Development of Mesenchymal Stem Cell Encoded with Myogenic Gene for Treating Radiation-Induced Muscle Fibrosis.

Radiation therapy (RT) is a typical treatment for head and neck cancers. However, prolonged irradiation of the esophagus can cause esophageal fibrosis due to increased reactive oxygen species and proinflammatory cytokines. The objective of this study was to determine whether myogenic gene-transfected mesenchymal stem cells (MSCs) could ameliorate damage to esophageal muscles in a mouse model of radiation-induced esophageal fibrosis. We cloned esophageal myogenic genes (MyoD, MyoG, and Myf6) using plasmid DNA. Afterward, myogenic genes were transfected into Human Mesenchymal Stem Cells (hMSCs) using electroporation. Gene transfer efficiency, stemness, and myogenic gene profile were examined using flow cytometry, quantitative polymerase chain reaction, and RNA sequencing. In vivo efficacy of gene-transfected hMSCs was demonstrated through histological and gene expression analyses using a radiation-induced esophageal fibrosis animal model. We have confirmed that the gene transfer efficiency was high (∼75%). Pluripotency levels in gene-transfected MSCs were significantly decreased compared with those in the control (vector). Particularly, myogenesis-related genes such as OAS2, OAS3, and HSPA1A were overexpressed in the group transfected with three genes. At 4 weeks after injection, it was found that thickness collagen layer and esophageal muscle in MSCs transfected with all three genes were significantly reduced compared to those in the saline group. Muscularis mucosa was observed prominently in the gene combination group. Moreover, expression levels of myogenin, Myf6, calponin, and SM22α known to be specific markers of esophageal muscles tended to increase in the group transfected with three genes. Therefore, using gene-transfected MSCs has the potential as a promising therapy against radiation-induced esophageal fibrosis.

TALEN-edited allogeneic inducible dual CAR T cells enable effective targeting of solid tumors while mitigating off-tumor toxicity.

Adoptive cell therapy using chimeric antigen receptor (CAR) T cells has proven to be lifesaving for many cancer patients. However, its therapeutic efficacy has been limited in solid tumors. One key factor for this is cancer-associated fibroblasts (CAFs) that modulate the tumor microenvironment (TME) to inhibit T cell infiltration and induce "T cell dysfunction." Additionally, the sparsity of tumor-specific antigens (TSA) and expression of CAR-directed tumor-associated antigens (TAA) on normal tissues often results in "on-target off-tumor" cytotoxicity, raising safety concerns. Using TALEN-mediated gene editing, we present here an innovative CAR T cell engineering strategy to overcome these challenges. Our allogeneic "Smart CAR T cells" are designed to express a constitutive CAR, targeting FAP+ CAFs in solid tumors. Additionally, a second CAR targeting a TAA such as mesothelin is specifically integrated at a TCR signaling-inducible locus like PDCD1. FAPCAR-mediated CAF targeting induces expression of the mesothelin CAR, establishing an IF/THEN-gated circuit sensitive to dual antigen sensing. Using this approach, we observe enhanced anti-tumor cytotoxicity, while limiting "on-target off-tumor" toxicity. Our study thus demonstrates TALEN-mediated gene editing capabilities for design of allogeneic IF/THEN-gated dual CAR T cells that efficiently target immunotherapy-recalcitrant solid tumors while mitigating potential safety risks, encouraging clinical development of this strategy.

Multi-armored allogeneic MUC1 CAR T cells enhance efficacy and safety in triple-negative breast cancer.

Solid tumors, such as triple-negative breast cancer (TNBC), are biologically complex due to cellular heterogeneity, lack of tumor-specific antigens, and an immunosuppressive tumor microenvironment (TME). These challenges restrain chimeric antigen receptor (CAR) T cell efficacy, underlining the importance of armoring. In solid cancers, a localized tumor mass allows alternative administration routes, such as intratumoral delivery with the potential to improve efficacy and safety but may compromise metastatic-site treatment. Using a multi-layered CAR T cell engineering strategy that allowed a synergy between attributes, we show enhanced cytotoxic activity of MUC1 CAR T cells armored with PD1KO, tumor-specific interleukin-12 release, and TGFBR2KO attributes catered towards the TNBC TME. Intratumoral treatment effectively reduced distant tumors, suggesting retention of antigen-recognition benefits at metastatic sites. Overall, we provide preclinical evidence of armored non-alloreactive MUC1 CAR T cells greatly reducing high TNBC tumor burden in a TGFB1- and PD-L1-rich TME both at local and distant sites while preserving safety.

Selective delivery of imaging probes and therapeutics to the endoplasmic reticulum or Golgi apparatus: Current strategies and beyond.

To maximize therapeutic effects and minimize unwanted effects, the interest in drug targeting to the endoplasmic reticulum (ER) or Golgi apparatus (GA) has been recently growing because two organelles are distributing hubs of cellular building/signaling components (e.g., proteins, lipids, Ca2+) to other organelles and the plasma membrane. Their structural or functional damages induce organelle stress (i.e., ER or GA stress), and their aggravation is strongly related to diseases (e.g., cancers, liver diseases, brain diseases). Many efforts have been developed to image (patho)physiological functions (e.g., oxidative stress, protein/lipid-related processing) and characteristics (e.g., pH, temperature, biothiols, reactive oxygen species) in the target organelles and to deliver drugs for organelle disruption using organelle-targeting moieties. Therefore, this review will overview the structure, (patho)physiological functions/characteristics, and related diseases of the organelles of interest. Future direction on ER or GA targeting will be discussed by understanding current strategies and investigations on targeting, imaging/sensing, and therapeutic systems.

Beyond nanoparticle-based oral drug delivery: transporter-mediated absorption and disease targeting.

Various strategies at the microscale/nanoscale have been developed to improve oral absorption of therapeutics. Among them, gastrointestinal (GI)-transporter/receptor-mediated nanosized drug delivery systems (NDDSs) have drawn attention due to their many benefits, such as improved water solubility, improved chemical/physical stability, improved oral absorption, and improved targetability of their payloads. Their therapeutic potential in disease animal models (e.g., solid tumors, virus-infected lungs, metastasis, diabetes, and so on) has been investigated, and could be expanded to disease targeting after systemic/lymphatic circulation, although the detailed paths and mechanisms of endocytosis, endosomal escape, intracellular trafficking, and exocytosis through the epithelial cell lining in the GI tract are still unclear. Thus, this review summarizes and discusses potential GI transporters/receptors, their absorption and distribution, in vivo studies, and potential sequential targeting (e.g., oral absorption and disease targeting in organs/tissues).

Association of periodontal disease treatment with mortality in patients with dementia: a population-based retrospective cohort study (2002-2018).

Dementia is one of the leading causes of death worldwide. In this study, we analyzed the association of periodontal treatment with the risk of death in patients with dementia. The analyzed data were obtained by linking the National Health Insurance Corporation claims data between 2002 and 2018 to the Statistics Korea death registry. In total, 1,131,406 patients with dementia aged ≥ 65 years had undergone dental treatment during the study period. Time-dependent Cox proportional hazards model was performed. The mortality rate was approximately 10% among the patients with dementia. The 17-years cumulative survival rates for patients who received periodontal treatment and their untreated counterparts were 83.5% and 71.5%, respectively. The crude hazard ratio of the periodontal group was approximately twice as high as that of the non-periodontal group (1.99; P < 0.001). Furthermore, in the regression model that was adjusted for socio-demographic variables and systematic chronic diseases, the risk of death in the non-periodontal group was approximately 1.83 times higher than that of the periodontal group (P < 0.00). These findings suggest that preventive periodontal treatment may decrease mortality risk in older people with dementia.

Functional Characterization of Circadian Nuclear Receptors REV-ERBα and REV-ERBß in Human Osteosarcoma Cell Cultures.

REV-ERBα and its paralog, REV-ERBß, encoded by NR1D1 and NR1D2 genes, are key nuclear receptors that link the circadian timing system and metabolic homeostasis. Since heme is an endogenous ligand, REV-ERBs have been considered key components of the circadian molecular clock and can be pharmacologically targeted to treat various circadian rhythm-related diseases, such as cardiometabolic, inflammatory, and neuropsychiatric diseases, as well as cancer. REV-ERBs are believed to be functionally redundant and compensatory, although they often affect the expression of gene subsets in an isoform-specific manner. Therefore, this study aimed to identify the redundant and distinct roles of each isoform in controlling its target genes by comparing the transcriptome profiles of a panel of mutant U2OS human osteosarcoma cells in which either NR1D1 or NR1D2 was ablated. Indeed, our transcriptomic analyses revealed that most REV-ERB-regulated genes are controlled by redundant or even additive actions. However, the RNA expression profiles of each single mutant cell line also provide strong evidence for isoform-dependent actions. For example, REV-ERBα is more responsible for regulating the NF-κΒ signaling pathway, whereas a group of extracellular matrix components requires REV-ERBß to maintain their expression. We found that REV-ERBs have isoform-selective functions in the regulation of certain circadian output pathways despite their overlapping roles in the circadian molecular clock. Thus, the development of isoform-selective REV-ERB modulators can help treat metabolic disturbances and certain types of cancer.

Serum Lipid Levels and Suicidal Ideation of Adults: A Cross-Sectional Study Using the Korea National Health and Nutrition Examination Survey.

Cholesterol plays a crucial role in the brain, which suggests that changes in its concentration levels may have an impact on the central nervous system. To examine the association between serum lipid levels and suicidal ideation according to sex, we performed a cross-sectional study using data from the Korea National Health and Nutrition Examination Survey 2014-2018. A total of 13,772 adults 19 years or older were analyzed. The ninth item of the Patient Health Questionnaire was used to evaluate the suicidal ideation of participants. After sorting by sex, a complex logistic regression was performed to measure the association between serum lipid indicators and suicidal ideation. The analysis adjusted for age, body mass index, smoking, heavy drinking, regular exercise, household income, education level, dyslipidemia medication, depression, and chronic diseases. Compared to the intermediated category, the lowest range of low-density lipoprotein cholesterol (LDL-C; <100 mg/dL) was associated with increased suicidal ideation in men (odds ratio [OR] = 1.97; 95% confidence interval [CI]: 1.30-3.01). The association between lipid levels and suicidal ideation was not clear in women. We found an association between lower LDL-C levels and an increased risk of suicidal ideation among Korean men aged 19 years or older.

PGC-1α senses the CBC of pre-mRNA to dictate the fate of promoter-proximally paused RNAPII.

PGC-1α is well established as a metazoan transcriptional coactivator of cellular adaptation in response to stress. However, the mechanisms by which PGC-1α activates gene transcription are incompletely understood. Here, we report that PGC-1α serves as a scaffold protein that physically and functionally connects the DNA-binding protein estrogen-related receptor α (ERRα), cap-binding protein 80 (CBP80), and Mediator to overcome promoter-proximal pausing of RNAPII and transcriptionally activate stress-response genes. We show that PGC-1α promotes pausing release in a two-arm mechanism (1) by recruiting the positive transcription elongation factor b (P-TEFb) and (2) by outcompeting the premature transcription termination complex Integrator. Using mice homozygous for five amino acid changes in the CBP80-binding motif (CBM) of PGC-1α that destroy CBM function, we show that efficient differentiation of primary myoblasts to myofibers and timely skeletal muscle regeneration after injury require PGC-1α binding to CBP80. Our findings reveal how PGC-1α activates stress-response gene transcription in a previously unanticipated pre-mRNA quality-control pathway.

Phase separation of low-complexity domains in cellular function and disease.

In this review, we discuss the ways in which recent studies of low-complexity (LC) domains have challenged our understanding of the mechanisms underlying cellular organization. LC sequences, long believed to function in the absence of a molecular structure, are abundant in the proteomes of all eukaryotic organisms. Over the past decade, the phase separation of LC domains has emerged as a fundamental mechanism driving dynamic multivalent interactions of many cellular processes. We review the key evidence showing the role of phase separation of individual proteins in organizing cellular assemblies and facilitating biological function while implicating the dynamics of phase separation as a key to biological validity and functional utility. We also highlight the evidence showing that pathogenic LC proteins alter various phase separation-dependent interactions to elicit debilitating human diseases, including cancer and neurodegenerative diseases. Progress in understanding the biology of phase separation may offer useful hints toward possible therapeutic interventions to combat the toxicity of pathogenic proteins.

Nanogel-mediated delivery of oncomodulin secreted from regeneration-associated macrophages promotes sensory axon regeneration in the spinal cord.

Preconditioning nerve injury enhances axonal regeneration of dorsal root ganglia (DRG) neurons in part by driving pro-regenerative perineuronal macrophage activation. How these macrophages influence the neuronal capacity of axon regeneration remains elusive. We report that oncomodulin (ONCM) is produced from the regeneration-associated macrophages and strongly influences regeneration of DRG sensory axons. We also attempted to promote sensory axon regeneration by nanogel-mediated delivery of ONCM to DRGs. Methods:In vitro neuron-macrophage interaction model and preconditioning sciatic nerve injury were used to verify the necessity of ONCM in preconditioning injury-induced neurite outgrowth. We developed a nanogel-mediated delivery system in which electrostatic encapsulation of ONCM by a reducible epsilon-poly(L-lysine)-nanogel (REPL-NG) enabled a controlled release of ONCM. Results: Sciatic nerve injury upregulated ONCM in DRG macrophages. ONCM in macrophages was necessary to produce pro-regenerative macrophages in the in vitro model of neuron-macrophage interaction and played an essential role in preconditioning-induced neurite outgrowth. ONCM increased neurite outgrowth in cultured DRG neurons by activating a distinct gene set, particularly neuropeptide-related genes. Increasing extracellularly secreted ONCM in DRGs sufficiently enhanced the capacity of neurite outgrowth. Intraganglionic injection of REPL-NG/ONCM complex allowed sustained ONCM activity in DRG tissue and achieved a remarkable long-range regeneration of dorsal column sensory axons beyond spinal cord lesion. Conclusion: NG-mediated ONCM delivery could be exploited as a therapeutic strategy for promoting sensory axon regeneration following spinal cord injury.

Impaired Proteolysis of Noncanonical RAS Proteins Drives Clonal Hematopoietic Transformation.

Recently, screens for mediators of resistance to FLT3 and ABL kinase inhibitors in leukemia resulted in the discovery of LZTR1 as an adapter of a Cullin-3 RING E3 ubiquitin ligase complex responsible for the degradation of RAS GTPases. In parallel, dysregulated LZTR1 expression via aberrant splicing and mutations was identified in clonal hematopoietic conditions. Here we identify that loss of LZTR1, or leukemia-associated mutants in the LZTR1 substrate and RAS GTPase RIT1 that escape degradation, drives hematopoietic stem cell (HSC) expansion and leukemia in vivo. Although RIT1 stabilization was sufficient to drive hematopoietic transformation, transformation mediated by LZTR1 loss required MRAS. Proteolysis targeting chimeras (PROTAC) against RAS or reduction of GTP-loaded RAS overcomes LZTR1 loss-mediated resistance to FLT3 inhibitors. These data reveal proteolysis of noncanonical RAS proteins as novel regulators of HSC self-renewal, define the function of RIT1 and LZTR1 mutations in leukemia, and identify means to overcome drug resistance due to LZTR1 downregulation. SIGNIFICANCE: Here we identify that impairing proteolysis of the noncanonical RAS GTPases RIT1 and MRAS via LZTR1 downregulation or leukemia-associated mutations stabilizing RIT1 enhances MAP kinase activation and drives leukemogenesis. Reducing the abundance of GTP-bound KRAS and NRAS overcomes the resistance to FLT3 kinase inhibitors associated with LZTR1 downregulation in leukemia. This article is highlighted in the In This Issue feature, p. 2221.

Aberrant EVI1 splicing contributes to EVI1-rearranged leukemia.

Detailed genomic and epigenomic analyses of MECOM (the MDS1 and EVI1 complex locus) have revealed that inversion or translocation of chromosome 3 drives inv(3)/t(3;3) myeloid leukemias via structural rearrangement of an enhancer that upregulates transcription of EVI1. Here, we identify a novel, previously unannotated oncogenic RNA-splicing derived isoform of EVI1 that is frequently present in inv(3)/t(3;3) acute myeloid leukemia (AML) and directly contributes to leukemic transformation. This EVI1 isoform is generated by oncogenic mutations in the core RNA splicing factor SF3B1, which is mutated in >30% of inv(3)/t(3;3) myeloid neoplasm patients and thereby represents the single most commonly cooccurring genomic alteration in inv(3)/t(3;3) patients. SF3B1 mutations are statistically uniquely enriched in inv(3)/t(3;3) myeloid neoplasm patients and patient-derived cell lines compared with other forms of AML and promote mis-splicing of EVI1 generating an in-frame insertion of 6 amino acids at the 3' end of the second zinc finger domain of EVI1. Expression of this EVI1 splice variant enhanced the self-renewal of hematopoietic stem cells, and introduction of mutant SF3B1 in mice bearing the humanized inv(3)(q21q26) allele resulted in generation of this novel EVI1 isoform in mice and hastened leukemogenesis in vivo. The mutant SF3B1 spliceosome depends upon an exonic splicing enhancer within EVI1 exon 13 to promote usage of a cryptic branch point and aberrant 3' splice site within intron 12 resulting in the generation of this isoform. These data provide a mechanistic basis for the frequent cooccurrence of SF3B1 mutations as well as new insights into the pathogenesis of myeloid leukemias harboring inv(3)/t(3;3).

AKT constitutes a signal-promoted alternative exon-junction complex that regulates nonsense-mediated mRNA decay.

Despite a long appreciation for the role of nonsense-mediated mRNA decay (NMD) in destroying faulty, disease-causing mRNAs and maintaining normal, physiologic mRNA abundance, additional effectors that regulate NMD activity in mammalian cells continue to be identified. Here, we describe a haploid-cell genetic screen for NMD effectors that has unexpectedly identified 13 proteins constituting the AKT signaling pathway. We show that AKT supersedes UPF2 in exon-junction complexes (EJCs) that are devoid of RNPS1 but contain CASC3, defining an unanticipated insulin-stimulated EJC. Without altering UPF1 RNA binding or ATPase activity, AKT-mediated phosphorylation of the UPF1 CH domain at T151 augments UPF1 helicase activity, which is critical for NMD and also decreases the dependence of helicase activity on ATP. We demonstrate that upregulation of AKT signaling contributes to the hyperactivation of NMD that typifies Fragile X syndrome, as exemplified using FMR1-KO neural stem cells derived from induced pluripotent stem cells.

Modulation of IKs channel-PIP2 interaction by PRMT1 plays a critical role in the control of cardiac repolarization.

Recent studies have shown that protein arginine methyltransferase 1 (PRMT1) is highly expressed in the human heart, and loss of PRMT1 contributes to cardiac remodeling in the heart failure. However, the functional importance of PRMT1 in cardiac ion channels remains uncertain. The slow activating delayed rectifier K+ (IKs ) channel is a cardiac K+ channel composed of KCNQ1 and KCNE1 subunits and is a new therapeutic target for treating lethal arrhythmias in many cardiac pathologies, especially heart failure. Here, we demonstrate that PRMT1 is a critical regulator of the IKs channel and cardiac rhythm. In the guinea pig ventricular myocytes, treatment with furamidine, a PRMT1-specific inhibitor, prolonged the action potential duration (APD). We further show that this APD prolongation was attributable to IKs reduction. In HEK293T cells expressing human KCNQ1 and KCNE1, inhibiting PRMT1 via furamidine reduced IKs and concurrently decreased the arginine methylation of KCNQ1, a pore-forming α-subunit. Evidence presented here indicates that furamidine decreased IKs mainly by lowering the affinity of IKs channels for the membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2 ), which is crucial for pore opening. Finally, applying exogenous PIP2 to cardiomyocytes prevented the furamidine-induced IKs reduction and APD prolongation. Taken together, these results indicate that PRMT1 positively regulated IKs activity through channel-PIP2 interaction, thereby restricting excessive cardiac action potential.

Synthetic introns enable splicing factor mutation-dependent targeting of cancer cells.

Many cancers carry recurrent, change-of-function mutations affecting RNA splicing factors. Here, we describe a method to harness this abnormal splicing activity to drive splicing factor mutation-dependent gene expression to selectively eliminate tumor cells. We engineered synthetic introns that were efficiently spliced in cancer cells bearing SF3B1 mutations, but unspliced in otherwise isogenic wild-type cells, to yield mutation-dependent protein production. A massively parallel screen of 8,878 introns delineated ideal intronic size and mapped elements underlying mutation-dependent splicing. Synthetic introns enabled mutation-dependent expression of herpes simplex virus-thymidine kinase (HSV-TK) and subsequent ganciclovir (GCV)-mediated killing of SF3B1-mutant leukemia, breast cancer, uveal melanoma and pancreatic cancer cells in vitro, while leaving wild-type cells unaffected. Delivery of synthetic intron-containing HSV-TK constructs to leukemia, breast cancer and uveal melanoma cells and GCV treatment in vivo significantly suppressed the growth of these otherwise lethal xenografts and improved mouse host survival. Synthetic introns provide a means to exploit tumor-specific changes in RNA splicing for cancer gene therapy.

Travel Time for Dental Care Services Based on Patient Preference in South Korea.

This study analyzed patient preferences using travel time from residence to dental institution when selecting dental care services. We used data from the Korean Health Panel from 2008 to 2017 and analyzed each dental service episode. Since the distribution of travel time was skewed to the left, median travel time was analyzed. The association of travel time with services was analyzed via the population-averaged generalized estimating equation (GEE) with the Poisson family. The median of the average travel time per episode was longer for non-National Health Insurance (NHI)-covered services and shorter for NHI-covered services. The first quintile of low-income subjects traveled the longest for all services and utilized dental care the most. In the GEE analysis, travel time was approximately three times longer for implant treatment and gold inlay/resin fillings and >2 times longer for orthodontic care than for NHI-covered services. Patients residing in rural counties traveled for longer than residents of large cities. Income was statistically significant; however, the coefficient was close to zero. Travel time was related to the type of service and reflected patient preference. This was more prominent for expensive non-NHI-covered services than for NHI-covered services. The findings suggest patients' subjective preferences for dental clinic selection are expressed as rational deliberation considering each individual's situation.

The effect of ultrasound-guided transversus abdominis plane block on pulmonary function in patients undergoing laparoscopic cholecystectomy: a prospective randomized study.

BACKGROUND: Transversus abdominis plane block (TAPB) is commonly used for postoperative pain control after laparoscopic cholecystectomy. However, few studies have analyzed its effect on pulmonary function. The goal of this study was to elucidate the effect of ultrasound-guided bilateral TAPB on pulmonary function preservation and analgesia after laparoscopic cholecystectomy. METHODS: We enrolled 58 patients who underwent laparoscopic cholecystectomy. Among them, 53 were randomized to group T (n = 27) and group C (n = 26). Group T and group C received ultrasound-guided bilateral TAPB with 40 ml of 0.375% ropivacaine and 40 ml of 0.9% normal saline, respectively. Visual analog scale (VAS) scores, patient-controlled analgesia (PCA) consumption, forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), FEV1/FVC, peak expiratory flow rate (PEF), and modified Borg scale scores were measured until 24 h post-surgery. RESULTS: The VAS scores were significantly lower in group T than in group C at 1 and 8 h after the surgery. PCA consumption was significantly lower in group T than in group C at all postoperative time points. FEV1, PEF, and FEV1/FVC were more preserved in group T than in group C at 1 h. Group T had significantly lower modified Borg scale scores than did group C at 1 and 8 h. CONCLUSION: Ultrasound-guided TAPB is effective in pulmonary function preservation and pain control after laparoscopic cholecystectomy. Therefore, it could be a great option for multimodal analgesia, preservation of pulmonary function, prevention of pulmonary complications including atelectasis, and promotion of postoperative recovery after laparoscopic cholecystectomy. CLINICAL REGISTRATION: This study was enrolled in the Clinical Research Information Service (Clinical Research Information Service, KCT0004435, Hwa Yong Shin, 2019-08-19).

Mechanisms of Resistance to Noncovalent Bruton's Tyrosine Kinase Inhibitors.

BACKGROUND: Covalent (irreversible) Bruton's tyrosine kinase (BTK) inhibitors have transformed the treatment of multiple B-cell cancers, especially chronic lymphocytic leukemia (CLL). However, resistance can arise through multiple mechanisms, including acquired mutations in BTK at residue C481, the binding site of covalent BTK inhibitors. Noncovalent (reversible) BTK inhibitors overcome this mechanism and other sources of resistance, but the mechanisms of resistance to these therapies are currently not well understood. METHODS: We performed genomic analyses of pretreatment specimens as well as specimens obtained at the time of disease progression from patients with CLL who had been treated with the noncovalent BTK inhibitor pirtobrutinib. Structural modeling, BTK-binding assays, and cell-based assays were conducted to study mutations that confer resistance to noncovalent BTK inhibitors. RESULTS: Among 55 treated patients, we identified 9 patients with relapsed or refractory CLL and acquired mechanisms of genetic resistance to pirtobrutinib. We found mutations (V416L, A428D, M437R, T474I, and L528W) that were clustered in the kinase domain of BTK and that conferred resistance to both noncovalent BTK inhibitors and certain covalent BTK inhibitors. Mutations in BTK or phospholipase C gamma 2 (PLCγ2), a signaling molecule and downstream substrate of BTK, were found in all 9 patients. Transcriptional activation reflecting B-cell-receptor signaling persisted despite continued therapy with noncovalent BTK inhibitors. CONCLUSIONS: Resistance to noncovalent BTK inhibitors arose through on-target BTK mutations and downstream PLCγ2 mutations that allowed escape from BTK inhibition. A proportion of these mutations also conferred resistance across clinically approved covalent BTK inhibitors. These data suggested new mechanisms of genomic escape from established covalent and novel noncovalent BTK inhibitors. (Funded by the American Society of Hematology and others.).

Beyond hydrophilic polymers in amphiphilic polymer-based self-assembled NanoCarriers: Small hydrophilic carboxylate-capped disulfide drug delivery system and its multifunctionality and multispatial targetability.

Due to increasing safety and intracellular delivery concerns about hydrophilic polymers in amphiphilic polymer-based nanoparticles (NPs), this study investigates small hydrophilic molecule-stabilized NPs for effective intracellular delivery with multiorganelle targetability and dual responsiveness to acidic pH/glutathione (GSH). In the construction of small hydrophilic molecule-stabilized NP (MSPCL-NP), the A-B-A-type amphiphilic polymer (MSPCL-P) is composed of two short hydrophilic carboxylate-capped disulfide derivatives (A) that replace hydrophilic polymers and assist in providing colloidal stability and preventing antibody (e.g., at least anti-PEG antibody)-mediated specific interactions and complement activation in the plasma and a hydrophobic multiple disulfide-containing poly(ε-caprolactone) block (B) that carries hydrophobic drugs. The carboxylates on the surface of MSPCL-NP target the acidic extratumoral/endolysosomal milieu by sensing and buffering acidic pH values, and the hydrophobic carboxylic acids improve adsorptive endocytosis and effective endosomal escape. Multiple disulfide linkages selectively target cytosolic GSH, resulting in rapid drug release from the destroyed MSPCL-NP via the cleavage of disulfide bonds in MSPCL-P. Doxorubicin (DOX)-loaded NP (DOX@MSPCL-NP) exerts strong effects on killing cells in vitro and inhibits tumor growth in HCT116 xenograft tumor-bearing mice. In conclusion, the multifunctionality and multispatial targetability of MSPCL-NP might effectively overcome various sequential drug delivery hurdles, ranging from blood circulation to drug release. Furthermore, the introduction of small hydrophilic molecules represents a potential strategy to make self-assembled NPs without the use of hydrophilic polymers.