Analysis of the Effector Functions of Vδ2 γδ T Cells and NK Cells against Cholangiocarcinoma Cells.

Cholangiocarcinoma (CCA) is a rare disease characterized by malignant cells derived from the epithelial cells of the biliary duct system. Despite extensive treatments, the prognosis for CCA remains poor, emphasizing the critical need for the development of novel treatments. Considerable attention has been directed towards innate immune effector cells, which can recognize tumor cells independently of the major histocompatibility complex, laying the foundation for the development of off-the-shelf drugs. In this study, we cultured innate immune cells obtained from the peripheral blood of healthy adults and conducted a comparative analysis of the effector functions against CCA cell lines by Vδ2 γδ T cells and NK cells. This analysis was performed using standard short- and long-term cytotoxicity assays, as well as ELISA for IFN-γ. Vδ2 γδ T cells demonstrated cytotoxicity and IFN-γ production in response to CCA cells in a TCR-dependent manner, particularly in the presence of tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1-bisphosphonate, a bisphosphonate prodrug. In contrast, direct killing and antibody-dependent cellular cytotoxicity were relatively slow and weak. Conversely, NK cells displayed potent, direct cytotoxicity against CCA cells. In summary, both Vδ2 γδ T cells and NK cells show promise as innate immune effector cells for adoptive transfer therapy in the context of CCA.

Longitudinal patterns of alcohol and cannabis use among US young adults: Correlates and implications for problematic health outcomes.

INTRODUCTION: Alcohol and cannabis use are common during young adulthood. Less is known regarding correlates of alcohol-cannabis use patterns and related problematic health outcomes. METHODS: Using longitudinal survey data (Fall 2018, 2019, 2020) from 2,194 young adults (YAs; ages 18-34), bivariate analyses and multivariable logistic regressions examined: (1) Wave 1 (W1) sociodemographics and psychosocial factors (i.e., adverse childhood experiences [ACEs], depressive symptoms, personality traits, parent and peer alcohol and cannabis use) in relation to W3 past-month use group (i.e., use of neither, alcohol only, cannabis only, both/co-use); and (2) W3 use group in relation to W5 problematic alcohol use (Alcohol Use Disorder Identification Test), problematic cannabis use (Cannabis Use Disorder Identification Test), and depressive/anxiety symptoms (Patient Health Questionnaire - 4 item). RESULTS: Overall, 42.3% reported W3 alcohol-only use, 34.9% co-use, 17.8% no use, and 5.0% cannabis-only use. Those reporting W3 co-use reported greater W1 extraversion, openness, friend alcohol/cannabis use, and were more likely to report parent cannabis use (vs. no use); reported less conscientiousness, greater friend cannabis use, and were more likely to report depressive symptoms and parent cannabis use (vs. alcohol-only use); and reported greater friend alcohol use, and were more likely to report parent alcohol use (vs. cannabis-only use). W3 co-use was associated with higher odds of W5 problematic alcohol use (vs. alcohol-only use) and problematic cannabis use (vs. cannabis-only use). CONCLUSIONS: Substance use messaging and interventions should consider YAs' alcohol-cannabis co-use and the unique correlates of such use.

Positive and Negative Gender Role Beliefs and Intimate Partner Violence.

An ethnically diverse sample of 384 male and female undergraduates was assessed for their gender role beliefs based on positive (family responsibility) vs. negative (male dominance and female submissiveness) aspects derived from Hispanic cultural traditions. Negative male and female gender role beliefs were significantly positively correlated with reported victimization by and perpetration of severe intimate partner violence (IPV) for both men and women. Positive male gender role beliefs were negatively correlated with reported victimization by and perpetration of IPV for both men and women, with women also providing some evidence that positive female gender role beliefs were associated with less IPV.

Proximity to humans is associated with antimicrobial-resistant enteric pathogens in wild bird microbiomes.

Humans are radically altering global ecology, and one of the most apparent human-induced effects is urbanization, where high-density human habitats disrupt long-established ecotones. Changes to these transitional areas between organisms, especially enhanced contact among humans and wild animals, provide new opportunities for the spread of zoonotic pathogens. This poses a serious threat to global public health, but little is known about how habitat disruption impacts cross-species pathogen spread. Here, we investigated variation in the zoonotic enteric pathogen Campylobacter jejuni. The ubiquity of C. jejuni in wild bird gut microbiomes makes it an ideal organism for understanding how host behavior and ecology influence pathogen transition and spread. We analyzed 700 C. jejuni isolate genomes from 30 bird species in eight countries using a scalable generalized linear model approach. Comparing multiple behavioral and ecological traits showed that proximity to human habitation promotes lineage diversity and is associated with antimicrobial-resistant (AMR) strains in natural populations. Specifically, wild birds from urban areas harbored up to three times more C. jejuni genotypes and AMR genes. This study provides novel methodology and much-needed quantitative evidence linking urbanization to gene pool spread and zoonoses.

Predictive Models for Long Term Survival of AML Patients Treated with Venetoclax and Azacitidine or 7+3 Based on Post Treatment Events and Responses: Retrospective Cohort Study.

BACKGROUND: The treatment of acute myeloid leukemia (AML) in older or unfit patients typically involves a regimen of venetoclax plus azacitidine (ven/aza). Toxicity and treatment responses are highly variable following treatment initiation and clinical decision-making continually evolves in response to these as treatment progresses. To improve clinical decision support (CDS) following treatment initiation, predictive models based on evolving and dynamic toxicities, disease responses, and other features should be developed. OBJECTIVE: This study aims to generate machine learning (ML)-based predictive models that incorporate individual predictors of overall survival (OS) for patients with AML, based on clinical events occurring after the initiation of ven/aza or 7+3 regimen. METHODS: Data from 221 patients with AML, who received either the ven/aza (n=101 patients) or 7+3 regimen (n=120 patients) as their initial induction therapy, were retrospectively analyzed. We performed stratified univariate and multivariate analyses to quantify the association between toxicities, hospital events, and short-term disease responses and OS for the 7+3 and ven/aza subgroups separately. We compared the estimates of confounders to assess potential effect modifications by treatment. 17 ML-based predictive models were developed. The optimal predictive models were selected based on their predictability and discriminability using cross-validation. Uncertainty in the estimation was assessed through bootstrapping. RESULTS: The cumulative incidence of posttreatment toxicities varies between the ven/aza and 7+3 regimen. A variety of laboratory features and clinical events during the first 30 days were differentially associated with OS for the two treatments. An initial transfer to intensive care unit (ICU) worsened OS for 7+3 patients (aHR 1.18, 95% CI 1.10-1.28), while ICU readmission adversely affected OS for those on ven/aza (aHR 1.24, 95% CI 1.12-1.37). At the initial follow-up, achieving a morphologic leukemia free state (MLFS) did not affect OS for ven/aza (aHR 0.99, 95% CI 0.94-1.05), but worsened OS following 7+3 (aHR 1.16, 95% CI 1.01-1.31) compared to that of complete remission (CR). Having blasts over 5% at the initial follow-up negatively impacted OS for both 7+3 (P<.001) and ven/aza (P<.001) treated patients. A best response of CR and CR with incomplete recovery (CRi) was superior to MLFS and refractory disease after ven/aza (P<.001), whereas for 7+3, CR was superior to CRi, MLFS, and refractory disease (P<.001), indicating unequal outcomes. Treatment-specific predictive models, trained on 120 7+3 and 101 ven/aza patients using over 114 features, achieved survival AUCs over 0.70. CONCLUSIONS: Our findings indicate that toxicities, clinical events, and responses evolve differently in patients receiving ven/aza compared with that of 7+3 regimen. ML-based predictive models were shown to be a feasible strategy for CDS in both forms of AML treatment. If validated with larger and more diverse data sets, these findings could offer valuable insights for developing AML-CDS tools that leverage posttreatment clinical data.

A Novel Role for the Histone Demethylase JMJD3 in Mediating Heroin-Induced Relapse-Like Behaviors.

BACKGROUND: Epigenetic changes that lead to long-term neuroadaptations following opioid exposure are not well understood. We examined how histone demethylase JMJD3 in the nucleus accumbens (NAc) influences heroin seeking after abstinence from self-administration. METHODS: Male Sprague Dawley rats were trained to self-administer heroin. Western blotting and quantitative polymerase chain reaction were performed to quantify JMJD3 and bone morphogenetic protein (BMP) pathway expression in the NAc (n = 7-11/group). Pharmacological inhibitors or viral expression vectors were microinfused into the NAc to manipulate JMJD3 or the BMP pathway member SMAD1 (n = 9-11/group). The RiboTag capture method (n = 3-5/group) and viral vectors (n = 7-8/group) were used in male transgenic rats to identify the contributions of D1- and D2-expressing medium spiny neurons in the NAc. Drug seeking was tested by cue-induced response previously paired with drug infusion. RESULTS: Levels of JMJD3 and phosphorylated SMAD1/5 in the NAc were increased after 14 days of abstinence from heroin self-administration. Pharmacological and virus-mediated inhibition of JMJD3 or the BMP pathway attenuated cue-induced seeking. Pharmacological inhibition of BMP signaling reduced JMJD3 expression and H3K27me3 levels. JMJD3 bidirectionally affected seeking: expression of the wild-type increased cue-induced seeking whereas expression of a catalytic dead mutant decreased it. JMJD3 expression was increased in D2+ but not D1+ medium spiny neurons. Expression of the mutant JMJD3 in D2+ neurons was sufficient to decrease cue-induced heroin seeking. CONCLUSIONS: JMJD3 mediates persistent cellular and behavioral adaptations that underlie heroin relapse, and this activity is regulated by the BMP pathway.

A new approach to RNA synthesis: immobilization of stably and functionally co-tethered promoter DNA and T7 RNA polymerase.

Current approaches to RNA synthesis/manufacturing require substantial (and incomplete) purification post-synthesis. We have previously demonstrated the synthesis of RNA from a complex in which T7 RNA polymerase is tethered to promoter DNA. In the current work, we extend this approach to demonstrate an extremely stable system of functional co-tethered complex to a solid support. Using the system attached to magnetic beads, we carry out more than 20 rounds of synthesis using the initial polymerase-DNA construct. We further demonstrate the wide utility of this system in the synthesis of short RNA, a CRISPR guide RNA, and a protein-coding mRNA. In all cases, the generation of self-templated double stranded RNA (dsRNA) impurities are greatly reduced, by both the tethering itself and by the salt-tolerance that local co-tethering provides. Transfection of the mRNA into HEK293T cells shows a correlation between added salt in the transcription reaction (which inhibits RNA rebinding that generates RNA-templated extensions) and significantly increased expression and reduced innate immune stimulation by the mRNA reaction product. These results point in the direction of streamlined processes for synthesis/manufacturing of high-quality RNA of any length, and at greatly reduced costs.

Lysosomal acid lipase A modulates leukemia stem cell response to venetoclax/tyrosine kinase inhibitor combination therapy in blast phase chronic myeloid leukemia.

The treatment of blast phase chronic myeloid leukemia (bpCML) remains a challenge due at least in part to drug resistance of leukemia stem cells (LSCs). Recent clinical evidence suggests that the BCL-2 inhibitor venetoclax in combination with ABL-targeting tyrosine kinase inhibitors (TKIs) can eradicate bpCML LSCs. In this report, we employed preclinical models of bpCML to investigate the efficacy and underlying mechanism of LSC-targeting with venetoclax/TKI combinations. Transcriptional analysis of LSCs exposed to venetoclax and dasatinib revealed upregulation of genes involved in lysosomal biology, in particular lysosomal acid lipase A (LIPA), a regulator of free fatty acids. Metabolomic analysis confirmed increased levels of free fatty acids in response to venetoclax/dasatinib. Pre-treatment of leukemia cells with bafilomycin, a specific lysosome inhibitor, or genetic perturbation of LIPA, resulted in increased sensitivity of leukemia cells toward venetoclax/dasatinib, implicating LIPA in treatment resistance. Importantly, venetoclax/dasatinib treatment does not affect normal stem cell function, suggestive of a leukemia-specific response. These results demonstrate that venetoclax/dasatinib is an LSCselective regimen in bpCML and that disrupting LIPA and fatty acid transport enhances venetoclax/dasatinib response in targeting LSCs, providing a rationale for exploring lysosomal disruption as an adjunct therapeutic strategy to prolong disease remission.

In silico predicted compound targeting the IQGAP1-GRD domain selectively inhibits growth of human acute myeloid leukemia.

Acute myeloid leukemia (AML) is fatal in the majority of adults. Identification of new therapeutic targets and their pharmacologic modulators are needed to improve outcomes. Previous studies had shown that immunization of rabbits with normal peripheral WBCs that had been incubated with fluorodinitrobenzene elicited high titer antibodies that bound to a spectrum of human leukemias. We report that proteomic analyses of immunoaffinity-purified lysates of primary AML cells showed enrichment of scaffolding protein IQGAP1. Immunohistochemistry and gene-expression analyses confirmed IQGAP1 mRNA overexpression in various cytogenetic subtypes of primary human AML compared to normal hematopoietic cells. shRNA knockdown of IQGAP1 blocked proliferation and clonogenicity of human leukemia cell-lines. To develop small molecules targeting IQGAP1 we performed in-silico screening of 212,966 compounds, selected 4 hits targeting the IQGAP1-GRD domain, and conducted SAR of the 'fittest hit' to identify UR778Br, a prototypical agent targeting IQGAP1. UR778Br inhibited proliferation, induced apoptosis, resulted in G2/M arrest, and inhibited colony formation by leukemia cell-lines and primary-AML while sparing normal marrow cells. UR778Br exhibited favorable ADME/T profiles and drug-likeness to treat AML. In summary, AML shows response to IQGAP1 inhibition, and UR778Br, identified through in-silico studies, selectively targeted AML cells while sparing normal marrow.

PKMYT1 is a Marker of Treatment Response and a Therapeutic Target for CDK4/6 Inhibitor-Resistance in ER+ Breast Cancer.

Endocrine therapies (ET) with CDK4/6 inhibition are the standard treatment for estrogen receptor-α-positive (ER+) breast cancer, however drug resistance is common. In this study, proteogenomic analyses of 22 ER+ breast cancer patient-derived xenografts (PDXs) demonstrated that PKMYT1, a WEE1 homolog, is estradiol (E2) regulated in E2-dependent PDXs and constitutively expressed when growth is E2-independent. In clinical samples, high PKMYT1 mRNA levels associated with resistance to both ET and CDK4/6 inhibition. The PKMYT1 inhibitor lunresertib (RP-6306) with gemcitabine selectively and synergistically reduced the viability of ET and palbociclib-resistant ER+ breast cancer cells without functional p53. In vitro the combination increased DNA damage and apoptosis. In palbociclib-resistant, TP53 mutant PDX organoids and xenografts, RP-6306 with low-dose gemcitabine induced greater tumor volume reduction compared to treatment with either single agent. Our study demonstrates the clinical potential of RP-6306 in combination with gemcitabine for ET and CDK4/6 inhibitor resistant TP53 mutant ER+ breast cancer.

MYC Inhibition Potentiates CD8+ T Cells Against Multiple Myeloma and Overcomes Immunomodulatory Drug Resistance.

PURPOSE: Immunomodulatory drugs (IMiDs), such as lenalidomide and pomalidomide, are a cornerstone of multiple myeloma (MM) therapies, yet the disease inevitably becomes refractory. IMiDs exert cytotoxicity by inducing cereblon-dependent proteasomal degradation of IKZF1 and IKZF3, resulting in downregulation of the oncogenic transcription factors IRF4 and MYC. To date, clinical IMiD resistance independent of cereblon or IKZF1/3 has not been well explored. Here, we investigated the roles of IRF4 and MYC in this context. EXPERIMENTAL DESIGN: Using bone marrow aspirates from patients with IMiD-naïve or refractory MM, we examined IKZF1/3 protein levels and IRF4/MYC gene expression following ex vivo pomalidomide treatment via flow cytometry and qPCR. We also assessed exvivo sensitivity to the MYC inhibitor MYCi975 using flow cytometry. RESULTS: We discovered that although pomalidomide frequently led to IKZF1/3 degradation in MM cells, it did not affect MYC gene expression in most IMiD-refractory samples. We subsequently demonstrated that MYCi975 exerted strong anti-MM effects in both IMiD-naïve and -refractory samples. Unexpectedly, we identified a cluster of differentiation 8+ (CD8+ T) cells from patients with MM as crucial effectors of MYCi975-induced cytotoxicity in primary MM samples, and we discovered that MYCi975 enhanced the cytotoxic functions of memory CD8+ T cells. We lastly observed synergy between MYCi975 and pomalidomide in IMiD-refractory samples, suggesting that restoring MYC downregulation can re-sensitize refractory MM to IMiDs. CONCLUSIONS: Our study supports the concept that MYC represents an Achilles' heel in MM across disease states and that MYCi975 may be a promising therapeutic for patients with MM, particularly in combination with IMiDs.

Targeting Acute Myeloid Leukemia Stem Cells Through Perturbation of Mitochondrial Calcium.

Acute myeloid leukemia stem cells (LSCs) are uniquely reliant on oxidative phosphorylation (OXPHOS) for survival. Moreover, maintenance of OXPHOS is dependent on BCL-2, creating a therapeutic opportunity to target LSCs using the BCL-2 inhibitor venetoclax. While venetoclax-based regimens have shown promising clinical activity, the emergence of drug resistance is prevalent. Thus, in the present study, we investigated how mitochondrial properties may influence venetoclax responsiveness. Our data show that utilization of mitochondrial calcium is fundamentally different between drug-responsive and non-responsive LSCs. By comparison, venetoclax-resistant LSCs demonstrate a more active metabolic (i.e. OXPHOS) status with relatively high levels of calcium. Consequently, we tested genetic and pharmacological approaches to target the mitochondrial calcium uniporter, MCU. We demonstrate that inhibition of calcium uptake reduces OXPHOS and leads to eradication of venetoclax-resistant LSCs. These findings demonstrate a central role for calcium signaling in LSCs and provide an avenue for clinical management of venetoclax resistance.

Temporal Single Cell Analysis of Leukemia Microenvironment Identifies Taurine-Taurine Transporter Axis as a Key Regulator of Myeloid Leukemia.

Signals from the microenvironment are known to be critical for development, sustaining adult stem cells, and for oncogenic progression. While candidate niche-driven signals that can promote cancer progression have been identified1-6, concerted efforts to comprehensively map microenvironmental ligands for cancer stem cell specific surface receptors have been lacking. Here, we use temporal single cell RNA-sequencing to identify molecular cues from the bone marrow stromal niche that engage leukemia stem cells (LSC) during oncogenic progression. We integrate these data with our RNA-seq analysis of human LSCs from distinct aggressive myeloid cancer subtypes and our CRISPR based in vivo LSC dependency map7 to develop a temporal receptor-ligand interactome essential for disease progression. These analyses identify the taurine transporter (TauT)-taurine axis as a critical dependency of myeloid malignancies. We show that taurine production is restricted to the osteolineage population during cancer initiation and expansion. Inhibiting taurine synthesis in osteolineage cells impairs LSC growth and survival. Our experiments with the TauT genetic loss of function murine model indicate that its loss significantly impairs the progression of aggressive myeloid leukemias in vivo by downregulating glycolysis. Further, TauT inhibition using a small molecule strongly impairs the growth and survival of patient derived myeloid leukemia cells. Finally, we show that TauT inhibition can synergize with the clinically approved oxidative phosphorylation inhibitor venetoclax8, 9 to block the growth of primary human leukemia cells. Given that aggressive myeloid leukemias continue to be refractory to current therapies and have poor prognosis, our work indicates targeting the taurine transporter may be of therapeutic significance. Collectively, our data establishes a temporal landscape of stromal signals during cancer progression and identifies taurine-taurine transporter signaling as an important new regulator of myeloid malignancies.

The dominance and growth of shallow groundwater resources in continuous permafrost environments.

Water is a limited resource in Arctic watersheds with continuous permafrost because freezing conditions in winter and the impermeability of permafrost limit storage and connectivity between surface water and deep groundwater. However, groundwater can still be an important source of surface water in such settings, feeding springs and large aufeis fields that are abundant in cold regions and generating runoff when precipitation is rare. Whether groundwater is sourced from suprapermafrost taliks or deeper regional aquifers will impact water availability as the Arctic continues to warm and thaw. Previous research is ambiguous about the role of deep groundwater, leading to uncertainty regarding Arctic water availability and changing water resources. We analyzed chemistry and residence times of spring, stream, and river waters in the continuous permafrost zone of Alaska, spanning the mountains to the coastal plain. Water chemistry and age tracers show that surface waters are predominately sourced from recent precipitation and have short (<50 y) subsurface residence times. Remote sensing indicates trends in the areal extent of aufeis over the last 37 y, and correlations between aufeis extent and previous year summer temperature. Together, these data indicate that surface waters in continuous permafrost regions may be impacted by short flow paths and shallow suprapermafrost aquifers that are highly sensitive to climatic and hydrologic change over annual timescales. Despite the lack of connection to regional aquifers, continued warming and permafrost thaw may promote deepening of the shallow subsurface aquifers and creation of shallow taliks, providing some resilience to Arctic freshwater ecosystems.

Bacterial community shifts of commercial apples, oranges, and peaches at different harvest points across multiple growing seasons.

Assessing the microbes present on tree fruit carpospheres as the fruit enters postharvest processing could have useful applications, as these microbes could have a major influence on spoilage, food safety, verification of packing process controls, or other aspects of processing. The goal of this study was to establish a baseline profile of bacterial communities associated with apple (pome fruit), peach (stone fruit), and Navel orange (citrus fruit) at harvest. We found that commercial peaches had the greatest bacterial richness followed by oranges then apples. Time of harvest significantly changed bacterial diversity in oranges and peaches, but not apples. Shifts in diversity varied by fruit type, where 70% of the variability in beta diversity on the apple carposphere was driven by the gain and loss of species (i.e., nestedness). The peach and orange carposphere bacterial community shifts were driven by nearly an even split between turnover (species replacement) and nestedness. We identified a small core microbiome for apples across and between growing seasons that included only Methylobacteriaceae and Sphingomonadaceae among the samples, while peaches had a larger core microbiome composed of five bacterial families: Bacillaceae, Geodermtophilaceae, Nocardioidaceae, Micrococcaeceae, and Trueperaceae. There was a relatively diverse core microbiome for oranges that shared all the families present on apples and peaches, except for Trueperaceae, but also included an additional nine bacterial families not shared including Oxalobacteraceae, Cytophagaceae, and Comamonadaceae. Overall, our findings illustrate the important temporal dynamics of bacterial communities found on major commercial tree fruit, but also the core bacterial families that constantly remain with both implications being important entering postharvest packing and processing.

Bacterial diversity and composition on the rinds of specific melon cultivars and hybrids from across different growing regions in the United States.

The goal of this study was to characterize the bacterial diversity on different melon varieties grown in different regions of the US, and determine the influence that region, rind netting, and variety of melon has on the composition of the melon microbiome. Assessing the bacterial diversity of the microbiome on the melon rind can identify antagonistic and protagonistic bacteria for foodborne pathogens and spoilage organisms to improve melon safety, prolong shelf-life, and/or improve overall plant health. Bacterial community composition of melons (n = 603) grown in seven locations over a four-year period were used for 16S rRNA gene amplicon sequencing and analysis to identify bacterial diversity and constituents. Statistically significant differences in alpha diversity based on the rind netting and growing region (p < 0.01) were found among the melon samples. Principal Coordinate Analysis based on the Bray-Curtis dissimilarity distance matrix found that the melon bacterial communities clustered more by region rather than melon variety (R2 value: 0.09 & R2 value: 0.02 respectively). Taxonomic profiling among the growing regions found Enterobacteriaceae, Bacillaceae, Microbacteriaceae, and Pseudomonadaceae present on the different melon rinds at an abundance of ≥ 0.1%, but no specific core microbiome was found for netted melons. However, a core of Pseudomonadaceae, Bacillaceae, and Exiguobacteraceae were found for non-netted melons. The results of this study indicate that bacterial diversity is driven more by the region that the melons were grown in compared to rind netting or melon type. Establishing the foundation for regional differences could improve melon safety, shelf-life, and quality as well as the consumers' health.

Expression of the chemokine receptor CCR1 decreases sensitivity to bortezomib in multiple myeloma cell lines.

BACKGROUND: The proteasome inhibitor bortezomib is one of the primary therapies used for the haematological malignancy multiple myeloma (MM). However, intrinsic or acquired resistance to bortezomib, via mechanisms that are not fully elucidated, is a barrier to successful treatment in many patients. Our previous studies have shown that elevated expression of the chemokine receptor CCR1 in MM plasma cells in newly diagnosed MM patients is associated with poor prognosis. Here, we hypothesised that the poor prognosis conferred by CCR1 expression is, in part, due to a CCR1-mediated decrease in MM plasma cell sensitivity to bortezomib. METHODS: In order to investigate the role of CCR1 in MM cells, CCR1 was knocked out in human myeloma cell lines OPM2 and U266 using CRISPR-Cas9. Additionally, CCR1 was overexpressed in the mouse MM cell line 5TGM1. The effect of bortezomib on CCR1 knockout or CCR1-overexpressing cells was then assessed by WST-1 assay, with or without CCL3 siRNA knockdown or addition of recombinant human CCL3. NSG mice were inoculated intratibially with OPM2-CCR1KO cells and were treated with 0.7 mg/kg bortezomib or vehicle twice per week for 3 weeks and GFP+ tumour cells in the bone marrow were quantitated by flow cytometry. The effect of CCR1 overexpression or knockout on unfolded protein response pathways was assessed using qPCR for ATF4, HSPA5, XBP1, ERN1 and CHOP and Western blot for IRE1α and p-Jnk. RESULTS: Using CCR1 overexpression or CRIPSR-Cas9-mediated CCR1 knockout in MM cell lines, we found that CCR1 expression significantly decreases sensitivity to bortezomib in vitro, independent of the CCR1 ligand CCL3. In addition, CCR1 knockout rendered the human MM cell line OPM2 more sensitive to bortezomib in an intratibial MM model in NSG mice in vivo. Moreover, CCR1 expression negatively regulated the expression of the unfolded protein response receptor IRE1 and downstream target gene XBP1, suggesting this pathway may be responsible for the decreased bortezomib sensitivity of CCR1-expressing cells. CONCLUSIONS: Taken together, these studies suggest that CCR1 expression may be associated with decreased response to bortezomib in MM cell lines.