 -6 ) in group A was 62.5% ( 15/24 ). Follow - up deadline is March 2023 On the 31st , the median progression-free survival ( PFS ) time and median overall survival ( OS ) time of patients in the three groups were not reached. After the application of carfilzomib, the incidence rate of grade 3-4 adverse events ( AE ) was 32.7% ( 18/55 ). The main hematological adverse reactions were anemia, thrombocytopenia, lymphopenia and neutropenia. Non-hematological adverse reactions were mainly infusion-related adverse reactions and infections.

in conclusion

Carfilzomib-based regimens in the treatment of multiple myeloma have good curative effect and safety, and the first-line and first-time relapse patients have better curative effect.
Selinexor Enhances Natural Killer Cell Function Against Multiple Myeloma Cells
Authors: Fisher Jack G , Doyle Amber DP , Graham Lara V , Forconi Francesco , Cragg Mark S , Walker Christopher J , Khakoo Salim I , Blunt Matthew D
Published: December 2023

Introduction: Natural killer (NK) cells are innate immune effector cells which can mediate antibody dependent cellular cytotoxicity (ADCC) and lyse cancer cells. Harnessing NK cell function against multiple myeloma (MM) is of high interest and multiple strategies are currently under development.

Exportin-1 (XPO1) is a nuclear export protein which inhibits the function of tumour suppressor proteins and facilitates oncogene mRNA translation. Selinexor is a first-in-class XPO1 inhibitor approved for the treatment of relapsed or refractory MM that induces cancer cell apoptosis. In addition, selinexor has recently been shown to promote NK cell cytotoxicity against B cell lymphoma cells. This study aimed to determine whether selinexor augments NK cell activation against MM cells both alone, and in combination with the anti-CD38 antibody daratumumab.

Methods: A panel of MM cell lines (MM.1S, U266 and RPMI-8226) were incubated with clinically relevant concentrations of selinexor for 24 hours, then either assessed for expression of ligands for NK cell receptors (KIR, NKG2A, NKG2D and NKp46), or co-cultured with primary human NK cells ± daratumumab. Following co-culture, NK-mediated lysis of MM cells was assessed by propidium iodide staining and NK cell degranulation assessed by CD107a expression.

Results: Selinexor pre-treatment of MM cells significantly increased NK cell degranulation (p<0.05) and enhanced NK specific lysis of MM targets (p<0.05). Furthermore, selinexor pre-treatment significantly (p<0.05) enhanced NK cell activation in combination with daratumumab compared to daratumumab alone. Importantly, selinexor did not alter surface expression of CD38 on MM cell lines. To investigate the mechanism behind this enhanced NK cell function, we assessed the effect of selinexor on the expression of NK cell ligands by MM cells. Selinexor increased the expression of ULBP1/2/5/6 (2-fold, p<0.001) which bind to the NK cell activating receptor NKG2D. In addition, selinexor decreased MM cell expression of HLA-E (30% decrease, p<0.05) which binds to the inhibitory NK receptor NKG2A.

Conclusions: These data suggest that selinexor may promote the efficacy of NK targeted therapeutics in multiple myeloma.
Post-Transplant Cyclophosphamide or ATG As Gvhd Prophylaxis in Mismatched Unrelated Stem Cell Transplantation? - a Registry Analysis By the EBMT Transplant Complications Working Party
Authors: Penack Olaf , Abouqateb Mouad , Peczynski Christophe , Boreland William , Gulbas Zafer , Gedde-Dahl Tobias , Castilla-Llorente Cristina , Kröger Nicolaus , Eder Matthias , Rambaldi Alessandro , Bonifazi Francesca , Wolfgang Blau Igor , Stelljes Matthias , Dreger Peter , Moiseev Ivan Sergeevich , Schoemans Helene , Koenecke Christian , Peric Zinaida
Published: December 2023

There is an increased risk of GVHD and non-relapse mortality (NRM) after allogeneic stem cell transplantations (alloSCT) when mismatched unrelated donors (MMUD) are used. Prophylaxis with either rabbit anti-thymocyte globulin (rATG, also termed anti-T-lymphocyte globulin) or post-transplantation Cyclophosphamide (PTCy) is often used to reduce the risk of NRM and GVHD. However, it is currently impossible to give general recommendations regarding preference for rATG or PTCy since comparative evidence from larger data sets is lacking.

To better understand the outcome of these GHVD prophylactic regimens, we analyzed outcomes of rATG vs. PTCy prophylaxis in adult patients with hematologic malignancies, who underwent peripheral blood alloSCT from 9/10 antigen MMUD between Jan 2018 and June 2021 in the EBMT database. We performed multivariate analyses using Cox cause-specific hazard models, adjusting for known risk factors, and variables that showed significant difference between the two comparison groups.

Overall 2123 patient were included, treatment- and patient characteristics are shown in Table 1. Patients receiving PTCy (n=583) were younger and had undergone alloSCT more recently compared to patients receiving rATG (n=1540). Median follow up was 2.0 years (PTCy) and 2.4 years (rATG). Outcome graphs are shown in figure 1. All outcomes are given starting from AlloSCT (with 95% CI).

NRM (fig 1A) was higher in the rATG arm (2-years NRM: PTCy 18% [14.6-21.6] vs. rATG 24.9% [22.7-27.3]; HR 0.74, p=0.03), Relapse incidence was slightly higher but not significant in the rATG arm (2-years RI: PTCy 22.9% [19.2 - 26.9] vs. rATG 26.2% [23.9 - 28.7]; HR 0.82, p=0.07), Overall survival (fig 1B) was better in the PTCy arm (2-years OS: PTCy 65.7% [61.2 - 69.9] vs. rATG 55.7% [52.9 - 58.3]; HR 0.77, p<0.001), same for Progression free survival (fig 1C) (2-years PFS: PTCy 59.1% [54.4 - 63.5] vs. rATG 48.8% [46.1 - 51.5]; HR 0.78, p=0.001). The incidences of chronic and acute GVHD were not significantly different between the groups; (2-years cGVHD: PTCy 31.7% [27.4 - 36] vs. rATG 30.3% [27.8 - 32.8]; HR 0.95, p=0.67. And 100-days aGVHD grades II-IV: PTCy 29.9% [25.9-34.1] vs. rATG 32.5% [30-34.9]; HR 0.83, p=0.11).

In summary, we found significantly higher survival and lower NRM in recipients of peripheral blood alloSCTs from MMUD receiving PTCy as compared to rATG. The current analysis improves the evidence base for decision making on GVHD prophylaxis in MMUD alloSCT.
Transcriptomic Profile of Peripheral Blood Mononuclear Cells in Sirolimus-Sensitive Acquired Pure Red Cell Aplasia Patients
Authors: Yang Yuan , Hu Qinglin , Yang Chen , Chen Miao , Zhuang Junling , Han Bing
Published: December 2023

Objective: Sirolimus is effective for treatment of acquired pure red cell aplasia (aPRCA). Nevertheless, the immunomodulatory effect of sirolimus on peripheral blood mononuclear cells (PBMCs) from aPRCA remains undetermined. This study aims to investigate the immunomodulatory effect of sirolimus using PBMCs transcriptomics of aPRCA patients.

Methods: PBMCs were isolated from treatment-naïve (n = 11) or sirolimus-sensitive aPRCA patients ((n = 11) and then underwent RNA-sequencing.

Results: A total of 4060 genes, including 1639 upregulated genes and 2421 downregulated genes were significantly dysregulated in sirolimus-sensitive aPRCA patients compared with treatment-naïve patients. The top ten dysregulated genes were SYTL5, VSNL1, LINC00707, BTBD17, LINC01954 AL445437.1, IGKV1-8, AC090826.1, PRAME and LINC00520, respectively. Among the identified biological processes significantly enriched by dysregulated genes, B-cell-mediated immunity and circulating immunoglobulin-mediated humoral immune responses are the top two dysregulated immune responses in aPRCA patients with sirolimus treatment. Additionally, TNF, CD40, HLA-DRB1, SERPING1, SERPING1, C1QA, IGLL5 and IGHV3-11 were the key modulator in B-cell-mediated immune or circulating immunoglobulin-mediated humoral immune responses in sirolimus-sensitive aPRCA patients.

Conclusion: We first provided evidence showing the immunomodulatory effect of sirolimus on PBMCs from patients with aPRCA. Sirolimus can modulate the B-cell-mediated immunity and circulating immunoglobulin-mediated humoral immune responses in aPRCA patients.
Serpin-PC in Persons with Severe Hemophilia (PwH): Updated Results from a Multicenter Multi-Part, First-in-Human Study
Authors: Baglin Trevor , Huntington James A. , Koch Annelize , Mocanu Irina , Makhaldiani Levani
Published: December 2023

Introduction

SerpinPC is an investigational serine protease inhibitor (SERPIN) engineered to specifically inhibit Activated Protein C (APC). The previously presented data from the completed parts of AP-0101 showed that administration of SerpinPC reduced bleeding in persons with severe hemophilia with no observations of unexplained chronic elevation in D-dimer. We will now present all results up to the end of Part 5 by which time we anticipate the median continuous exposure will be more than 3 years.

Methods

AP-0101 is an ongoing first-in-human open-label multicenter study utilizing an adaptive design to investigate the safety, tolerability, pharmacokinetics and efficacy of SerpinPC in subjects with severe hemophilia A and B.

Part 1a was a Single Ascending Dose Study of SerpinPC in 15 healthy male volunteers and 12 males with severe hemophilia.

Part 2 enrolled 23 males with severe hemophilia (19 hemophilia A and 4 hemophilia B), who were not on replacement factor prophylaxis, to receive SerpinPC at 0.3, 0.6 or 1.2 mg/kg, administered as a subcutaneous (SC) injection once every 4 weeks over a 24-week period (6 total doses).

In Part 3, subjects who completed Part 2 received a flat dose of 60 mg of SerpinPC once every 4 weeks for 48 weeks.

Part 4 was a further extension in which subjects who completed Part 3 received 1.2 mg/kg of SerpinPC once every 2 weeks for 24 weeks.

Part 5 was a further extension in which subjects who completed Part 4 continued to receive 1.2 mg/kg of SerpinPC once every 2 weeks for 52 weeks.

Results

Annualized bleed rates, safety and tolerability for Part 5 will be available and a complete summary of all results to the end of Part 5 will be presented, including available pharmacokinetic and anti-drug antibody data.
Prevalence and Proportion of Transcranial Doppler Screenings in a Population Children with Sickle Cell Disease and Association with Hydroxyurea Adherence
Authors: Knight La'Kita , Tanabe Paula Jean , Blewer Audrey , Goodrich James , King Allison A. , Reuter-Rice Karin , Crego Nancy
Published: December 2023

Sickle cell disease (SCD) is an inherited or genetic hemoglobinopathy in the United States and approximately 100,000 Americans are affected. The prevalence of stroke in unscreened children with sickle cell anemia (SCA) is 10% and can begin at infancy. National guidelines strongly recommend annual Transcranial Doppler (TCD) screenings as well as hydroxyurea (HU) administration in children with SS and Sβ o thalassemia (SCA) genotypes. The purpose of this project was to examine the prevalence and proportion of TCD screenings in North Carolina (NC) Medicaid enrollees and to identify associations with sociodemographic factors (age, sex, and rurality) and HU adherence. A longitudinal analysis of NC Medicaid claims data (2016-2019) was conducted for children with SCD ages 2-16 years. Prevalence of TCD screening claims was calculated to determine the number of children with TCD claims for each of the three, 12-month enrollment periods. The proportion of TCD screening claims was calculated based on NC Medicaid enrollees who had increasing TCD claims (0, 1, 2, or 3+) for 12, 24, and 36 months of Medicaid enrollment. HU adherence was measured using Proportion of Days Covered (PDC). HU PDC is a measure of HU prescription fills over 365 days within a 12-month period. HU PDC was used to categorize enrollees into good (≥80%), moderate (60-79%), or poor (<60%) adherence categories. Age (years) was declared as the age in years of each participant during each study observation period and was categorized into three groups: preschoolers (2-5 years), school aged (6-11 years), and adolescents (12-16 years). Rurality was identified by converting residential zip codes to rural/urban county codes (RUCC). An adjusted multivariable Poisson regression model was fit to assess the rate of TCD screenings by HU adherence while controlling for age, sex, and rurality. The prevalence of children with annual TCD screening claims ranged from 39.5% to 40.1%. The percentage of children with 2 or more TCD claims increased when they had extended months of NC Medicaid enrollment (10.5% at 12 months, 33.7% at 24 months, and 52.6% at 36 months). Overall, children with Good HU adherence were 2.48 (95% CI 2.00, 3.06) times more likely to have TCD claims than children with poor HU adherence. In conclusion, the prevalence of TCD screenings of children with SCD on NC Medicaid was extremely low. Children with better HU adherence and extended NC Medicaid enrollment time were associated with higher number of TCD screenings.
Venous Thromboembolism Outcomes Among Cancer and Non-Cancer Patients Managed with Patient-Centric Guideline-Driven Protocol
Authors: Cassianni Claire E. , McBane Robert D. , Vlazny Danielle T. , Hodge David O. , Casanegra Ana I. , Houghton Damon E. , Wysokinski Waldemar E.
Published: December 2023

Purpose/Introduction: Patients with cancer and venous thromboembolism (VTE) have higher complication rates including thrombosis recurrence and bleeding. Real world prospective clinical outcome estimates of VTE management comparing cancer and non-cancer patients are limited. To assess VTE recurrence, major bleeding, and clinically relevant non-major bleeding (CRNMB) in patients with cancer and without cancer, the prospective Mayo Clinic Thrombophilia Clinic Registry was analyzed.

Methods: Upon recruitment, consecutive patients with confirmed acute VTE (03/01/2013 - 04/30/2023) were treated in a standardized, guideline-sanctioned, protocol driven strategy incorporating shared patient-decision making. Patients were divided into groups based on cancer status. After enrollment, patients were actively followed at 3 month intervals, in person whenever feasible, by mailed questionnaire or scripted phone interview to assess vital status, medication compliance, VTE recurrence, major bleeding and CRNMB.

Results: Over the study time-frame, 2,064 patients (53.8% male, 46.2% female) with a cancer and 2,647 patients (54.9% male, 45.1% female) without cancer were enrolled. The most common cancers were gastrointestinal (n=423, 20.5%), pancreatic (n=287, 13.9%), genitourinary (n=198, 9.6%), hematologic (n=171, 8.3%), and lung cancer (n=170, 8.2%). Patients with cancer were older, had lower weight and lower platelet counts compared to non-cancer patients (Table1). Pulmonary embolism (PE, 52.6% vs 43.7%, p<0.001), upper extremity DVT (9.1% vs 6.0%, p<0.001), and splanchnic DVT (11.1% vs 6.9%, p<0.001) were more frequent among cancer patients. In contrast, leg DVT was more frequent among non-cancer patients (65.4% vs.47.2%, p<0.001). While mean duration of anticoagulation was similar between groups, notable differences in 3 month and > 9 month durations were evident as were initial anticoagulant choices (Table 1). Despite a well-organized protocol driven and guideline sanctioned management strategy, patients with active cancer experienced a 2.2-fold higher rate of VTE recurrence (p<0.001) and a 1.8 fold higher rate of major bleeding (p<001) compared to non-cancer patients (Table 2). CRNMB rates did not differ by cancer status.

Conclusions: In this large prospective, guideline-driven and patient-centric registry of VTE management, patients with cancer had significantly higher rate of VTE recurrence and major bleeding, compared to non-cancer patients. These data provide important estimates for power calculations for future randomized trials of VTE treatment.
Efficacy and Safety of Zanubrutinib-Containing Regimens in Patients with Secondary CNS Relapse in Diffuse Large B-Cell Lymphoma
Authors: Bao Li , HU Kai , Ke Xiaoyan , Wang Yutong , Wang Mengzhen , Shi Lei , Sun Kai
Published: December 2023

Background: In the era of immune-targeted therapy represented by rituximab, despite improved remission and survival rates in patients with diffuse large B-cell lymphoma (DLBCL), central nervous system (CNS) relapse, occurring in some DLBCL patients after achieving remission, remains a clinical challenge. Zanubrutinib is one of a new generation of BTK inhibitors that have higher blood-brain barrier permeability compared to other BTK inhibitors.

Research Design and Methods: We performed a multicenter retrospective study to evaluate the efficacy and safety of Zanubrutinib-containing regimens for salvage treatment in 28 relapsed DLBCL patients with CNS involvement treated in 6 centers from January 2020 to December 2021.

Results: The IPI score and CNS-IPI score did not have good enough predictive significance for lack of consistency of the median time from the initial diagnosis to CNS recurrence. Of the 28 patients, 16 had targeted next-generation sequencing (NGS) data. NGS test was performed on their tissue samples that were obtained at initial diagnosis and/or at the timepoint of CNS relapse in DLBCL. 8 at the time of initial diagnosis and 10 at the time of recurrence, with 2 of them having undergone NGS sequencing at both initial diagnosis and recurrence. Median study follow-up from CNS relapse was 8.55 months, all patients were evaluated for response with 20/28 (71.4 %) showing a response: 11 CR, 9 PR, and 4 SD, 4 PD. The median PFS is 6.6 months, and the median OS has not yet been reached. Notably, MYD88, CD79B, or TP53 mutations tested at diagnosis in these patients were associated with significantly more favorable treatment response, while patients with a dual rearrangement of MYC and BCL2 and/or BCL6 genes did not get a treatment response to Zanubrutinib-containing regimens. It has well-tolerated toxicity with few adverse events requiring treatment discontinuation or dose reduction.

Conclusion: Zanubrutinib-containing regimens extended PFS and OS in relapsed DLBCL patients with CNS involvement.
The Iron Chaperone Poly C Binding Protein 1 Regulates Iron Efflux through Intestinal Ferroportin in Mice
Authors: Wang Yubo , Protchenko Olga , Huber Kari , Shakoury-Elizeh Minoo , Philpott Caroline
Published: December 2023

Iron is an essential nutrient required by all cells but used primarily for red blood cell production. Because humans have no effective mechanism for ridding the body of excess iron, the absorption of dietary iron must be precisely regulated. The critical site of regulation is the transfer of iron from the absorptive enterocyte to the portal circulation via the sole iron efflux transporter, ferroportin (FPN). Here we report that poly(rC)-binding protein 1 (PCBP1), the major cytosolic iron chaperone, is necessary for the regulation of iron flux through FPN in the intestine of mice. PCBP1 binds iron-glutathione complexes in the cytosol of cells and delivers the iron to ferritin, the iron storage protein, to iron centers in enzymes, and to iron-sulfur cluster chaperones. PCBP1 also limits the chemical reactivity of iron, thereby limiting iron-catalyzed oxidative damage in cells.

To explore the role of PCBP1 in dietary iron absorption, we developed mice with an inducible, enterocyte-specific deletion of PCBP1. Mice lacking PCBP1 in the intestinal epithelium exhibit low levels of enterocyte iron, poor retention of dietary iron in enterocyte ferritin, and excess efflux of iron through FPN. Kinetic studies of iron transport using a stable isotope ( 57Fe) coupled with a mini-hepcidin that blocks FPN activity demonstrated that PCBP1 deletion had no effect of intestinal iron uptake but caused high rates of iron efflux through FPN. Surprisingly, excess iron efflux occurred despite lower levels of FPN protein in enterocytes and upregulation of the iron regulatory hormone hepcidin. PCBP1 deletion and the resulting unregulated dietary iron absorption led to poor growth, severe anemia on a low iron diet and liver oxidative stress with iron loading on a high iron diet.

To investigate the roles of PCBP1 more precisely in enterocytes, we developed 3-D organoid cultures from intestinal epithelium which functionally expressed the major iron trafficking proteins: divalent metal transporter 1, PCBP1, ferritin, transferrin receptor 1 and FPN. Ex vivo culture of wild-type and PCBP1-depleted enteroids demonstrated normal patterns of FPN expression and hepcidin-mediated turnover. However, fluorescence measurements of kinetically labile iron pools in enteroids competent or blocked for FPN iron efflux indicated that PCBP1 dramatically altered efflux activity. PCBP1 functioned as a cytosolic buffer for iron, binding and retaining iron and thereby keeping “free” iron at very low levels. This low concentration of free iron limited its availability for FPN-mediated efflux and allowed hepcidin-mediated changes in FPN levels to regulate the amount of iron exported from the enterocyte. Thus, by binding enterocyte iron, PCBP1 reduces the concentration of unchaperoned, “free” iron to a low level. This low level of cytosolic free iron is necessary for hepcidin-mediated regulation of FPN activity.
Spectrum and Clinical Features of Gene Mutations in Chinese Follicular Lymphoma
Authors: Li Chunyuan , Yin Jingjing , Yuan Yue , Yang Ping , Liu Hui , Jing Hongmei
Published: December 2023

Background: Follicular lymphoma (FL) is the most common indolent lymphoma with variable biologic presentation and heterogeneous clinical outcomes, and some of patients experiences early progression and have inferior survival outcomes. Next-generation sequencing (NGS) identified recurrently mutated genes in FL. While few studies on gene mutations in Chinese FL patients have been identified in the real world, this present study aimed to characterize the genomic landscape and provide insights into the biomarkers of high-risk FL populations.

Methods: 590 FL patients were reviewed, and 100 patients with sufficient samples were enrolled in our cohort. We performed targeted sequencing of 521 lymphoma-related genes based on NGS in this study.

Results: A total of 207 genetic alterations were detected in 99/100 patients. T NFRSF14, CARD11, and NOTCH3 mutations were common in FL1-2 patients compared to FL3a patients (p = 0.025, 0.0036, and 0.0047, respectively). SOCS1 mutations were more common in patients with Ki-67 index > 30% (p =0.324), and PRDM1 mutations were more common in patients who experienced progression of disease within 24 months (POD24) (33.3% vs. 0%, p =0.008). We observed a trend that patients with a high mutation load (with ≥ 6 mutated genes) showed decreased PFS compared with patients with < 6 mutated genes (p=0.068). For patients who received R-CHOP-like regimens, the multivariate COX proportional hazard modeling identified TP53, TNFAIP3, and SOCS1 mutations as independent risk factors of PFS (HR 6.76, 95% CI 1.81 to 25.18, p =0.004; HR 3.68, 95% CI 1.06 to 12.82, p =0.041; HR 5.07, 95% CI 1.81 to 21.73, p =0.029). TP53 and TNFAIP3 were significant independent predictors of PFS when testing with either FLIPI (p=0.029) or FLIPI2 (p=0.023) in multivariable analysis.

Conclusion: Our study depicted genomic characterization of real-world Chinses FL patients and demonstrated TP53, TNFAIP3 and SOCS1 mutations can help identify high-risk patients.
Survival Outcomes of Patients with Diffuse Large B-Cell Lymphoma Who Received Autologous Stem Cell Transplantation in Germany - Real World Evidence from an Administrative Database
Authors: Borchmann Peter , Heger Jan-Michel , Mahlich Joerg , Papadimitrious Micheal , Riou Sybille , Werner Barbara
Published: December 2023

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma with increasing incidence. Autologous stem cell transplantation (ASCT) is a treatment option for patients who relapse or progress after up-front chemoimmunotherapy if they suitable for ASCT. However, patients may not qualify for this treatment because of age and/or comorbidity or because they are non-responder to second-line chemoimmunotherapy. Limited real-world data of the ASCT patient population is available for Germany. Therefore, this retrospective database analysis was conducted to describe real-world survival outcomes of patients with DLBCL who received ASCT in Germany.

Patients and Methods: Using a large claims database of the German statutory health insurance with 6.7 million enrollees between 2010 and 2019, we identified patients who were newly diagnosed with DLBCL, had no other cancer co-morbidity, and received ASCT (index date). Pre-defined treatment lines were used to identify the utilization of ASCT. Unadjusted overall survival (OS) from index was plotted by means of the Kaplan-Meier estimator for the overall population and for those patients who had/had not a subsequent treatment line after ASCT. Furthermore, we performed a Cox proportional hazard survival regression with the following co-variates: age, sex, Charlson Comorbidity Index (CCI), time from start of first line treatment to ASCT, and presence of subsequent treatment after ASCT.

Results: 112patients received ASCT. Among them, 39.3% received subsequent treatment after ASCT. Time to ASCT was on average 11.7 months for the entire cohort. Median OS estimated by Kaplan-Meier was 83.4 months for the entire cohort and 27.8 months for patients who received subsequent treatment. Median OS was not estimated for the group without subsequent treatment, for whom the Kaplan-Meier curve reached a plateau at just under 90%, assuming a potential cure. Finally, the only variable significantly reducing the OS was the presence of subsequent treatment after ASCT in a time-dependent model.

Conclusion: Given the high survival outcomes, ASCT is a valuable treatment option for relapsed/refractory DLBCL. However, only a small subset of patients is fit enough to benefit from this treatment and patients who fail ASCT have a poor prognosis. Therefore, novel therapeutic approaches are required to address these unmet medical needs.
Epigenetic Control of B Cell Autoimmunity By Jmjd1c
Authors: Zhang Yongguang , Yu Mei , Zheng Yongwei , Chen Yuhong , Izaguirre-Carbonell Jesus , Zhu Nan , Wen Renren , Wang Demin
Published: December 2023

Emerging evidence underscores the importance of e pigenetic modifications in controlling B-cell development and function. While Jmjd1c, a member of the l ysine - specific histone demethylase 3 subfamily, is known to regulate the self-renewal of normal hematopoietic stem cells and leukemia stem cells, its impact on B cell biology is unknown. To explore Jmjd1c's role in B cells, we developed B-cell-specific Jmjd1c-deficient mice through crossbreeding Jmjd1c “floxed” mice with B cell-specific Mb1-Cre mice. The absence of Jmjd1c did not impact B-cell development, but it did lead to significant alterations in basal B cell antibody production. Specifically, we observed skewed isotypes from IgG2c and IgG3 to IgG1 and IgG2b. Additionally, Jmjd1c deficiency affected antigen-specific immune responses upon T-dependent and T-independent antigen challenges, implying a crucial role of Jmjd1c in B cell function. Furthermore, auto-antibody array assays revealed increased self-reactive antibody production in Jmjd1c-deficient animals, indicating the role of Jmjd1c in regulating self-reactive B cells. At a cellular level, Jmjd1c-deficient B cells showed elevated expression of CD86, a canonical marker of B cell activation. Moreover, these B cells exhibited increased BCR-induced cell proliferation, suggesting hyperactivity to antigens, including self-antigens. Notably, Jmjd1c deficiency induced systemic autoimmune disorder in a BM12-induced systemic lupus erythematosus (SLE) animal model, resulting in amplified auto-antibody production and CD86 expression.

To investigate the underlying molecular mechanisms, RNA sequencing analysis demonstrated upregulation of B cell receptor signaling related genes, NF-κB pathway signature genes, and cell cycle-related genes in Jmjd1c-deficient B cells upon anti-IgM stimulation, consistent with their hyperresponsiveness. Of particular interest, we observed upregulation of genes encoding the 26S proteasome complex, a pivotal regulator of diverse cellular processes including NF-κB pathway activation and self-antigen presentation. To explore the epigenetic aspect of these genes, we employed CUT&Tag sequencing and found that Jmjd1c deficiency correlated with increased levels of H3K36me1, a marker of active chromatin and gene transcription. Moreover, the H3K36me1 levels of B cell receptor signaling related genes, NF-κB pathway signature genes, and cell cycle-related genes were significantly augmented in Jmjd1c-deficient B cells upon anti-IgM stimulation. Notably, 26S proteasome subunit genes that were upregulated in Jmjd1c-deficient B cells exhibited higher H3K36me1 modification at the promoter regions.

Collectively, our study provides valuable insights into the epigenetic control of B cell autoimmunity through the regulation of H3K36 mono-methylation by Jmjd1c. This mechanism plays a critical role in B cell activation and function, including the regulation of key genes such as those encoding the 26S proteasome complex.
HT-6184 Inhibits the Formation of the NLRP3 Inflammasome in Human Monocytic THP-1 Cells
Authors: Bearss Benjamin , Bursey Devan , Avei Taylor , Mollard Alexis , Bearss Jared , Janat-Amsbury Margit Maria , Bearss David J.
Published: December 2023

Background

The NLRP3 inflammasome is a multiprotein complex composed of several proteins, including NLRP3, ASC, Nek-7, and pro-caspase-1. Many immune cell populations express the NLRP3 inflammasome. In the presence of various exogenous stimuli such as lipopolysaccharide (LPS) or extracellular ATP, the NLRP3 inflammasome components assemble and become activated, releasing pro-inflammatory cytokines IL-1β and IL-18. One of the critical steps in inflammasome assembly is the recruitment of ASC and the formation of ASC protein aggregates called specks. These specks are large enough to be measured through microscopy, and the ASC speck count indicates NLRP3 activation. The release of IL-1β and IL-18 and the upregulation of NLRP3 inflammasome component genes have been observed in many inflammatory and hematological disorders, suggesting the NLRP3 pathway is involved in the disease pathogenesis of many diseases.

Purpose

HT-6184 is a small molecule that blocks the assembly of the NLRP3 inflammasome through allosteric binding to the protein Nek7. Here we demonstrate the ability of HT-6184 to modulate NLRP3 inflammasome formation by measuring ASC specks using a human monocytic THP-1 cell line engineered to express a GFP-labeled ASC protein.

Methods

THP1-ASC-GFP cells were cultured in RPMI 1640 with 10% heat-inactivated FBS, 100 μg/mL normocin, and 100 μg/mL Pen-Strep. Cells were seeded at 400,000 cells per well in a 96-well plate and left overnight. Cells were then treated with vehicle or LPS for 3 hours. Following LPS stimulation, cells were treated with vehicle or HT-6184 for 2 hours. After incubation, cells were treated with 5 μM Nigericin and imaged in 3-minute intervals for 1 hour. The images were analyzed for specks.

Results

By treating THP1-ASC-GFP with LPS and nigericin, we induced the formation of ASC specks and visualized them using fluorescent microscopy. Cells treated with 200 nM HT-6184 had significantly fewer ASC specks than the LPS and nigericin-treated control. ASC speck inhibition by HT-6184 was dose-dependent, as inhibition of speck formation decreased with lower doses of HT-6184.

Conclusions

HT-6184 effectively prevents the formation of ASC specks in THP-1-ASC-GFP cells. Since THP-1 cells are a monocytic cell line, this data suggests our ability to inhibit the NLRP3 pathway in hematological disorders effectively. By preventing inflammasome formation and activation, we expect to be able to reduce disease burden in hematological diseases.
Ex-Vivo Human Whole Blood Assay for Evaluating the Pharmacodynamics of HT-6184: Reduction in NLRP3 Inflammasome Mediated Cytokine Production
Authors: Avei Taylor , Bursey Devan , Bearss Benjamin , Mollard Alexis , Bearss Jared , Janat-Amsbury Margit Maria , Bearss David J.
Published: December 2023

The NLRP3 inflammasome is an intracellular multiprotein complex that plays a crucial role in the innate immune response by mediating the maturation and release of pro-inflammatory cytokines IL-1 beta, and IL-18. The release of these cytokines leads to the downstream release of TNF-alpha, IL-6, which propagate a robust inflammatory response. Dysregulation of the NLRP3 inflammasome pathway has been linked to various inflammatory diseases, suggesting the possibility for novel therapeutic strategies targeting NLRP3 activation. NLRP3 has been linked to regulating hematopoietic stem progenitor cell (HSPCs) development, expansion, release, and mobility. In addition, various hematological disorders such as myelodysplastic syndrome, myeloproliferative neoplasms, acute and chronic leukemias, and graft-versus-host disease (GvHD) have all been shown to involve NLRP3 inflammasome activation. Given its significant role in these conditions, inhibiting the NLRP3 inflammasome may be a novel therapeutic strategy in hematology and hemato-oncology

This study utilized an ex-vivo human whole blood assay to investigate the pharmacodynamics of HT-6184, an allosteric Nek7 inhibitor. We used LPS and ATP to prime and activate the NLRP3 inflammasome and examined the effects of pre-treatment with various concentrations of HT-6184 (starting at 1 mM) on the production of critical pro-inflammatory cytokines, such as IL-1b, IL-18, IL-6 and TNF-a. Notably, preincubation with HT-6184 at concentrations as low as 4.1 nM led to a significant reduction in cytokine production (p<0.0001). Specifically, TNF-α and IL-6 were reduced by 38% and 58%, respectively, while IL-1β and IL-18 were decreased by 44% and 53% respectively.

These results provide further evidence of the ability of HT-6184 to modulate the activity of the NLRP3 inflammasome in a complex biological milieu. Our study suggests that HT-6184 has significant therapeutic potential in treating a broad spectrum of inflammasome-mediated diseases. In addition, this assay has the potential to be used to measure the pharmacodynamic properties and the efficacy of HT-6184 and other inflammasome inhibitors in future healthy volunteer clinical trials. In conclusion, this study highlights the importance of innovative and physiologically relevant assay systems in identifying and evaluating potential therapeutic agents for inflammatory diseases.
Cryo-EM Structure of Coagulation Factor VIII Bound to a Patient-Derived Anti-A2 Domain Antibody Inhibitor
Authors: Childers Kenneth C , Vaughan Jordan , Lollar John S. , Doering Christopher B , Coxon Carmen Helena , Spiegel P. Clint
Published: December 2023

Coagulation factor VIII (FVIII) promotes hemostasis by binding to activated factor IX on activated platelet surfaces to form the intrinsic tenase complex. The formation of inhibitory antibodies against FVIII occurs in approximately 30% of congenital hemophilia A patients receiving FVIII replacement therapy, significantly complicating treatments. By identifying the FVIII amino acids which bind to pathogenic inhibitory antibodies, FVIII replacement therapies can be engineered with reduced immunogenicity. Here, we aimed to structurally characterize FVIII bound to NB11B2, a patient-derived antibody inhibitor which binds to the A2 domain and disrupts tenase complex activity, using cryogenic electron microscopy (cryo-EM). The FVIII:NB11B2 structure was solved to a global resolution of 4.0 Å, representing the first structure of FVIII bound to an anti-A2 domain antibody inhibitor. Structural analysis revealed that NB11B2 binds to a discontinuous epitope encompassing FVIII residues S470-P472, R490-K493, and P502-E507. Furthermore, our structure identified a 20° swivel-like movement adopted by the C2 domain while the C1 domain remains immobile, consistent with a previous X-ray crystal structure of FVIII bound to an anti-C1 domain inhibitor. These results provide the structural basis for inhibition of FVIII by NB11B2 and elucidate a novel role for portions of the FVIII A2 domain in regulating tenase complex assembly and/or activity.
Biophysical Characterization of the Thermodynamic Basis and Binding Orientation for Factor VIII Membrane Association
Authors: Avery Nathan G , Childers Kenneth C , Lu Selena , Korus Patrick , McGlone Justin , Jacobs Mady , Richardson Tera , McGill Juliet , Croney Kayla , Vaughan Jordan , McCarty James , Spiegel P. Clint
Published: December 2023

After vesicular damage occurs, factor VIII (fVIII) is proteolytically activated by thrombin and binds to activated factor IX (fIX) on the surface of activated platelets during the blood coagulation cascade to form the intrinsic “tenase” complex. Once activated, fVIII dissociates from von Willebrand factor and C1 and C2 domains bind to activated platelet surfaces. Previous studies have identified that the C1 and C2 domains bind to negatively charged membranes through both surface exposed hydrophobic residues located in the beta hairpin loops and adjacent positively charged residues. The thermodynamics of fVIII lipid binding are currently uncharacterized and our current understanding of the binding orientation of the C1 and C2 domains of fVIII on lipid membranes is incomplete. In this study, we investigate the thermodynamics and binding orientation of the C domains via mutagenesis to both surface exposed hydrophobic residues and adjacent charged residues. Binding measurements between phosphatidylserine (PS) containing lipid nanodiscs and fVIII C domains have revealed that the C1 domain lipid binding is driven by enthalpic interactions, whereas the C2 domain binding is driven by entropic interactions. Binding experiments on fVIII C2 domain mutants for both surface exposed hydrophobic and positively charged residues have revealed differential disruption to lipid binding. Lastly, we have utilized molecular dynamics to perform simulations on the C1 and C2 domains binding to lipid nanodiscs to elucidate the binding orientation of the C domains and identify novel residues that interact with the membrane. Our results from these simulations agree with previous work, showing that C domain lipid binding centers on R2163 (C1) and R2320 (C2).