bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel Anti-TMPRSS6 Monoclonal Antibody Provides a Potential Therapeutic Approach for the Treatment of Polycythemia Vera
Authors: Chen Buxin , Wang Jean , Huang Lei , Bujold Matthew , Du Xin
Published: December 2023

Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) triggered by activating mutations in JAK2, an important kinase in the principal signaling pathway of the erythropoietin receptor. Most of the clinical manifestations of PV are caused by increased erythrocyte count, leading to increased risk of pulmonary hypertension and thrombosis. The mainstay of therapy for PV is therapeutic phlebotomy to reduce the hematocrit (HCT) level and minimize the risk of thrombosis, however, the effect of phlebotomy is transient until patients become iron deficient. Therefore, systemic iron restriction could be effective in treating PV patients.

Transmembrane serine protease 6 (TMPRSS6) is a negative regulator hepcidin, the master regulator of iron homeostasis. Increasing genetic, preclinical and clinical evidence has demonstrated that inhibition of TMPRSS6 can potently elevated hepcidin levels, thereby achieving systemic iron restriction.

Previously, we reported the generation of anti-TMPRSS6 antibody MWTx-003 and its efficacy in reducing iron overload and improving ineffective erythropoiesis in a mouse model of β-thalassemia. In this study, we explored therapeutic application of MWTx-003 in PV using a Jak2 V617F bone marrow transplantation mouse model. The mouse model exhibits typical PV phenotypes of erythrocytosis and elevated HCT levels. In the PV mice, treatment with MWTx-003 (2-10 mg/kg, i.p., every 4 days for 3 weeks) led to significantly reduced HCT levels, red blood cell (RBC) counts and hemoglobin (HGB) concentration that were comparable to that of wildtype control mice. These effects were evident in a dose-dependent manner and observed as early as two weeks into treatment. It is worth noting that in the 10 mg/kg dose group, a worsening splenomegaly and expansion of early erythroid progenitors were observed after 3 weeks of treatment, and these findings are typical of iron-restricted erythropoiesis in mice. Taken together, the anti-TMPRSS6 antibody offers a promising therapeutic approach in the management of PV, where erythrocytosis and high HCT levels are associated with poor outcomes. A Phase I clinical trial with MWTx-003 in healthy volunteers has been initiated in China.
The Selective CDK9 Inhibitor VIP152 Overcame Therapeutic Resistance in Mantle Cell Lymphoma
Authors: Lee William , Jiang Vivian Changying , Zhang Tianci , Yan Fangfang , Cai Qingsong , McIntosh Joseph , Liu Yang , Wang Michael L.
Published: December 2023

Introduction

Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin lymphoma. Even most advanced therapies are generally followed by resistance and relapse, creating an urgent need for novel drugs to overcome resistance. Our and other studies have shown transcriptome is abnormally reprogrammed in patients with therapeutic resistance. CDK9, as a master transcriptional regulator, is overexpressed in tumors after subsequent relapses to BTK inhibitors (BTKi) and CAR-T therapy. VIP152 is a highly potent and selective CDK9 inhibitor under preclinical and early clinical development. As yet, its potency and safety in patients with MCL, especially those with multiple therapeutic resistances, has not been addressed.

Method

We performed single-cell RNA sequencing and bulk RNA sequencing to investigate enriched cancer hallmarks in MCL. Cell viability assays were conducted in a time- and dose-dependent manner to assess the in vitro efficacy of VIP152. Apoptosis assays and western blots were used to identify relevant pathways in bypassing resistances. Cell line-derived xenograft and patient-derived xenograft models of MCL created in immunodeficient mice were used to monitor tumor growth and thus determine the in vivo efficacy of VIP152 in overcoming therapeutic resistances.

Results

Based on single-cell RNA sequencing and bulk RNA sequencing, we found that the cancer hallmark MYC-TARGETS is progressively enriched in MCL patients with subsequent failures to BTKi and CAR-T therapy. CDK9 was found to be overexpressed in CAR-T-relapsed patients compared to CAR-T-naïve but BTKi-refractory/relapsed patients. We hypothesized that CDK9 overexpression and activity may greatly contribute to the transcriptomic reprogramming in patients with therapeutic resistance. Targeting CDK9-mediated transcription with the specific CDK9 inhibitor VIP152 was highly effective in MCL cell lines at low nanomolar range (55-172 nM). VIP152 robustly induced apoptosis in a time- and dose-dependent manner, which was largely dependent on caspase-3 activity. It markedly inhibited CDK9 phosphorylation and down streaming signaling, and suppressed the expression of short-lived proteins like MYC, MCL-1, and cyclin D1 that are critical for MCL cell survival and proliferation. VIP152 effectively inhibited the tumor growth of MCL cell line-derived xenograft models in vivo (P<0.001) with no apparent adverse effects in mice. Moreover, VIP152 markedly inhibited the growth of three patient-derived xenograft tumor models, overcoming BTKi resistance (P<0.001), BTKi-venetoclax dual resistance (P<0.01), and BTKi-CAR-T dual resistance (P<0.001).

Conclusion

These data showed that CDK9 was a promising target for treating MCL and using VIP152 to specifically target it was efficacious in overcoming a variety of therapeutic resistances in MCL.
Molecular Determinants of Sensitivity to Polatuzumab-Vedotin in Diffuse Large B-Cell Lymphoma
Authors: Corcoran Sean R , Choi Jaewoo , Fenner Rachel E , Yu Xin , Scheich Sebastian , Hsiao Tony , Schevchenko Galina , Morris Vivian M , Phelan James D , Papachristou Evangelia K , Kishore Kamal , D'Santos Clive S , Ji Yanlong , Pittaluga Stefania , Wright George , Urlaub Henning , Pan Kuan-Ting , Oellerich Thomas , Muppidi Jagan R. , Hodson Daniel James , Staudt Louis M.
Published: December 2023

Purpose: Polatuzumab Vedotin (Pola-V) is an antibody-drug conjugate directed to the B cell surface antigen CD79B. When combined with conventional immunochemotherapy, Pola-V improves outcomes in DLBCL overall; however, there is noted heterogeneity in response to Pola-V, with germinal center b-cell (GCB) DLBCL showing no added benefit to the addition of Pola-V compared to standard immunochemotherapy. We aimed to identify molecular determinants of sensitivity or resistance to Pola-V. We hypothesized that these might lead us to innovative strategies to improve on-target tumor killing by Pola-V, or to find novel biomarkers that predict drug resistance.

Methods: We employed combined drug-sensitization and CD79B-sorted CRISPR-Cas9 screening to identify molecular determinants of sensitivity to CD79B-directed, tumor killing by Pola-V in 9 cell lines representing different molecular subtypes of DLBCL.

Results: Our results reveal the striking impact of epitope glycosylation, specifically a2,6 sialylation, on the binding of Pola-V to CD79B and thereby its ability to kill tumor cells. Specifically, we identify the exact glycosylated residues on CD79A and CD79B which create a glycan shield around the Pola-V binding site, precluding binding to its target. We show how genetic, pharmacological and enzymatic approaches that remove terminal sialic acid residues from these N-linked glycans lead to enhanced tumor killing by Pola-V. We hypothesize and test multiple methods of targeting this pathway in order to enhance Pola-V killing both in vitro and in vivo. Finally, we reveal a previously unappreciated role for the ubiquitin ligase KLHL6 in regulating CD79B protein abundance and surface expression of the B cell antigen receptor (BCR), including how this pathway is used by physiological germinal center B cells and how it is corrupted to enhance BCR expression in GCB DLBCL.

Conclusions: These findings unravel the molecular basis of response heterogeneity to Pola-V and identify approaches that might be deployed therapeutically to enhance the efficacy of CD79B-specific tumor killing. In addition, we identify how KLHL6 determines expression of the BCR in both physiological and malignant germinal center B cells, and how KLHL6 mutation may modulate sensitivity of GCB DLBCL tumors to Pola-V.
Clinical Effects of Tacrolimus Blood Concentrations Early after Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Kubo Hiroyuki , Imataki Osamu , Fukumoto Tetsuya , Ishida Tomoya , Hamasaki Kubo Yukiko , Yoshida Shunsuke , Uchida Shumpei , Uemura Makiko , Fujita Haruyuki , Kadowaki Norimitsu
Published: December 2023

Tacrolimus (TAC) plus short-term methotrexate (stMTX) is used for graft-versus-host disease (GVHD) prophylaxis after allogeneic hematopoietic stem cell transplantation (allo-HSCT). TAC blood concentrations are frequently adjusted to enhance the graft-versus-leukemia/lymphoma effect or attenuate severe GVHD. Limited information is available on the clinical impact of these adjustments and the optimal time to perform them in order to achieve good clinical outcomes. We retrospectively analyzed 211 patients who underwent allo-HSCT at our institutes. Higher TAC concentrations in week 3 correlated with a significantly higher cumulative incidence of relapse (CIR) (p=0.03) and lower non-relapse mortality (p=0.04). The clinical impact of high TAC concentrations in week 3 on CIR was detected in the refined disease risk index: low/intermediate (p=0.04) and high (p<0.01), and conditioning regimens other than cyclophosphamide/total body irradiation and busulfan/cyclophosphamide (p=0.07). Higher TAC concentrations in week 1 correlated with a lower grade 2-4 acute GVHD rate (p=0.01). Higher TAC concentrations in weeks 2 and 3 correlated with slightly lower (p=0.05) and significantly lower (p=0.02) grade 3-4 acute GVHD rates, respectively. Higher TAC concentrations in weeks 1 and 3 were beneficial for severe acute GVHD in patients with a human leukocyte antigen-matched donor (p=0.03 and p<0.01, respectively), not treated with anti-thymocyte globulin (p=0.02 and p=0.02, respectively), and receiving three stMTX doses (p=0.03 and p=0.02, respectively). The clinical impact of TAC concentrations varied according to patient characteristics, including disease malignancy, conditioning regimens, donor sources, and GVHD prophylaxis. These results suggest that TAC management needs to be based on patient profiles.
Hijacking of the Fibrinolytic System By B-Cell Acute Lymphoblastic Leukaemia and Its Therapeutic Targeting
Authors: Minciacchi Valentina , Karantanou Christina , Zanetti Costanza , Kumar Rahul , Krack Theresa , Thomasberger Nathalie , Llavona Pablo , Hartmann Sylvia , Maguer-Satta Veronique , Lefort Sylvain , Putyrski Mateusz , Ernst Andreas , Meduri Eshwar , Huntly Brian , Ruf Wolfram , Krause Daniela S.
Published: December 2023

The fibrinolytic system plays a role in the mobilization of hematopoietic stem cells from their bone marrow microenvironment (BMM), but its function in leukemias is not well understood. The fibrinolytic system consists of the proenzyme plasminogen and its active form, plasmin, a serine protease. The activation of plasmin is tightly regulated by activators such as tissue plasminogen activator (tPA) and various inhibitors. A role of this plasminogen activation system in the microenvironment of solid tumors is being unravelled, but a function of this pathway in the BMM, where hematological cancers usually originate, has so far been elusive.

Transcriptome analysis of mesenchymal cells in the BMM of mice with different leukemias had revealed annexin A2 ( Anxa2), an initiator of fibrinolysis, to be differentially expressed. Using an ANXA2-deficient BMM as a model for impaired fibrinolysis, we showed that ANXA2 promotes the activation of plasmin, leading to decreased levels of the extracellular matrix (ECM) protein fibronectin in the BMM and accelerating the progression of BCR-ABL1 + B-cell acute lymphoblastic (B-ALL), but not MLL-AF9 + acute myeloid leukemia. Consistently, induction of B-ALL in tPA-deficient mice led to fibronectin accumulation in the BMM and significant survival prolongation.

The dense ECM in an ANXA2-deficient BMM is shown to entrap insulin-like growth factor (IGF) and to specifically reduce mTORC2-dependent signalling and proliferation in B-ALL cells. Conversely, B-ALL cell-derived interleukin (IL)-6 conditions hepatic generation of plasminogen, the precursor of plasmin, perpetuating this circuit. Treatment with -aminocaproic acid (EACA), an anti-hemorrhagic drug, which inhibits plasmin activation, reduces tumor burden and prolongs survival in syngeneic and xenogeneic murine models via increased fibronectin in the BMM. Human data confirm that IGF1 and fibronectin staining in trephine biopsies are correlated and that the mTORC2 pathway is distinctly activated in B-ALL. Higher expression of SERPINF2 (α2-antiplasmin), an inhibitor of plasmin, in human B-ALL cells leads to superior outcome. In summary, B-ALL-derived IL-6 impacts hepatic generation of fibrinolytic agents influencing B-ALL progression via ECM remodeling and regulation of the availability of B-ALL-supportive growth factors. Inhibition of plasmin-mediated degradation of the ECM by EACA may be beneficial as adjunct therapy in B-ALL.
A Phase 1 Study to Evaluate the Pharmacokinetics of Single High Doses of Iptacopan
Authors: Schmouder Robert , Kaetterer Noemi , Kulmatycki Kenneth , Nidamarthy Prasanna Kumar
Published: December 2023

Introduction: Iptacopan is a first-in-class, oral, selective inhibitor of factor B, a key component of the complement system alternative pathway. Inhibition of the complement system represents a potential mechanism for treatment of several diseases including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. At the current clinical dose of 200 mg twice daily, the steady-state area under the concentration curve (AUC) and peak concentration (C max) are 25,600 ng•hr/L and 4,120 ng/mL, respectively. In this randomized, participant-blinded, placebo-controlled phase 1 study, we evaluated the pharmacokinetics (PK) of single high doses of iptacopan.

Methods: Iptacopan single doses of 400 mg (n=6), 800 mg (n=9), and 1200 mg (n=9) were included in this study, representing ≤6-fold higher doses than the clinical dose of 200 mg twice daily. The study population included healthy men and women aged 18-55 years. The log-transformed PK parameters AUC and C max were analyzed separately using a linear model with treatment as fixed effect.

Results: Twenty-four patients received iptacopan and 8 received placebo. The median age of participants in the 400-, 800-, and 1200-mg groups was 51.5, 35, and 36 years, respectively. Following a single oral administration, iptacopan was rapidly absorbed with C max at 1.06, 2.00, and 2.52 hours for 400-mg, 800-mg, and 1200-mg doses, respectively ( Table). Variability (CV% geometric-mean) in iptacopan plasma concentrations were low to moderate (16.6%-30.6%) at C max. After administration, iptacopan plasma concentration-time profiles increased over the dose range of 400 to 1200 mg and were biphasic, with an inflection point at approximately 12 hours ( Figure). Compared to the 200-mg twice daily steady-state PK, the 1200-mg single dose resulted in C max and AUC exposure multiples of 4.0- and 6.5-fold, respectively. The mean apparent elimination half-life was similar across all doses, ranging from 20 to 21 hours. Iptacopan was well tolerated with no moderate or severe treatment-emergent adverse events reported.

Conclusion: Iptacopan was rapidly absorbed after administration of single oral doses ranging from 400 to 1200 mg. Variability in iptacopan plasma concentrations were low to moderate over the dose range studied and half-lives were similar. Supratherapeutic, single doses up to 1200 mg were well tolerated and resulted in substantial multiples of iptacopan systemic exposure.
CRIP1 Involves the Pathogenesis of Multiple Myeloma Via Dual-Regulation of Proteasome and Autophagy
Authors: Yu Zhen , Tang Peixia , Xu Zhenshu , Qiu Lugui , Hao Mu
Published: December 2023

Multiple myeloma (MM) is an incurable hematological malignancy of plasma cells. The maintenance of protein homeostasis is critical for the survival of MM cells. Elevated paraprotein in MM cells would be cleared by proteasomes or lysosomes, which are independent but inter-connected with each other. Proteasome inhibitors (PIs) work as the backbone agent and successfully improved the outcome of patients, but the increasing activity of autophagy suppresses the sensitivity to PIs treatment. In the present study, we elucidated that CRIP1 was a critical player in protein homeostasis via the dual regulation of proteasome and autophagy in MM. A combination analysis of RNA-seq, Co-IP and Tandem affinity purification/mass spectrometry identified that high-level CRIP1 represents a biomarker for relapsed/refractory MM with shorter overall-survival of patients. CRIP1 plays a critical role in MM progression and PIs resistance by binding to deubiquitinase USP7 and proteasome coactivator PA200. CRIP1 promotes proteasome activity and autophagosome maturation in MM by stabilizing PA200. Therefore, our findings clarified the pivotal roles of CRIP1/USP7/PA200 complex formation in ubiquitin-dependent proteasome degradation and autophagy maturation involved in the progression of MM.
Lbl-034, a Highly Differentiated T-Cell Engaging Bispecific Antibody Targeting GPRC5D for the Treatment of Relapsed or Refractory Multiple Myeloma
Authors: Wang Baohui , Huang Xiao , Sun Jianming , Qin Yurong , Shang Hongyan , Li Tingting , Wu Guojin , Chen Min , Wang Hailin , Zhang Peng , Jiang Duqing , Liu Tongjun , Ye Mi , Cai Shengli , Lai Shoupeng , Kang Xiaoqiang , Ling Hong
Published: December 2023

G protein-coupled receptor, class C group 5 member D (GPRC5D) is a type-C 7-pass transmembrane receptor protein that selectively overexpresses on malignant plasma cells in multiple myeloma (MM). The expression of GPRC5D is rarely seen in normal tissues or cells, and is not detectable on other hematopoietic derived cells, therefore indicating that GPRC5D may be a novel target for anti-multiple myeloma therapy.

LBL-034, a novel T-cell engaging bispecific antibody targeting GPRC5D, is developed with a specifically designed molecular format and unique 2:1 structure. It showed a potent binding on cell lines with various levels of GPRC5D expression, while its binding to CD3 on Jurkat cells was much lower examined by flow cytometry. Furthermore, LBL-034 re-directed engagement of GPRC5D+ cells and T cells was validated by gating engaged cell population with flow cytometry and confirmed visually with laser scanning confocal microscopy.

LBL-034 induced potent T cell dependent cell killing (TDCC) of various types of GPRC5D expressing cells (high to low expression: MM.1R, NCI-H929, MOLP-8 and RPMI 8226 cells), and elevated activation marker (CD25, CD69) expression and cytokine release (IFN-γ, TNF-α, IL-6). LBL-034 demonstrated weak binding to CD3 and minimal effects on cytokine release in the absence of GPRC5D expressing cells and it indicates that LBL-034 activates T cells only in the presence of GPRC5D target cells. LBL-034 was further examined in the MC38-GPRC5D syngeneic model and NCI-H929 xenograft model. Robust antitumor efficacy was observed at the low dose of LBL-034 at 1 mg/kg and 0.3mg/kg respectively.

LBL-034 GLP toxicology study with repeated doses was conducted in cynomolgus monkeys. 5, 15 and 50 mg/kg of LBL-034 was given intravenously with Q1W for up to 5 doses. LBL-034 was well tolerated with a good safety profile as determined by pharmacology, pathology and biochemistry analysis. The NOAEL of LBL-034 in cynomolgus monkeys was confirmed at 50 mg/kg.

In summary, LBL-034 is a novel T cell engaging bispecific antibody targeting GPRC5D expressing R/R MM with well differentiated binding of GPRC5D and CD3 to enhance anti-tumor activity, while mitigate the risk of CD3-induced CRS, showing a great in vitro potency and in vivo anti-tumor efficacy. LBL-034 IND has been approved by both FDA and NMPA and a FIH study in patients with R/R MM will begin in the 2 nd half of 2023.
Evaluating Chatgpt As an Adjunct for Analyzing Challenging Case
Authors: Liu Xiaoni , Song Ying , Lin Hai , Xu Yiying , Chen Chao , Yan Changjian , Yuan Xiaoliang , Bao Fang , Jing Hongmei , Wu Chaoling , Zhang Weilong
Published: December 2023

Introduction

Despite the rapid development of medicine, clinicians still faced with many challenging cases in practice. For challenging cases, most inexperienced doctors are unable to make a clear diagnosis in the first place, resulting in misdiagnosis and delayed disease. Recently, OpenAI released a new Chatbot model, called ChatGPT (Generation Pre training Converter), which is an artificial intelligence (AI) system that uses reinforcement learning model based on human feedback for training. There have been many research reports on the application of ChatGPT in the medical field, and it has been proven to have a wide range of clinical applications. In our study, we aimed to assess the accuracy and reliability of ChatGPT in diagnosing and treating challenging case.

Methods

We provided 2 challenging cases. Case 1 provided a medical history and auxiliary test results for ChatGPT, and then ChatGPT answered the possible diagnosis, diagnostic basis, further auxiliary examination and treatment. Case 2 provides negative symptoms and auxiliary test results for ChatGPT, which answers all possible diagnoses. Conduct a comprehensive evaluation and scoring of the results of ChatGPT responses by 5 senior physicians. A score of 10 is the perfect score.

Results

The analysis results of 2 challenging cases by ChatGPT were comprehensively evaluated by senior experienced professional doctors and scored 9 points. We found that ChatGPT's analysis of challenging case is comprehensive. Especially for the diagnosis and treatment of diseases, ChatGPT analyzes all possible diagnoses and provides accurate treatment plans through the provided medical history, auxiliary examination results, and treatment processes. ChatGPT can directly interpret the auxiliary examination results, such as high level of procalcitonin (PCT), abnormal kidney function, and with negative results can rule out certain diseases, such as ANA, ENA, and ANCA negative, ruling out the possibility of autoimmune diseases. ChatGPT can summarize all the diagnoses including rare diseases, based on the results of an auxiliary test, and explain the cause of the disease and the pathogenesis in detail.

Conclusions

Our study shows that ChatGPT can provide important reference value as an adjunct to clinicians in challenging case analysis. In particular, it can assist inexperienced doctors in remote areas and underdeveloped medical conditions to make clear diagnosis and treatment as soon as possible, avoid unnecessary referral to higher-level hospitals, reduce medical burden, and reduce waste of medical resources. In the medical field, ChatGPT needs more research to evaluate in clinical applications and become a better adjunct tool in clinical work.
Comprehensive Geriatric Assessment and Geriatric Intervention in Older Patients with Newly Diagnosed Lymphoma: A Prospective Study
Authors: Lopez Garcia Alberto , Hormigo Ana Isabel , Mahillo Ignacio , Askari Elham , Morillo Daniel , Perez Saez Maria Angeles , Prieto Elena , Riesco Miriam , Urrutia Celia , Martinez Peromingo Francisco Javier , Gonzalez Montalvo Juan Ignacio , Cordoba Raul
Published: December 2023

Introduction: Aggressive and indolent non-Hodgkin lymphomas have a median age at diagnosis of 70 years. Above this age, this population is very heterogeneous and clinical outcomes may be worsened due to patient's frailty phenotype. Very few studies of comprehensive geriatric assessment (CGA) guided approach to care have been published.

Methods: A prospective follow-up with baseline CGA were performed in a cohort of consecutive patients older than 70 years with newly diagnosed Hodgkin and non-Hodgkin lymphoma as standard of care. CGA tools are described in Table 1. Clinical outcome has been analyzed in patients diagnosed between May 2016 and March 2021.

Results: Ninety-three consecutive patients (55.9% of them were women) were included in this analysis, with a median age of 81.1 years (+/- 5.7). 23 patients (24.7%) were classified as robust (type I), 30 patients (32.3%) as pre-frail (type II) with potentially reversable impairments, 38 patients (40.9%) as frail (type III), and only 2 patients (2.2%) as unfit (type IV). Patients' characteristics are shown in Table 1. All patients received antineoplastic therapy. Treatment modifications were carried out in 64.5% of patients based on CGA results. In the follow-up, with a median follow-up 27.3 months (range 18-74 months), the relapse rate was of 25.8%, with no differences between groups. Overall survival was around 2-fold higher among the fit patients (type I) (42.5 ±19.6 months) than in the disabled patient's group (types III-IV) (23.7 ±20.5 months) (p 0.002) Statistically significant differences in overall survival (p 0.002), response to treatment (p<0.001) and likelihood of increased frailty at the end of treatment (p 0.024) were observed among groups, with type III-IV patients showing worse overall survival (Figure 1), lower response rates, and higher incidence of frailty.

Conclusion: Systematic CGA allows physicians to address the functional reserve or resilience in older patients with lymphoma in the need of antineoplastic treatment. A frailty-guide approach to care may allow to improve clinical outcomes in this group of high-risk patients.
Bio-Orthogonally Redirected Bispecific Lentiviral Vectors for In Vivo CAR-T Cell Generation
Authors: Mei Heng , Chen Zhaozhao , Hu Yu , Mei Heng
Published: December 2023

Background: Chimeric antigen receptor T-cell (CAR-T) therapy has emerged as a remarkably efficacious treatment modality in recent years for refractory and relapsed hematopoietic malignancies. However, the exorbitant cost and intricate manufacturing procedure of ex vivo CAR-T cell generation have impeded its broader application. In vivo induced CAR-T therapy offers the potential to circumvent cumbersome manufacturing logistics, thereby providing novel insights into the field of CAR-T treatment. In this study, we have developed a bio-orthogonally redirected bispecific lentiviral vector platform for the reprogramming of circulating T lymphocytes into CAR-T cells and subsequent elimination of tumor cells in vivo.

Methods: To engineer dual-targeting lentiviral vectors (D-LVs), the packing cell lines 293T was labelled with azide groups by glycometabolic bio-orthogonal chemistry (designated N 3-293T) firstly, and a CD3 promoter (CD3p) was fused to CAR transfer plasmid. Secondly, azide groups modified LVs were further surface engineered with anti-CD3 antibody (OKT3) via click chemistry. The conjunction of OKT3 and was analyzed using dot immunobinding assay, confocal microscopy and the in vitro transduction efficiency was evaluated using flow cytometry. To demonstrate the ability of targeted transduction and specific cytotoxicity in vivo, D-LVs were intravenously infused into humanized NOD-scid-IL2Rγnull (huNSG) mice engrafted with Nalm6-luc cells. Subsequently, the tumor burden was monitored using a noninvasive bioluminescence imaging system and the in-vivo CD19 -CAR-T cells existence were detected by flow cytometry.

Results: By displaying OKT3 on the single lentiviral surface and fused CD3p into the key construct, we could achieve targeted delivery of CD19-CAR genes to T cells both in vitro and in vivo.

Conclusion: These results demonstrate the great potential applications of this engineered lentiviral system as a new strategy for inducing CAR-T immunotherapy in vivo and a promising approach for leading personalized treatment.
Preclinical Evaluation of the Efficacy of Human Sirpα Antibodies for B-Cell Lymphoma Immunotherapy in Humanized Mouse Models
Authors: Saito Yasuyuki , Tania Afroj , Komori Satomi , Takai Tomoko , Oduori Okechi S , Kotani Takenori , Funakoshi Yohei , Murata Yoji , Yakushijin Kimikazu , Matsuoka Hiroshi , Minami Hironobu , Manz Markus G. , Matozaki Takashi
Published: December 2023

Tumor-associated macrophages (TAMs) are abundant in the tumor microenvironment and are considered potential targets to enhance cancer immunotherapy against B-cell lymphomas. However, faithful and predictive preclinical tumor models of human B-cell lymphomas for the evaluation of therapeutics targeting human TAMs have not been established yet. To examine the antitumor effects of agents targeting human TAMs in vivo, we here established preclinical tumor xenograft models based on Rag2-/-Il2rg-/- immunodeficient mice that express multiple human cytokines, such as M-CSF, IL-3, GM-CSF, and THPO (MITRG mice), and have been reconstituted with a human immune system (HIS) by transplantation of human CD34+ hematopoietic stem and progenitor cells (HIS-MITRG mice). HIS-MITRG mice supported the growth of both the human cell line (Raji)-derived and diffuse large B cell lymphoma (DLBCL) patient-derived xenograft tumors as well as the infiltration of human macrophages into these tumors. We examined the potential antitumor effect of an antibody to human SIRPα (SE12C3) that inhibits the interaction of CD47 on tumor cells with SIRPα on human macrophages and thereby promotes Fcγ receptor-mediated phagocytosis of tumor cells by human macrophages. Combined treatment with rituximab and SE12C3 inhibited Raji tumor growth in HIS-MITRG mice to a markedly greater extent than did rituximab monotherapy in a therapeutic model started after the engraftment of the tumor. This enhanced antitumor effect was dependent on human macrophages and attributable to enhanced rituximab-dependent phagocytosis of lymphoma cells by human macrophages. Treatment with rituximab and SE12C3 also induced reprogramming of human TAMs towards a proinflammatory phenotype. Furthermore, the combination treatment essentially prevented the growth of DLBCL patient-derived xenograft tumors in HIS-MITRG mice. Our findings thus support the utility of HIS-MITRG mice as a model for the preclinical in vivo evaluation of potential therapeutics, such as antibodies to human SIRPα, that target human TAMs.
Homoharringtonine Enhances the Anti-Leukemia Effect of Venetoclax in Acute Myeloid Leukemia with t(8;21)(q22;q22) Via Regulation of c-Myc/Bim Axis
Authors: Yu Guopan , Yin Zhao , Yao Zurong , Nie Xiaqi , Jin Hua , Zhang Yu , Xu Dan , Jiang Xuejie , Ping Baohong
Published: December 2023

Purpose: Recently, small cases reports showed that acute myeloid leukemia (AML) with translocation (8;21)(q22;q22) [t(8;21) AML] responded poorly to venetoclax (VEN) plus azacitidine (AZA) (VA) regimen. Our previous study revealed homoharringtonine (HHT) enhanced the the killing effect of VA in AML. This study went further to explore the mechanism of VEN-resistance and the synergetic effect of VEN and HHT (VH) in overcoming resistance in t(8;21) AML.

Methods: Patients with/without t(8;21) AML, being treated with VA with/without HHT were included for response assessment. Synergetic effect and mechanism of VEN combined with HHT or AZA was studied in cell lines, primary cells and mice.

Results: Patients with t(8;21) AML acquired significantly lower CR/CRi rate than those without, with VA regimen as frontline (1/5 vs. 56/82, P=0.027) or salvage treatment (1/10 vs. 60/127, P=0.023), while the CR/CRi rate was comparable between the two cohorts with VA plus HHT (VAH) as salvage treatment (9/13 vs. 102/152, P>0.05). Synergetic effect in anti-leukemia was observed in VH but not VA in t(8;21) AML cell lines, primary cells and cell line-derived xenograft mice. Transcriptome RNA-sequencing and western blot detection found c-Myc and its downstring signialing were significantly decreased with the treatment of VH but not VA as compared to the controls. In addition, VEN induced accumulation of c-Myc in t(8;21) cells. Genetic silencing or pharmacological inhibition of c-Myc restored VEN sensitivity in t(8;21) cells. Co-immunoprecipitation assay found Bim, the crucial BH3-only protein, participated in the course of VH-inducing apoptosis. Direct or indirect inhibition of c-Myc increased expression of Bim, while overexpression of c-Myc significantly inhibited the transcription of Bim in t(8;21) cells. CUT&RUN assay revealed two functional sites of c-Myc binded to the promoter of Bim gene.

Conclusion: Abnormal activation of c-Myc might contribute to the resistance of VEN via suppression of Bim in t(8;21) AML. HHT might enhance the killing effect of VEN by regulation of c-Myc/Bim axis.

Key words: acute myeloid leukemia, t(8;21), homoharringtonine, venetoclax, c-Myc
Myopathy with Lactic Acidosis and Sideroblastic Anemia Caused By a Novel PUS1mutation
Authors: Xiong Youzhen , Yang Wenrui , Fan Huihui , Zhang Li , Zhao Xin , Jing Liping , Zhang Fengkui
Published: December 2023

Mutations in the mitochondrial aminoacyl tRNA synthetases (ARSs) are associated with various clinical phenotypes, including myopathy, lactic acidosis, and sideroblastic anemia (MLASA), which is a rare autosomal recessive disorder. Due to oxidative phosphorylation disorder and abnormal iron metabolism in the skeletal muscle and erythroid bone marrow lines, patients show progressive muscle weakness, exercise intolerance, lactic acidosis, siderocyte anemia, and growth retardation, etc. Herein, we report a case of MLASA caused by a novel homozygous missense mutation (c.597G>T p.R199S) in the pseudouridine synthase 1 ( PUS1) gene, causing the loss of protein function. The patient's parents had a consanguineous marriage, with a grandmother in common. All mutations were heterozygous without disease. While the patient was homozygous for the mutation and developed symptoms of anemia, no specific treatment exists for this genetic disease. Deferrization therapy can reduce iron overload, which leads to reduced heme synthesis in such patients but cannot treat the disease. Consanguineous marriage often causes autosomal recessive genetic diseases and prohibiting it can reduce the incidence of these diseases.

The present patient's condition was caused by a mutation in nuclear PUS1, which manifested as SA, hyperlactatemia, iron overload, and muscle damage. PUS1 mutations can be missense, nonsense, or frameshift. We identified a novel missense mutation type in PUS1 in this patient, located on chromosome 12, c.597G>T (p.R199S), which is a homozygous mutation. This was confirmed by the PUS1 analysis revealing homozygosity for the novel mutation. Both the parents and grandmothers were heterozygous for the mutation, but none of them had a clinical manifestation. Combining the patient's family history, medical history, laboratory examination, and genetic characteristics of the disease, the mutation was identified as a pathogenic variant, and the clinical manifestations and genetic findings of the patient expanded the genotype-phenotype spectrum of MLASA.

The possibility of MLASA should be suspected in cases of progressive muscle weakness, growth retardation, exercise intolerance in childhood, SA, and lactic acidosis before or after puberty. With the recognition of MLASA and the advent of NGS, identifying patients with PUS1 mutations characterizing incomplete MLASA syndrome became easier, thus further expanding the phenotypic and genotypic heterogeneities of these mitochondrial disorders. Targeting NGS may also significantly improve the prenatal and preimplantation genetic diagnosis of MLASA, providing better genetic counseling for parents. Genetic diseases lack effective treatment measures, and the curative effect is unsatisfactory. Therefore, prevention is more important. Prevention strategies include avoiding consanguineous marriage, genetic counseling, genetic testing for carriers, and a prenatal diagnosis.
Retrospective Analysis of Clofarabine Salvage Therapy in Refractory Multifocal Langerhans Cell Histiocytosis (LCH)
Authors: Parekh Deevyashali , Lin Howard , McClain Kenneth L. , Gulati Nitya , Eckstein Olive S. , El-Mallawany Nader Kim , Agrusa Jennifer Elise , Allen Carl E
Published: December 2023

Purpose: LCH is a neoplastic inflammatory disorder driven by recurrent somatic mutations in the MAPK pathway in myeloid precursors. Over 50% of patients with systemic LCH are not cured with front-line therapies and data to guide salvage options are limited. In this study, we describe 58 patients with relapsed/refractory LCH who were treated with clofarabine.

Methods: Data were extracted from clinical records of patients treated with clofarabine for LCH by Texas Children's Hospital physicians or collaborators between May 2011 and 2023.

Results: Patients were treated with a median of two chemotherapeutic regimens prior to receiving clofarabine; median age was 3.49 (4 months to 61 years). The typical treatment course was 25mg/m2 daily for 5 consecutive days per month; 19%(11/58) received treatment for less than 6 months; 26%(15/58) for 6-9 months; and 55%(32/58) for 9-24 months. OS in this cohort was 100%(58/58) and PFS was 75% with median 2-year follow-up (range: 5 months to 11 years); The objective response rate (ORR) for all patients was 84% with 9%(5/58) maintaining stable disease and 7%(4/58) developing progressive disease during the study period. Patients treated for LCH-associated neurodegeneration had relatively worse outcomes (ORR: 63%) compared to patients with systemic disease without LCH-ND (ORR: 88%). Patients treated with clofarabine showed significant decreases in the amount of detectable BRAFV600E alleles in their circulating peripheral blood mononuclear cells. Toxicities included cytopenia, vomiting, and bacterial infections, but the majority tolerated chemotherapy in the outpatient setting.

Conclusion: Clofarabine monotherapy has activity against LCH in heavily pretreated patients, the majority of whom achieved durable remission. Prospective multi-center trials are warranted to determine optimal dosing as well as long-term efficacy, late toxicities, relative cost and patient reported outcomes of clofarabine compared to alternative salvage therapy strategies (e.g. MAPK inhibitors) in patients with relapsed/refractory LCH.
The Efficacy and Safety of CLAG±Ida/Mito+Ven As Salvage Therapy of Relapsed/Refractory Acute Myeloid Leukemia
Authors: Yu Guopan , Yin Zhao , Zhang Yu , Jiang Xuejie , Nie Xiaqi , Yao Zurong , Xu Dan , Shi Pengcheng
Published: December 2023

Purpose: As known CLAG±Ida/Mito regimen is recommended by different guidelines as a chioce for salvage therapy of relapsed/refractory acute myeloid leukemia (RR-AML). Recent study showed venetoclax (Ven) added to CLAG+Ida as frontline treatment of AML acquired a good response and well toleration. Yet, how CLAG±Ida/Mito+Ven works in the salvage therapy of RR-AML remains unclear. Aim of this study was to analyze the efficacy and safety of CLAG±Ida/Mito+Ven as salvage therapy of RR-AML.

Methods: A single center, retrospective study was performed. Patients with RR-AML, who were salvagedly treated with CLAG±Ida/Mito+Ven during July 2021 to June 2023, were included. CLAG±Ida/Mito+Ven: cladribine 5mg/m 2 day 1-5, cytarabine 1-1.5g/m 2 day 1-5, PEG-G-CSF 6mg day 0, idarubicin 6mg/m 2 day 1-3 or mitoxantrone 6mg/m 2 day 1-3 (lipo-Mito 20mg/m 2 day 1), Ven 400mg day 2-8.

Results: 33 patients were included, with a median age of 42(15-72) years old, male to female 17/16, de novo/sAML 31/2, refractory/relapsed 17/16, ELN-low /intermediate/high risk 7/10/16. They previously received median 2(1-12) cycles of chemotherapy, including 2 relapsed after allogeneic hematopoietic stem cell transplantation (allo-HSCT), 14 with prior hypomethylating agents (HMA). Among the 33 patients with CLAG+Ven, 3 were combined with Mito, 1 with Ida, 15 with FLT3 inhibitors. After one cycle of salvage therapy, 24(72.7%) acquired response, including 22(66.7%) with CR/CRi, 15(45.5%) with MRD-negative (accounting for 68.2% of the patients with CR/CRi). Multivariate analysis showed that prior HMA, FLT3 mutation and ELN-high risk were the independently poor factors for CR/CRi. With a median follow-up of 3(1-23) months, 15(45.5%) patients were bridged to allo-HSCT, 3(12.5%) relapsed, 5(15.2%) died, including 3 died of leukemia progression and 2 with transplant-related motality. Patients obtaining CR/CRi had significantly longer overall survival (mOS, 19.8(15.8-23.8) months) than those without (mOS, 5.1(2.7-7.5) months, P=0.013). Patients being bridged to allo-HSCT also had significantly better OS than those without. During the salvage therapy, the median duration of agranulocytosis was 19(11-40) days. 25(75.6%) patients underwent manageable infection. The motality of the 30 and 60 day were 0.

Conclusion: CLAG±Ida/Mito+Ven might be a chioce for salvage therapy of RR-AML, with the CR/CRi rate of 66.7%, MRD-negative rate of 68.2% and well toleration.

Key words: CLAG, venetoclax, salvage therapy, relapsed/refractory, acute myeloid leukemia
CSF1R Inhibition Promotes Neuroinflammation and Behavioural Deficits during Graft-Versus-Host Disease in Mice
Authors: Adams Rachael C , Carter-Cusack Dylan , Llanes Genesis T , Hunter Christopher R , Vinnakota Janaki Manoja , Ruitenberg Marc , Vukovic Jana , Bertolino Patrick , Chand Kirat K. , Wixey Julie A. , Nayler Samuel P , Hill Geoffrey R , Furlan Scott N , Zeiser Robert , MacDonald Kelli PA
Published: December 2023

Chronic graft-versus-host disease (cGVHD) remains a significant complication of allogeneic hematopoietic stem cell transplantation. Central nervous system (CNS) involvement is becoming increasingly recognised, where brain-infiltrating donor MHC class II + bone marrow-derived macrophages (BMDM) are implicated mediators of pathology. Colony-stimulating factor 1 receptor (CSF1R)-dependent BMDM are established mediators of cutaneous and pulmonary cGVHD, and clinical trials assessing the efficacy of CSF1R antibody blockade to deplete macrophages are promising. We hypothesized that CSF1R antibody blockade may also be a useful strategy to prevent/treat CNS cGVHD.

We leveraged studies in acute GVHD (aGVHD) to determine the feasibility of an antibody-based approach for depleting brain microglia and macrophages. Moreover, we queried whether CSF1R blockade may be a viable therapy for CNS aGVHD, which has been shown to be mediated by host microglia. Increased blood-brain barrier permeability during aGVHD facilitated CNS antibody access. However, CSF1R blockade early post-transplant induced region-specific microglia depletion and was associated with unexpected exacerbation of aGVHD neuroinflammation, including increased T cell and inflammatory monocyte infiltration, and whole brain cytokine levels. In established cGVHD, vascular changes and anti-CSF1R efficacy was more limited. Anti-CSF1R-treated mice retained donor BMDM, activated microglia, CD8 + and CD4 + T cells, and local cytokine expression in the brain. These findings were recapitulated in GVHD recipients where CSF1R was conditionally depleted in donor CX3CR1 + BMDM. Notably, inhibition of CSF1R signalling post-transplant failed to reverse either acute or chronic GVHD-induced behavioral changes. Taken together, these data suggest that ongoing clinical trials of CSF1R inhibition should assess neurological adverse events in patients.

In contrast to the lack of efficacy with CSF1R inhibition, transfer of Ifngr-/- grafts reduced MHC class II + BMDM infiltration, resulting in improved behaviour. Furthermore, single nuclei RNA sequencing of brains from mice receiving wild-type or Ifngr-/- allografts revealed a disease-associated interferon-inducible mature oligodendrocyte population in the cGVHD brain. This was markedly reduced in recipients of Ifngr-/- grafts, indicating that targeting this axis can promote brain repair beyond the myeloid compartment during cGVHD. In summary, our findings reveal unexpected neurological immune toxicity during CSF1R blockade and highlight the IFNGR pathway as an alternative target for the treatment of cGVHD within the CNS.
TIF1γ Counteracts Ferroptosis to Drive Erythroid Progenitor Differentiation
Authors: Rossmann Marlies P. , Yang Song , Abraham Brian J. , Wang Ying , Young Richard A. , Hekimi Siegfried , Zon Leonard I.
Published: December 2023

Understanding in-vivo mechanisms of hematopoiesis is critical for developing directed blood differentiation approaches to treat blood disorders such as leukemias. Zebrafish moonshine ( mon) mutant embryos defective for transcriptional intermediary factor 1 gamma ( tif1γ), a conserved transcription elongation and chromatin factor, lack red blood cells due to a block in hematopoietic stem cell differentiation along the erythroid lineage. We recently showed that TIF1γ plays a critical role in mitochondrial metabolism, including maintaining adequate coenzyme Q levels. Through a chemical suppressor screen for the mon mutant, we identified inhibitors of the essential mitochondrial pyrimidine synthesis enzyme dihydroorotate dehydrogenase to promote erythroid differentiation in mon mutants due to its functional link to the electron transport chain. In agreement, our in-vivo metabolomics analyses identified nucleotide metabolism as the most significantly altered processes in mon mutants, with elevated levels of uridine monophosphate and low levels of N-carbamoyl-L-aspartate. Interestingly, imbalances in nucleotide metabolism similar to those in mon mutants have also been reported in the presence of active ferroptosis, a pathway of programmed cell death due to iron-dependent lipid peroxidation. Through transcriptome profiling upon tif1γ loss of function in zebrafish embryos at the onset of hematopoiesis, we uncovered an expression signature indicative of activated ferroptosis. gpx4 and fsp1, inhibitors of ferroptosis acting in parallel pathways, were both downregulated, and chac1 and ptgs2, an enhancer and marker of ferroptosis, respectively, were upregulated. In addition, parallel genome-wide expression and chromatin immunoprecipitation analyses identified anti-ferroptotic genes as direct TIF1γ targets in human cells. In support of these results, we found mon mutants to exhibit increased levels of lipid peroxidation. Functionally, tif1γ loss of function synergizes with loss of gpx4 in blocking erythroid differentiation. These results demonstrate a tight coordination of nucleotide metabolism, mitochondrial respiration, and the regulation of lipid peroxidation as a key function of tif1γ-dependent transcription that drives cell fate decisions in the early erythroid lineage. Our work highlights the importance of the plasticity achieved by transcription regulatory processes such as transcription elongation for metabolic processes during lineage differentiation and could have therapeutic potential for blood diseases.