The gut microbiome’s role in the pathogenesis of hematologic malignancies is actively being explored; yet studies on chronic lymphocytic leukemia (CLL) are limited. Using the Eμ-TCL1 murine model of CLL, we identify a unique and dysbiotic disease-associated gut microbiome that develops in mice over time. Leukemic mice show an increase in abundance of pathogenic bacteria, specifically members of the Proteobacteria phylum. We found that this dysbiotic microenvironment is associated with CLL involvement within the intestinal tract and high levels of markers indicative of altered tight junction permeability (e.g., claudin-2, soluble CD14, and zonulin). Moreover, utilizing the syngeneic model of CLL in tandem with an antibiotic-mediated microflora ablation approach, we found that leukemic mice receiving microflora-ablating antibiotics show marked changes to the gut microbiome and a delayed disease onset compared with mice receiving antibiotic-free water. Immunophenotyping of murine spleens showed that this delay in disease was accompanied by more tumor-reactive CD8+ T cells that coexpressed fewer inhibitory receptors (e.g., PD-1, LAG-3, and TIM-3). These findings confirm that a dysbiotic gut microbiome develops during CLL disease and demonstrate unique intestinal involvement and potential immune dysregulation occurring during CLL progression that may be influencing the overall microbial signature. Significance: There is a growing appreciation for the gut microbiome’s role in hematologic malignancies. Despite this, its role in CLL remains obscure. This study demonstrates a dysbiotic microbiome within CLL that may contribute to further intestinal and immune dysregulation present during CLL progression.

Background: B-cell receptor (BCR) signaling is a central driver in chronic lymphocytic leukemia (CLL), along with the activation of pro-survival pathways (e.g., NF-κB) and aberrant anti-apoptotic mechanisms (e.g., BCL2) culminating to CLL cell survival and drug resistance. Front-line targeted therapies such as ibrutinib (BTK inhibitor) and venetoclax (BCL2 inhibitor) have radically improved CLL management. Yet, persisting CLL cells lead to relapse in ~20% of patients, signifying the unmet need of inhibitor-resistant refractory CLL. SpiD3 is a novel spirocyclic dimer of analog 19 that displays NF-κB inhibitory activity and preclinical anti-cancer properties. Recently, we have shown that SpiD3 inhibits CLL cell proliferation and induces cytotoxicity by promoting futile activation of the unfolded protein response (UPR) pathway and generation of reactive oxygen species (ROS), resulting in the inhibition of protein synthesis in CLL cells. Methods: We performed RNA-sequencing using CLL cells rendered resistant to ibrutinib and venetoclax to explore potential vulnerabilities in inhibitor-resistant and SpiD3-treated CLL cells. Results: The transcriptomic analysis of ibrutinib- or venetoclax-resistant CLL cell lines revealed ferroptosis, UPR signaling, and oxidative stress to be among the top pathways modulated by SpiD3 treatment. By examining SpiD3-induced protein aggregation, ROS production, and ferroptosis in inhibitor-resistant CLL cells, our findings demonstrate cytotoxicity following SpiD3 treatment in cell lines resistant to current front-line CLL therapeutics. Conclusions: Our results substantiate the development of SpiD3 as a novel therapeutic agent for relapsed/refractory CLL disease.

Background: Chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia in the Western world. Targeted therapies have made CLL manageable for many patients, but the ongoing threat of disease relapse or transformation beckons a deeper understanding of CLL pathogenesis, and thus, its durable eradication. This study identifies bile acids (BAs) as elevated in the peripheral blood of CLL patients and a murine model of CLL, in comparison to healthy controls. Elevated BA concentrations have been associated with intestinal malignancies and immunomodulation; however, their role in CLL is relatively unknown. Methods: Metabolomic analysis was performed on murine and human plasma. Flow cytometry analysis of CLL patient B-cells and healthy donor T-cells were utilized to evaluate the immunomodulatory impact of differentially abundant BAs. Results: Herein, BAs were found to be differentially abundant in CLL. Elevated BAs demonstrated minimal impact on CLL cell proliferation or CLL-associated T-cell function. Conclusions: Future studies are needed to determine the mechanistic influence of BAs on CLL pathogenesis.

Background: Targeted therapies (e.g., ibrutinib) have markedly improved chronic lymphocytic leukemia (CLL) management; however, ~20% of patients experience disease relapse, suggesting the inadequate depth and durability of these front-line strategies. Moreover, immunotherapeutic success in CLL has been stifled by its pro-tumor microenvironment milieu and low mutational burden, cultivating poor antigenicity and limited ability to generate anti-tumor immunity through adaptive immune cell engagement. Previously, we have demonstrated how a three-carbon-linker spirocyclic dimer (SpiD3) promotes futile activation of the unfolded protein response (UPR) in CLL cells through immense misfolded-protein mimicry, culminating in insurmountable ER stress and programmed CLL cell death. Method: Herein, we used flow cytometry and cell-based assays to capture the kinetics and magnitude of SpiD3-induced damage-associated molecular patterns (DAMPs) in CLL cell lines and primary samples. Result: SpiD3 treatment, in vitro and in vivo, demonstrated the capacity to propagate immunogenic cell death through emissions of classically immunogenic DAMPs (CALR, ATP, HMGB1) and establish a chemotactic gradient for bone marrow-derived dendritic cells. Conclusions: Thus, this study supports future investigation into the relationship between novel therapeutics, manners of cancer cell death, and their contributions to adaptive immune cell engagement as a means for improving anti-cancer therapy in CLL.

Background: Myeloid-derived suppressor cells (MDSCs) contribute to immune suppression observed in chronic lymphocytic leukemia (CLL). MDSCs are immature myeloid cells that are hijacked during development and further reprogrammed by the tumor microenvironment (TME) to harbor immune-suppressive properties and inhibit T-cell functions. Bromodomain and extraterminal domain (BET) proteins, including BRD4, are epigenetic modulators that regulate genes implicated in CLL pathogenesis and TME interactions. Previously, we investigated how the novel BET inhibitor OPN-51107 (OPN5) prevents CLL disease expansion, modulates T-cell immune function, and alters gene expression related to MDSCs. In turn, we hypothesize that BET proteins such as BRD4 regulate MDSC functions, and subsequent pharmacological inhibition of BRD4 will alleviate MDSC-mediated immune suppression in CLL. Methods: Utilizing the Eµ-TCL1 mouse model of CLL, we evaluated BRD4 protein expression in MDSCs derived from the bone marrow of transgenic and age-matched wild-type (WT) mice. We then investigated the ex vivo functionality of OPN5-treated MDSCs, expanded from Eµ-TCL1 and WT bone marrow in MDSC-supportive medium. Finally, we conducted an in vivo study utilizing the Eµ-TCL1 adoptive transfer mouse model to determine the in vivo effects of OPN5 on MDSCs and other immune populations. Results: Through the course of this study, we found that MDSCs isolated from Eμ-TCL1 mice upregulate BRD4 expression and are more immune-suppressive than their WT counterparts. Furthermore, we demonstrated ex vivo OPN5 treatment reverses the immune-suppressive capacity of MDSCs isolated from leukemic mice, evident via enhanced T-cell proliferation and IFNγ production. Finally, we showed in vivo OPN5 treatment slows CLL disease progression and modulates immune cell populations, including MDSCs. Conclusions: Altogether, these data support BET inhibition as a useful therapeutic approach to reverse MDSC-mediated immune suppression in CLL.

Dysbiotic microbiome is observed in the aggressive syngeneic model of CLL. A, Study schematic illustrating WT B6 mice were engrafted intravenously via the tail vein with 1e7 spleen-derived lymphocytes from a moribund Eμ-TCL1 mouse (CLL engraftment). Engrafted mice (n = 10) were monitored with serial blood and fecal pellet sample collections over the study course of 7 weeks. B, CLL disease burden was measured by the percentage of CD45+/CD19+/CD5+ cells in the peripheral blood via flow cytometric analysis starting 1 week after engraftment. Asterisks denote the significance between leukemic mice over time (***, P < 0.001). One-way ANOVA with Dunnett multiple comparisons was applied for testing. C, Alpha diversity measured by the Shannon index in leukemic mice over 7 weeks. Data are presented as box-and-whisker plots, with box edges representing the 25th and 75th percentiles, the center line showing the median value, and whiskers extending to minimum and maximum values. One-way ANOVA with Dunnett multiple comparisons was applied for testing. D, PCoA plot depicting Bray–Curis distance as a measure of beta diversity in leukemic mice over 7 weeks. E, Relative abundance bar plots of bacterial taxa present in the gut microbiome of diseased mice over 7 weeks following engraftment. Unassigned includes all remaining taxa present in the microbiome at decreased abundance. F, LEfSe analysis demonstrating enrichment of specific bacterial taxa at the phylum level in leukemic mice at the time of engraftment and 1 and 3 weeks after engraftment. Eng./engraft., engraftment. LDA, linear discriminant analysis.