bluuebunny/crossref_metadata_2025 huggingface dataset:
JAK2V617F Activates IL-10R Signalling Potentially through Transactivation of JAK1
Authors: Wadley Lucas , Lai Hew Yeng , Arora Eshika , Huang Helen , Jing Dennis , Fleischman Angela G.
Published: December 2023

Classical myeloproliferative neoplasms (MPNs) arise from somatic mutations in haematopoietic stem cells (HSCs) that lead to unregulated proliferation of mature myeloid cells. The most common mutation associated with MPNs is JAK2 V617F, this causes constitutive JAK-STAT signalling leading to excessive production of mature myeloid cells. In humans, for MPN to arise, a JAK2 V617F HSC must have a selective advantage over WT HSC. However, in mouse MPN models Jak2 V617F HSCs do not display an overt selective advantage. This suggests that unique selective pressures may be present among those predisposed to acquire MPN that allow for the emergence of Jak2 V617F clones.

We have identified defects in the IL-10R signalling pathway among MPN patients, with evidence that this defect is an intrinsic not acquired abnormality. In mice, we found that the blockade of IL-10R extends the cycling of WT but not Jak2 V617FHSC in response to inflammatory stimuli and hastens proliferation-induced WT HSC exhaustion. We hypothesized that IL-10R blockade may create an environment that affords Jak2 V617F HSC a selective advantage, indeed we found that IL-10R blocking antibody allowed Jak2 V617F cells to outcompete WT cells in mouse transplant experiments.

From these mouse data, we hypothesized that Jak2 V617Fenhances IL-10R signalling. To test this hypothesis, we created Ba/F3 cell lines co-expressing IL-10Rα and Jak2 V617F. We found that Jak2 V617F enhances growth at limiting concentrations of IL-10. We also found that cytokine-independent IL-10Rα Jak2 V617F clones (but never IL-10Rα Jak2 WTor IL-10Rα cells) emerge at day 8-10 in culture after withdrawal of cytokines. We performed a limiting dilution assay to pinpoint the proportion of IL-10R Jak2 V617F cells capable of surviving IL-3 withdrawal. One in 126 IL-10Rα Jak2 V617F cells has transforming ability, whereas 1 in 1.4 MPL Jak2 V617F has transforming ability. Together, these data support our hypothesis that Jak2 V617F enhances IL-10R signalling and can activate IL-10Rα, however with weaker transformation potential compared to Jak2V617F's activation of MPL signalling. JAK2 canonically does not participate in the IL-10R pathway.

It has been previously shown that JAK1 can transactivate JAK2 when JAK2 V617F cells are under the stress of persistent JAK2 inhibitor exposure, and this is the purported mechanism of JAK inhibitor resistance among MPN patients. We hypothesized that JAK2 V617F can transactivate JAK1, leading to the activation of IL-10R. The same transfected BaF3 cells aforementioned were serum starved for 4 hours and stimulated with mIL-10 for 15 minutes to induce signalling. By way of Western Blot analysis, cytokine-independent Jak2 V617F IL-10Rα Ba/F3 cells showed constitutive activation of phosphorylated JAK1 and JAK2 as well as STAT3 and STAT5, consistent with transactivation. Studies to demonstrate direct interactions between JAK2 V617F and JAK1 are currently ongoing.
SUMO E3 Ligase ZMIZ1 Drives Leukemogenesis Via Transcriptional Deregulation in Acute Myeloid Leukemia
Authors: Ziyu Fu , Tan Yun , Xu Xiaoguang , Xu Jie , Chen Sai-Juan
Published: December 2023

Transcriptional deregulation is one of the hallmarks of acute myeloid leukemia (AML), which causes the aberrant expression of genes essential for hematopoiesis, proliferation, stemness et, al. Emerging evidence has demonstrated that transcription is regulated by post-translational modification of transcription factors, such as SUMOylation, and the recruitment of co-regulators, such as epigenetic regulators.

Here, we conducted a CRISPR-Cas9 screen using sgRNAs targeting epigenetic regulators and a SUMO E3 ligase to explore essential regulators for hematopoiesis and leukemogenesis. Intriguingly, sgRNAs targeting ZMIZ1 was identified to be significantly decreased in proliferated leukemia cells and enriched in differentiated ones. ZMIZ1 (also known as ZIMP10 or RAI17) is a SUMO E3 ligase and transcriptional co-regulator of the Protein Inhibitor of Activated STAT (PIAS)-like family. Moreover, we found ZMIZ1 was highly expressed in AML and related to unfavorable outcomes, suggesting a potential diagnostic and prognostic target for AML.

Next, we analyzed the function of ZMIZ1 in AML using both in vivo and in vitro approaches. We knocked down the expression of ZMIZ1 in MOLM-13, THP1, OCI-AML3, HL60, and NB4 cells and found that knockdown of ZMIZ1 suppressed cell viability and induced apoptosis in all five cell lines. Meanwhile, knockdown of ZMIZ1 enhanced the sensitivity to chemotherapy (such as Ara-C). Additionally, knockdown of ZMIZ1 induced cell differentiation in NB4 cells. Moreover, we established conditional knockout mouse models (Zmiz1 flox/floxMx1-Cre), in which Zmiz1 was deleted with the Mx1-Cre transgene. We isolated the bone marrow of these mice and transfected it with the MLL-AF9 fusion gene, followed by transplantation into recipient mice. The conditional knockout was induced after two weeks of transfection. We found that inactivation of Zmiz1 significantly prolonged survival and relieved the leukemic burden.

To examine the regulatory function of ZMIZ1 in AML, we conducted ChIP-seq analysis in MOLM-13, THP1, OCI-AML3, HL60, and NB4 cells using antibodies against ZMIZ1. The binding peaks of ZMIZ1 differ in AML cell lines, as fewer than 30% of binding sites were overlapped between different cells. However, the binding motifs for ZMIZ1 showed a similar pattern. Motifs bound by MYB, CENPE, and RUNX1 were enriched in all five cell lines. To test the regulatory effects of ZMIZ1 on these transcription factors, we conducted luciferase reporter assays and found that ZMIZ1 enhanced the transcriptional activity of MYB.

Taken together, our study suggests an essential function of ZMIZ1 in leukemogenesis, presumably via SUMOylating essential transcription factors, and sheds new light on AML targeting therapy probably by targeting ZMIZ1.
Β-Lactam Antibiotic Ceftriaxone As a Potential Therapeutic Intervention for Chronic Pain in Sickle Cell Disease
Authors: He Ying , Guo Xiao , Molokie Robert , Wang Zaijie Jim
Published: December 2023

Sickle cell disease (SCD) features the presence of complex physiologic changes beyond a simple vasculopathy. Among its most debilitating neurological complications, chronic pain remains a challenging condition with limited effective treatments. Patients with SCD suffer tremendous pain every day. β-Lactam antibiotics have been shown to offer neuroprotection by promoting glutamate transporter GLT1 expression in the nervous system. This study aimed to investigate the effect of ceftriaxone, a prototype beta-lactam antibiotic, on chronic pain in SCD.

In a humanized mouse model of SCD, spontaneous ongoing pain as well as evoked hypersensitivity to mechanical and thermal stimuli were present in mice carrying human sickle hemoglobin, but not non-sickle control littermates. Notably, prominent activation of astrocytes was observed in the spinal cord dorsal horn region in the mice with SCD. As compared with non-sickle littermates, the spinal expression of GLT1 was dramatically decreased in sickle cell mice. Repeated intraperitoneal administration of ceftriaxone effectively attenuated spontaneous pain, mechanical allodynia, and heat hyperalgesia in mice with SCD. We found the pain reversal effect by ceftriaxone (200 mg/kg, i.p., for 7 days) lasted for at least 4 weeks. Correlating with the behavioral manifestations, ceftriaxone significantly deactivated astrocytes and elevated the level of GLT1 in the spinal cord in the mice with SCD.

These findings suggest that ceftriaxone alleviates both non-evoke ongoing pain and evoked pain in SCD by up-regulating spinal GLT1 expression, which highlights the possibility of a new clinical strategy to treat chronic pain in SCD. This study sheds light on the role of astrocyte activation in SCD and lays the foundation for the development of a potential new intervention for chronic pain in sickle cell disease.
Efficacy and Safety of Herombopag for the Treatment of Secondary Failure of Platelet Recovery after Allogeneic Hematopoietic Stem Cell Transplantation: The Single Center Prospective Study from China
Authors: Liu Huanfeng , Zhu Lidan , Liu Jia , Gao Shichun , Wang Lu , Kong Peiyan , Chen Ting , Feng Yimei , Liu Yuqing , Gao Lei , Zhang Xi
Published: December 2023

Background Thrombocytopenia is a frequent and serious complication after Allogeneic hematopoietic stem cell transplantation (allo-HSCT). It often has a multifactorial etiology, including poor graft function, GVHD, drugs, infections, and microangiopathy. Prolonged thrombocytopenia after HSCT, which is an independent adverse prognostic factor for transplant-related mortality (TRM) and overall survival, has not yet been systematically investigated. Among then, secondary failure of platelet recovery (SFPR) is the most common type of thrombocytopenia after allo-HSCT. SFPR refers to thrombocytopenia that develops after initial platelet engraftment and is not due to graft rejection or relapse. It defined as a decline in platelet count of < 20×10 9/L for 7 consecutive days or requiring transfusion support after achieving a sustained platelet count > 50×10 9/L without transfusion for 7 consecutive days after HSCT. Methods Here, we conduct a retrospective study to the efficacy and safety of herombopag in 81 patients with SFPR after allo-HSCT. Patients were administered the following treatment regimen: 5 mg/d herombopag; if the PLT count was less than 50×10^9/L for at least 2 weeks, the dose was increased to 7.5 mg/d; if the PLT count was 200-400×10 9/L, the dose was reduced; and if the PLT count was greater than 400×10 9/L, herombopag was terminated. Results Baseline platelet count of patients before treatment is 12 (2-20)×10 9/L. The initial time of treatment is 5 (3-14) months after allo-HSCT, and the median duration of treatment is 103 (45-276) days. Among the 81 patients, 51 patients (62.9%) had complete response (CR, defined as PLT≥50×10 9/L without PLT transfusion for 7 continuous days), 9 patients (11.1%) had a partial response (PR, defined as PLT of [20-50] ×10 9/L without PLT transfusion for 7 continuous days), and 21 patient (26.0%) had no response (NR, defined as the application of the maximum tolerated dose for 8 weeks and PLT<20×10 9/L or the need for PLT transfusion). The median time to obtain CR was 49 (18-229) days after treatment. The time to reach 20×10 9/L<PLT<50×10 9/L in the 9 patients with PR was 74 (52-276) days after treatment, respectively. One patient died of intracranial hemorrhage and two patients died of severe infection. Conclusion Our results indicated that herombopag can promote platelet recovery for patients with SFPR after allo-HSCT, thereby improving the survival rate of patients and improving the quality of life of patients after transplantation, and providing a new method and strategy for the treatment of thrombocytopenia after allo-HSCT.
Plasmin-Cleaved Von Willebrand Factor As a Biomarker for Microvascular Thrombosis
Authors: El Otmani Hinde , Frunt Rowan , Smits Simone , De Maat Steven , Fijnheer Rob , Barendrecht Arjan , Lenting Peter J , Tersteeg Claudia
Published: December 2023

Von Willebrand factor (VWF) is an essential contributor to microvascular thrombosis. Physiological cleavage by ADAMTS13 limits its prothrombotic properties, explaining why ADAMTS13 deficiency leads to attacks of microthrombosis in patients with thrombotic thrombocytopenic purpura (TTP). We previously reported that plasminogen activation takes place during TTP attacks in these patients. Furthermore, stimulation of plasminogen activation attenuates pathogenesis in preclinical TTP models in vivo. This suggests that plasmin is an endogenous regulator of VWF thrombogenicity, in particular when ADAMTS13 falls short to prevent microvascular occlusions. VWF cleavage by plasmin is biochemically distinct from cleavage by ADAMTS13. We hypothesized that plasmin-cleaved VWF (cVWF) holds value as a biomarker of microvascular thrombosis. We developed a V HH-based bioassay that can distinguish cVWF from intact and ADAMTS13-cleaved VWF in plasma. We validate this assay by tracking cVWF release during degradation of microthombi in vitro. We demonstrate that endogenous cVWF formation takes place in TTP patients during acute attacks of thrombotic microangiopathy, but not in remission. Finally, we show that therapeutic plasminogen activation in a mouse model for TTP amplifies cVWF formation, which is accompanied by VWF clearance. Our combined findings indicate that cVWF is released from microthrombi in the context of microvascular occlusion.
A Completely Oral Regimen Including Oral Etoposide As Cytoreductive Chemotherapy Combined with All Trans Retinoic Acid and Oral Arsenic for Induction Therapy in Patients with High-Risk APL：Comparable to Intravenous Cytoreductive Therapy
Authors: Tang Feifei , Duan Wen-Bing , Liu Xiao-Hong , Lu Sheng-Ye , Zhao Xiao-Su , Qin Ya-Zhen , Jia Jin-Song , Wang Jing , Gong Li-Zhong , Jiang Qian , Ting Zhao , Shi Hong-Xia , Chang Ying-Jun , Huang Xiao-Jun , Jiang Hao
Published: December 2023

Background ：Under the pandemic of coronavirus disease 2019 (COVID-19), in addition to all trans retinoic acid (ATRA) and oral arsenic(RIF), high-risk acute promyelocytic leukemia (APL) patients might lack of the necessity of intravenous cytotoxic chemotherapy infusions timely. Therefore, there is an urgent need for an oral, efficient and safe regimen for high-risk APL.

Methods ：We retrospectively analyzed 66 high-risk APL patients from January 2017 to to July 2022, of which 6 (9.1%, 6/66) patients died in the emergency room without treatment within 24 hours of arriving at our hospital (cerebral hemorrhage in 4 cases and multiple organ failure in 2 cases) and were excluded from the final analysis. A total of 60 patients received anti-leukemia treatment: 22 inpatients receiving oral etoposide (VP16) as cytoreductive chemotherapy from February 2020 to July 2022, and 38 inpatients receiving intravenous cytoreductive chemotherapy as historical control group from January 2017 to December 2019.

Results ：The median age of oral VP16 group and the control group were 35 years (21-65) and 34.5 years (16-62) (P=0.75). The median dose of oral VP16 was 1000mg (range, 400 to 1550). Differentiation syndrome occurred in 4 patients (18.2%) without severe forms in oral VP16 group and 7 patients (18.4%) in intravenous cytoreductive group(P=0.98). Except for one patient died of early death during induction therapy in the control group, all 59 evaluable patients (100%) achieved complete hematological remission(CHR) after induction therapy and complete molecular remission (CMR) after consolidation therapy. The median time to CHR and CMR was 36 days (range, 28-55) vs. 35 days (range, 28-52) (P=0.75) and 3 months (range, 1.1-5.5) vs. 3.3 months (range, 0.9-5.8) (P=0.58) in oral VP16 group and the control group, respectively. The median follow-up time was 56.6 months (range,10.3-76.4). Two (9.1%) patients in oral VP16 group and 3 (7.9%) patients in the control group experienced molecular relapse. The 2-year estimated OS and EFS was 100% vs. 94.7%(P=0.37) and 90.9% vs. 89.5%(P=0.88), respectively.

Conclusions ：A completely oral, efficient and safe induction regimen including oral VP16 as cytoreductive chemotherapy combined with ATRA and RIF is more convenient to administer for patients with high-risk APL.
A Model of Cumulative Risk of Disease Progression Among Patients with Indolent Systemic Mastocytosis
Authors: Mukherjee Sudipto , Cattie Douglas J , Shaheen Daniel , Green Teresa , Powell Dakota
Published: December 2023

Introduction

Indolent systemic mastocytosis (ISM), a clonal mast cell disease driven by the KIT D816V mutation in ~95% of cases, can cause lifelong debilitating cutaneous, gastrointestinal, and neurocognitive symptoms with some cases progressing to advanced systemic mastocytosis (AdvSM). While several studies have quantified variable rates of disease progression from ISM to AdvSM based on the absolute frequency of these events, these studies were limited by short median follow-up time considering the long clinical course of this condition sometimes in decades, thereby leading to underestimation of the cumulate rates of progression from ISM to AdvSM. We conducted a Markov analysis using population level data in the US to estimate the lifetime risk of progression for ISM patients.

Methods

We modeled the total course of SM as a discrete-time Markov chain, enabling us to estimate cumulative rates of disease progression based on fixed-interval empirical observations from the literature and, where there is no literature available, expert opinion. The Markov model assumes that patients enter the model as at-risk ISM, not-at-risk ISM, or de novo AdvSM, based on the assumed relative incidence of each subtype. Not-at-risk patients will then move to death after a period of time, with an annualized risk of death from disease. At-risk patients with ISM will remain in that state, progress to AdvSM (which may be tyrosine kinase inhibitor treated or not) or die. Finally, those who progress to AdvSM will either remain in their state or die. See Figure 1 for details, where each model parameter is reflected as a line between states and modeled based on an annualized rate of state transition. See Figure 2 for a full list of model parameters. Two hundred trials were performed, in which the model parameters were statistically sampled from a range of likely values, based on a triangular distribution between minimum, mode, and maximum. For each trial, the disease course for 5000 patients with SM was then simulated from initial SM diagnosis until death.

Results

Based on the rates of disease incidence, progression, treatment, and survival documented within various studies, we estimate the cumulative risk (± standard deviation) of disease progression from ISM to AdvSM to be 18.0% ± 3.1%, based on a probabilistic sampling of the underlying assumptions.

Conclusions

This study suggests that the cumulative rate of disease progression among patients with ISM may be higher than previously recognized. In our earlier work, we showed a higher rate of disease progression in ISM patients with high symptom burden (Mukherjee S, et al. ASH 2022). These data highlight the importance of close monitoring for signs of progression and warrant further research to identify subsets of ISM patients who may have a higher underlying risk of progression.
Ceftazidime-Avibactam in the Treatment of Bacteremia Due to Carbapenem-Resistant Gram-Negative Bacteria in Hematological Patients: Experience in a Single Center
Authors: Zhen Sisi , Lin Qingsong , Zhai Weihua , Jiang Erlie , Feng Sizhou
Published: December 2023

Background: Experience with ceftazidime-avibactam (CAZ-AVI) for bacteremia caused by carbapenem-resistant Enterobacterales (CRE) and Pseudomonas aeruginosa (CRPA) in hematological patients is limited.

Methods: We performed a single-center, retrospective, observational study including patients receiving CAZ-AVI for bacteremia due to CRE or CRPA between 2018 and 2022. The primary outcome was 30-day survival. To identify predictors of survival, multivariable analysis was performed.

Results: 56 patients were included and 57 (41 CRE and 16 CRPA) strains were isolated. 35 strains produced carbapenemase (25 MBL, 10 serine-beta-lactamase). 48 (85.7%) patients received combination therapy. All patients with MBL-CRE bacteremia (n=24) received combination therapy with aztreonam. The susceptibility rates to CAZ-AVI were only 25.0% (10/40) in CRE and 80.0% (8/10) in CRPA. The 30-day survival rates were 85.0% (34/40) and 81.3% (13/16) in CRE and CRPA group, respectively. In patients with MBL-CRE bacteremia, 30-day survival was high to 91.7% (22/24) due to combination with aztreonam. Ceftazidime did not influence the activity of aztreonam-avibactam against MBL-CRE in-vitro. Multivariable cox analysis revealed neutropenia > 14 days (P=0.002, HR: 0.029, 95%CI: 0.003~0.260) and higher Pitt bacteremia score (P=0.005, HR: 0.482, 95%CI: 0.291~0.798) were risk factors of 30-day survival.

Conclusions: CAZ-AVI is highly effective for bacteremia due to CRPA and serine-beta-lactamase CRE. Avibactam in combination with aztreonam is highly active for bacteremia due to aztreonam-resistant MBL producers.
Development of an Evidence-Based Clinical Tool for Childhood Cancer Survivorship Care in Tanzania
Authors: Spiegl Rosie , Ajuaye Gaudence , Noyd David , Chapman Hutton , Kashaigili Heronima J , Schroeder Kristin
Published: December 2023

CONCLUSIONS

Three in every four childhood cancer survivors experience late effects from treatment. As childhood cancer outcomes improve globally, it is important for providers to screen for these potential late effects to reduce morbidity. The present study aims to increase survivorship care capacity in low-resource settings by developing a digital clinical tool that integrates patient diagnosis and treatment exposure with evidenced-based surveillance recommendations to provide comprehensive childhood cancer follow-up guidelines.

Surveys with key stakeholders at each of the four childhood cancer treatment centers in Tanzania were completed to determine the current capacity to provide off-therapy surveillance. This informed the selection of late-effect screening tests by the International Late Effects of Childhood Cancer Guideline Harmonization Group's 2023 guidelines that were both evidenced-based and feasible in our setting. Qualtrics software was utilized to create a provider-facing digital survey to enter patient diagnosis, treatment exposures, and cumulative dosing.

The clinical tool was successfully created and implemented in our setting. In addition to entering patient demographic data, the survey burden for providers is anywhere from 5 to 11 questions in length. Using entered treatment exposures, the clinical tool uses a programmed algorithm to determine the late effect risk category, cardiovascular risk score, and personalized screening recommendations. Recommendations include both screening tests and frequency, to improve provider compliance with current best practice guidelines.

This tool can be adapted for other settings based on current capacity and provides personalized surveillance recommendations. This can help standardize treatment guidelines by increasing provider capacity and knowledge, with the potential to improve adherence to follow-up guidelines among childhood cancer survivors in LMICs, like Tanzania.
Rps19 and Rpl5 Play Distinct Roles in hematopoietic Stem Cell maintenance and Erythroid Differentiation
Authors: Tang Yuefeng , Ling Te , Khan Mushran , Rao Riaz , Schulz Vincent P , Papoin Julien , Narla Anupama , Lipton Jeffrey Michael , Palis James , Steiner Laurie A. , Gallagher Patrick G , Narla Mohandas , Crispino John D. , Blanc Lionel
Published: December 2023

Mutations in ribosomal proteins (RPs) lead to ribosomopathies. Mechanistic basis underlying the failure of erythropoiesis due to mutations in various RPs is yet to be fully defined. Lack of relevant murine models of ribosomapathies has hindered significant progress. To fill this gap, we generated Rps19 and Rpl5 floxed mice using CRISPR/Cas9 technologies and characterized their hematologic phenotype following crossing them to a Vav-iCremice. Vav-iCre;Rps19 fl/+mice are born at expected Mendelian rates with severe macrocytic anemia and reticulocytopenia. These mice die by post-natal day 10 (P10), due to bone marrow failure and absence of splenic stress erythropoiesis. Analysis of fetal erythropoiesis revealed a 40% reduction in the total number of erythroid cells in Vav-iCre;Rps19 fl/+, however erythroblasts at all stages of terminal erythroid differentiation were found. Intriguingly, studies of hematopoietic stem and progenitor cell s demonstrated a progressive exhaustion of the stem cell compartment.

scRNAseq studies of Vav-iCre;Rps19 fl/+ fetal livers demonstrated increased expression of RUNX1, a transcription factor implicated in the regulation of ribosome biogenesis. Accordingly, we observed that hematopoietic progenitors of Vav-iCre,Rps19 fl/+had enhanced global protein synthesis along with increased RUNX1 protein, despite ribosomal protein haploinsufficiency demonstrated by polysome profiling.

In marked contrast, Vav-iCre;Rpl5 fl/+ mice presented with a more severe phenotype and died perinatally from anemia. Further analysis revealed a failure of erythropoiesis at erythroid progenitor stages in Vav-iCre;Rpl5 fl/+. In striking contrast with the Rps19 model, Vav-iCre;Rpl5 fl/+ mice had an expansion of the hematopoietic stem and progenitor cell compartment that also had an increased protein translation rate . This increased translation occurred in the context of normal expression levels of RUNX1. Interestingly Vav-iCre;Rpl5 fl/+ micehad reduced mTORC1 activity and a dramatic reduction in the expression of other ribosomal proteins. Consistent with previous studies about the pivotal role of mTORC1 in erythropoiesis Vav-iCre,Raptor fl/fl(mTORC1 conditional knockout) had reduced expressions of RPs in fetal liver.

In summary, we report different requirements for Rps19 and Rpl5 during fetal hematopoiesis in regulating erythroid progenitor biology. While Rps19 plays an essential role at the HSC level to maintain the generation of erythroid progenitors, Rpl5 is critical once progenitors are committed towards an erythroid fate. Along with the findings that translation is increased in RP haploinsufficient models, our study unravels potential compensatory mechanisms during fetal hematopoiesis.
Hospital Associated Disability Among Older Adults with Plasma Cell Disorders Receiving Autologous Stem Cell Transplant
Authors: Kidwell Kyle , Zhu Rui , Bal Susan , Godby Kelly , Ravi Gayathri , Costa Luciano , Shrestha Sadeep , Williams Grant , Bhatia Smita , Giri Smith
Published: December 2023

Background: An increasing number of older adults with Plasma Cell Disorders (PCDs) are receiving autologous stem cell transplant (ASCT) in the US. Hospital associated disability (HAD) is a loss in independence in one or more activities of daily living associated with acute care hospitalization and is a common complication among older adults. The prevalence and prognostic significance of HAD among older adults with PCDs undergoing ASCT is unknown.

Methods: This retrospective cohort study used consecutive adults receiving ASCT at a large U.S. comprehensive medical center between 1/2013 and 5/2023. Trained nursing staff assessed Katz Activities of Daily Living (ADL) at admission and every 3 days thereafter under our Virtual Acute Care for Elders program. The primary outcome was development of HAD defined as ≥1 point decline on the Katz ADL scale from hospital admission to discharge. We also examined the association between development of HAD and various predictors including age, Karnofsky Performance Status (KPS), and Hematopoietic Cell Transplantation-Specific Comorbidity Index (HCT-CI) using modified Poisson regression models with robust variance estimators. Using similar models, we studied the impact of HAD on unplanned 30-day readmission rates.

Results: Of 870 patients undergoing ASCT during the study period, 778 had at least two Katz ADL assessments and were included. The study population was comprised of 56% Caucasian/white ethnicity and 56% male with a median age of 62y (IQR 55-68y). In the overall population, 112 (14.4%) developed HAD. The incidence of HAD was much higher among adults ≥65y compared to their younger counterparts (22% vs. 9%; p=<0.01). In multivariate analysis, age ≥65y (RR 2.22; 95% CI 1.56-3.15; p=<0.001) and baseline KPS <=70 (RR 1.83; 95% CI 1.15-2.92; p=0.011) was associated with increased risk of developing HAD; HCT-CI scores did not have a significant association with HAD. Those with HAD had a two-fold higher risk of 30-day readmission as compared to those without HAD (RR 2.18; 95% CI 1.20-3.66; p=0.003).

Conclusions: Nearly 1 in 4 adults ≥65y developed HAD while undergoing ASCT, which was associated with a two-fold increased risk of 30-day readmission. Interventions to prevent HAD and its downstream consequences are critically needed.
HDAC6 Inhibition Activates Proteasomes to Modulate the MHC Class I Immunopeptidome and Promote Antimyeloma Immunity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Malek Ehsan , Driscoll James J.
Published: December 2023

The evasion of host immunity is a hallmark of cancer. Tumor cells develop strategies to evade host immunity and to resist currently available immunotherapies, while others present a low tumor mutation burden limiting the presence of tumor antigenicity. Proteasomes are exquisitely complex, intricately regulated protein-degrading machines that are responsible for the overwhelming majority of intracellular proteolysis in eukaryotic cells. Proteasomes are also essential for the efficient processing of intracellular proteins to generate antigenic epitopes that complex with MHC-class I molecules and are displayed on antigen-presenting cells (APCs) to activate CD8 + cytotoxic T-lymphocytes (CTLs). We hypothesized that pharmacologic activation of proteasomes would modulate the tumor immunopeptidome to increase MHC class I antigen presentation and promote CTL activity. Here, we developed a cell-based, high-throughput screen (HTS) to identify FDA-approved pharmacologics and bioactive molecules that increased proteasomal activity to promote the presentation of MHC class I antigens. The HTS revealed that the histone deacetylase 6 (HDAC6)-selective inhibitors tubastatin-A, ACY-738 and ACY-1215 increased proteasome activity in a panel of multiple myeloma (MM) cell lines. The HDAC6 inhibitors also significantly enhanced MHC-class I antigen presentation pan-MHC class-I expression in a diverse panel of cancer cell lines. Pretreatment of MM patient CD138 + tumor cells with HDAC6 inhibitors promoted the cytotoxic activity of autologous patient-derived T-cells. Mechanistic studies revealed that pharmacologic blockade or genetic ablation of HDAC6 or the HDAC6 ubiquitin-binding (BUZ) domain disrupted the association of HDAC6 with the ubiquitin-like protein HR23B, a substrate-shuttle factor that delivers ubiquitinylated cargo to proteasomes. Taken together, the results validate the HDAC6-BUZ domain as an attractive drug discovery target and demonstrate the immediate translational impact of proteasome activators to modulate the immunopeptidome and enhance antitumor immunity. Proteasome activators also represent a paradigm-shifting approach to overcome mechanisms of immune escape. FDA-approved drugs that increase proteasome activity and boost antigen presentation can now be repositioned as cancer immunotherapeutics to overcome the existing bottlenecks in drug development.
MAIT Cell Proliferation Is a Potential Early Diagnosis Marker for Blood Stream Infection in AML Patients
Authors: Feng Sizhou , Fan Yuping , Zhang Sudong , Feng Xiaomeng , Xu Yuanfu , Jiang Erlie
Published: December 2023

Objectives

To explore the role and dynamics of MAIT (Mucosal-associated invariant T) cells in blood stream infection of AML (Acute myeloid leukemia) patients.

Methods

Healthy adults and hospitalized adult AML patients that with blood stream infection, non-infection, or in completely remission (CR) were prospectively enrolled. Characteristics of MAIT cells in peripheral blood of enrolled subjects were analyzed by flow cytometry.

Results

Both gram negative and gram positive bacteria could induce sepsis in AML patients. MAIT cells significantly increased in AML patients with blood stream infection comparing with non-infected AML patients, healthy subjects and AML patients in completely remission. Increased MAIT cells were mainly consists of CD8+ T cells and were activated. There is no significant difference in the recruitment and death of MAIT cells, while their proliferation is significantly increased. The proliferation of MAIT cells occurs before traditional infection indicators such as CRP, PCT, endotoxin in plasma and fever. MAIT cells in neutropenic blood stream infected mice were increased as we observed in AML patients.

Conclusions

MAIT cells increased without bacterial specificity, and were activated and proliferated in AML patients with blood stream infection that caused by various bacteria. They may enhance host defense and play a vital role in fighting against pathogenic microbial infections. Proliferation of MAIT cells can be applied as a monitoring indicator for blood stream infection in AML patients, which helps to reduce the mortality rate of them.
Combined Deficiency of Chromosome 7 Myeloid Tumor Suppressors Enhances Chemotherapy Resistance
Authors: Jotte Matthew R. M. , Martinez Tanner C. , Stoddart Angela , Imgruet Molly K. , Nam Henna S. , Hu Bonnie , Khan Saira , An Ningfei , McNerney Megan E.
Published: December 2023

Monosomy 7 and del(7q) [-7/del(7q)] are recurrent in myeloid neoplasms and associated with chemoresistance. -7/del(7q) is identified in up to half of therapy-related myeloid neoplasms (t-MN), high-risk secondary malignancies arising after prior exposure to chemotherapy or radiation. -7/del(7q) is also detected in clonal hematopoiesis, suggesting chromosome 7 aberrations can be early events in disease etiology. Despite this prevalence, the pathogenesis of -7/del(7q) in leukemogenesis remains unclear. We previously reported that deficiency of the transcription factor CUX1, a 7q-encoded tumor suppressor gene, promotes hematopoietic stem and progenitor cell drug resistance and t-MN transformation. Herein, we determined the combined impact of CUX1 loss with additional 7q tumor suppressor genes. To this end, we established a CRISPR/Cas9-based murine model of del(7q) clonal hematopoiesis and drug resistance. After targeting four 7q genes simultaneously, combined deficiency of CUX1 and the histone methyltransferase EZH2 uniquely promoted clonal outgrowth under genotoxic pressure in vivo and in vitro. Mechanistically, clonal selection is due, in part, to decreased apoptosis after chemotherapy exposure. RNA-seq in the absence of genotoxic insult revealed that Cux1 and Ezh2 loss has an additive transcriptional impact that is enriched for -7/del(7q) patient-derived gene signatures. Overall, we reveal a previously unknown genetic interaction between the 7q genes CUX1 and EZH2, supporting the concept of 7q as a contiguous gene syndrome region . A refined understanding of the molecular pathways driving del(7q) pathogenesis and drug resistance will enable development of therapies designed to counter or prevent these high-risk malignancies. In addition, we report a tractable approach for interrogating the pathogenesis of aneuploid events more broadly in cancer.
The Optimal Busulfan Dose of Conditioning Regimens for Allogeneic Hematopoietic Cell Transplantation in Younger Patients with Acute Myeloid Leukemia
Authors: Choi Yunsuk , Choi Eun-Ji , Park Han-Seung , Lee Jung-Hee , Lee Kyoo-Hyung , Lee Young-Shin , Kang Young-A , Jeon Mijin , Woo Ji Min , Kang Hyeran , Lee Je-Hwan
Published: December 2023

Background: In allogeneic hematopoietic cell transplantation (HCT) for younger patients with acute myeloid leukemia (AML), myeloablative conditioning (MAC) regimens have been considered as standard conditioning. MAC can reduce the risk of relapse, but increase non-relapse mortality, including organ toxicity and severe graft versus host disease. Therefore, the optimal busulfan dose in conditioning regimens has not been established in younger patients with AML. Thus, we aimed to investigate post-HCT outcomes according to the busulfan dose of conditioning regimens in younger patients with AML.

Methods: We retrospectively analyzed long-term outcomes according to the busulfan dose (2 days vs. 3 days vs. 4 days) in 686 adult patients (age <55 years) with AML who had undergone HCT between 2000 and 2020 at the Asan Medical Center, Seoul, Korea. The clinical data was collected from the Asan Medical Center HCT registry. Patients with AML who had undergone a second or third HCT were excluded from this study.

Results: The median age was 41 (range 15-54). The 5-year overall survival (OS) and event-free survival (EFS) rate was 52.2% and 46.4%, respectively. The 3 days of busulfan (Bu3) (n=88) showed significantly higher OS than 4 days of busulfan (Bu4) (n=229) and 2 days of busulfan (Bu2) (n=369) (2-year OS; 76.1% vs. 65.1% and 56.3%, respectively; P<0.001). The EFS rate of Bu3 and Bu4 was significantly higher than that of Bu2 (2-year EFS; 60.2% and 59.8% vs. 46.9%, respectively; P<0.001). Among the three groups, there was no significant difference in GRFS (P=0.952). The cumulative incidence of relapse was significantly lower in Bu4 (26.2% at 2 years) than in Bu2 (40.4%) and Bu3 (36.4%) (P<0.001). However, the NRM was significantly higher in Bu4 (13.9% at 2 years) and Bu2 (12.7%) than in Bu3 (3.4%) (P=0.005). In HCT at complete remission (CR) status, Bu3 showed significantly better OS than Bu4 and Bu2 (P=0.039), whereas there was no significant difference in EFS according to the busulfan dose (P=0.121). In patients with refractory disease, however, the higher dose of busulfan (Bu3 or Bu4) did not show survival advantages in terms of OS (P=0.153) and EFS (P=0.829) over Bu2. Regarding donor types, Bu3 showed significantly better OS than Bu2 in HCT from matched sibling donors (MSD) (P=0.030) and haploidentical family donors (HFD) (P<0.001). However, in HCT from unrelated donors (URD), the Bu2 group showed the best GRFS, and OS in Bu2 or Bu3 was significantly longer than in Bu4 (P=0.028).

Conclusion: For younger patients with AML, Bu3 would be the optimal conditioning intensity in HCT from MSD or HFD at CR status. In URD, Bu2 or Bu3 would be appropriate. In refractory disease, increasing busulfan dose intensity could not improve post-HCT outcomes.
Genetic Variants Associated with High Plasma Levels of Factor VIII and Von Willebrand Factor in a Family with Thrombotic Episodes
Authors: Spena Silvia , Cairo Andrea , Gianniello Francesca , Pappalardo Emanuela , Mortarino Mimosa , Garagiola Isabella , Martinelli Ida , Peyvandi Flora
Published: December 2023

Background: High levels of factor VIII (FVIII) and von Willebrand factor (vWF) have been indicated as independent risk factors for venous thromboembolism. However, the genetic factors responsible for their increase remain still poorly known. In a woman referred to our Thrombosis unit following a venous thrombotic event, very high levels of FVIII and vWF were found at the acute episode and in subsequent measurements. High levels of FVIII and vWF were also detected in other family members, suggesting a possible genetic cause (Figure 1).

Aim: To identify variants/genes associated with high levels of FVIII and vWF.

Methods: In a large Italian family with high levels of FVIII/vWF and thrombotic episodes, whole-exome sequencing was performed in 12 family members (SureSelect-Agilent). Data were analyzed according to the guidelines of the Broad Institute (https://software.broadinstitute.org/gatk/best-practices/). Variants were annotated (Annovar) and filtered (KGGseq) according to an autosomal dominant inheritance model of high levels of FVIII or vWF. RNA levels were evaluated in PBMCs by qPCR (Thermo Fisher).

Results: 20 variants, spread over a 8300-Kb region on chromosome 5, were identified in 12 genes, including the low frequency rs13158382 located upstream of the MIR143/145 genes. Since variants in the promoter of MIR143/145 have been reported to affect their expression and reduced levels of miR-145 have been associated with high vWF, the potential correlation between rs13158382 and vWF levels was evaluated. Lower levels of both miRNAs and higher level of vWF mRNA were measured in family members carrying the variant (n=3) compared to family members without the variant (n=3). Analysis of genetic and expression data from of a larger cohort of individuals from the 1000 Genomes and GEUVADIS project confirmed a statistically significant reduction (p=0.023) of miR-143 in heterozygous subjects (n=35) compared to homozygous wild-type individuals (n=386). Since low levels of pri-miRNA-143/145 were measured in heterozygous family members and structural changes of pre-miR-143 with rs13158382 was predicted by miRVaS tool, the reduction of mature miRNAs might result from a lowered synthesis of pri-miRNAs and/or from an aberrant processing of pre-miRNA.

Conclusion: This family-based study revealed a new genetic variant potentially involved in vWF increase by reducing the expression of miR-143/145.
Thrombotic Complications in Inflammatory Bowel Disease: The Role of Coagulation Markers and Family History
Authors: Algahtani Farjah , Altuwaijri Mansour , Saeed Maria , Aljebreen Abdulrahman , Aleem Aamer , Sewaralthahab Sarah , Alotaibi Ghazi S , Alanazi Abdulelah , Azzam Nahla , Almadi Majid , AlHarbi Othman
Published: December 2023

Background: Inflammatory bowel disease (IBD) is a chronic inflammatory disorder characterized mainly by intestinal pathology. The risk of thrombosis increases in IBD patients, increasing overall disease morbidity and mortality. We aim to investigate the role of coagulation markers in determining the risk of thrombotic complications in IBD patients compared to healthy controls.

Methods: The study was approved by the institutional review board. All patients aged 16 or over, diagnosed with IBD and followed at our center from January 2012 -2022, were included. We investigated the family history and the hemostatic profiles of these patients. Descriptive statistics were computed for continuous variables, including means, standard deviations (SDs), minimum and maximum values, as well as 95% confidence intervals (CIs), where appropriate, and frequencies for categorical variables. Fisher exact test was used for categorical variables. Student t-test was used for continuous variables.

Results: A total of 100 patients were included in the study, comprising of 51 (24%) females and 158 (76%) males. 73% of patients were diagnosed with Crohn's disease (CD), and 27% with ulcerative colitis (UC). Deep vein thrombosis (DVT) was observed in 5 (2.3%) of patients. In patients with a family history of DVT, 1.9% had developed a thrombotic event. Among continuous variables, prothrombin time, thrombin clotting time, and fibrinogen levels had mean values significantly different for patients with thrombosis than those without thrombosis ( p-values 0.01, 0.01, and 0.03, respectively).

Conclusions: Patients with both UC and CD are at an increased risk of thrombosis. A family history of IBD can increase the risk of these events. The coagulation markers, including PT, TTC, and fibrinogen levels, can be used as biomarkers to predict the risk of thrombosis and the evaluation of the hemostatic profiles of IBD patients regularly, which can help prevent serious thrombotic complications and thus reduce morbidity and mortality resulting from these events. Therefore, such evaluation should be included in the routine examination protocols of high-risk IBD patients. This study may help better understand the biology of disease.
Development of a Novel, Allogeneic GPRC5D-Directed CAR for Treatment of Multiple Myeloma Patients
Authors: Kinder Jeremy , Estrada Chanel Athena , Granger Brian , O'Rourke Allison , Liang Ouwen , Lampano Aaron , Zipp Garrett , Chaturvedi Vandana , Hu Xiaomeng , Young Chi , van Hoeven Neal , Schrepfer Sonja , Fry Terry J.
Published: December 2023

Despite the success of BCMA-targeted therapy in advanced multiple myeloma, the disease is still considered incurable. Relapse following BCMA-directed cellular and antibody-based therapies is commonly associated with decreased surface expression of BCMA on malignant plasma cells. The G protein-coupled receptor, class C group 5 member D (GPRC5D) represents an additional target whose expression is limited to bone marrow and hair follicles, with selectively increased expression on malignant plasma cells. Thus far, the use of GPRC5D-targeted CAR T cells has provided a strong clinical benefit in multiple myeloma with limited on-target, off-tumor effects. To this end, we have validated novel, fully human GPRC5D-specific CARs for use with our hypoimmune (HIP), allogeneic CAR T cell platform. This will allow rapid delivery of allogeneic GPRC5D CAR T cells from healthy donors that simultaneously circumvent “non-self” recognition through TCR, B2M and CIITA gene disruption and promote persistence through innate immune evasion by leveraging CD47 overexpression.

For early selection of candidate GPRC5D binders, T cells from healthy donors were transduced with VSV-G pseudotyped lentivirus containing second-generation, fully-human GPRC5D CARs. A clinically validated GPRC5D CAR was produced in similar fashion and used as benchmark control for initial characterization experiments. Transduction efficiency of GPRC5D CARs was analyzed via flow cytometry. GPRC5D CAR efficacy was analyzed in vitro via 24-hour luciferase or long-term Incucyte cytotoxicity assays against RPMI-8226 and MM.1S multiple myeloma target cells. Systemic MM.1S in vivo tumor models were performed in NSG mice to further assess efficacy of lead CAR candidates.

Here we show our candidate GPRC5D CARs demonstrate in vitro cytotoxicity and effector cytokine production that is comparable to clinically validated benchmark controls. In 5-day Incucyte cytotoxicity assays, GPRC5D CAR T cells expand up to 10-fold and reduce tumor cell counts at starting E:T ratios of 1:8. Furthermore, in MM.1S in vivo tumor challenge models, these GPRC5D CARs significantly reduce flux and prolonged survival relative to tumor only controls with tumor clearance that was in line with clinical benchmark controls.

Using ex vivo VSV-G production, our GPRC5D CAR T cells control multiple myeloma tumor cells both in vitro and in vivo, demonstrating efficacy that is on par with GPRC5D clinical benchmark CAR T cells. Studies to further define the efficacy of HIP-modified GPRC5D CAR T cells are ongoing. Moving forward, further interrogating GPRC5D CAR activity against multiple myeloma tumor cells with varying levels of target antigen expression will be critical to understanding the benefit of deploying GPRC5D CAR T cells within the multiple myeloma clinical landscape.